Characterisation of human adipose tissue : ceramide metabolism, depot differences and evaluation of dysfunctionality

Normal adipose tissue function is necessary for maintaining proper energy balance, as both excess and absence of this tissue lead to metabolic disturbances, such as insulin resistance. Adipose tissue can be dysfunctional in many ways, including disturbances in adipocyte size, lipolysis rate, adipokine secretion, inflammation, fibrosis or oxidative stress. Recently ceramides have been proposed as candidate molecules that mediate the development of adipocyte insulin resistance. The aim of this thesis is to evaluate different aspects of dysfunctionality in human adipose depots in relation to their potential contribution to insulin resistance and cardiovascular disease. Paper I and II focus on the role of ceramides in human adipose tissue. We show that in obese women with high liver fat increased ceramide content in the subcutaneous adipose depot is most probably due to the increased sphingomyelin hydrolysis rather than de novo production. Moreover, sphingomyelinases, that are responsible for this reaction, are present in areas rich in apolipoprotein B, which may suggest that circulating lipoproteins may be a source of sphingomyelin for the local ceramide production within adipose tissue. Additionally, we show increased ceramide levels in the mediastinal as compared to the subcutaneous adipose tissue and show that ceramides in this depot are associated with inflammatory processes. In our unpublished data we demonstrate that ceramide induces inflammatory cytokine expression in both macrophages and adipocytes. Paper III investigates whether the mediastinal depot shows characteristics of brown adipose tissue. A comparison of several markers of brown and white fat between subcutaneous and mediastinal adipose tissue reveals that the mediastinal fat has higher expression levels of some brown (UCP1, PPARGC1A) and lower expression levels of white (SHOX9, HOXC8) markers. Gene ontology analysis indicates that mediastinal depot is enriched in genes related to mitochondrial function. In some sections of mediastinal fat positive UCP1 staining and presence of multilocular cells are observed. In Paper IV we investigate whether adipose tissue in patients with chronic kidney disease is dysfunctional. We report that subcutaneous adipose tissue in patients with kidney failure is characterized by higher numbers of phagocytic cells and smaller adipocytes, but shows no signs of fibrosis as compared to healthy subjects. Additionally, proteomic analysis shows differential expression patterns between the patients and controls. Among the proteins expressed at higher levels in the patients, alpha-1-microglobulin/bikunin precursor is the most significant and among those expressed at lower levels in the patients, the most significant is vimentin – a protein known to be involved in lipid droplet metabolism. In summary, the work presented in this thesis demonstrates that adipose tissue ceramides can promote local inflammation, a process strongly linked to insulin resistance. Moreover, the mediastinal adipose depot shows some signs of brown fat, however the functional consequences remain to be evaluated. Finally, uremic adipose tissue shows adverse protein composition, which together with an increased number of phagocytic cells and smaller adipocyte size indicates that uremic adipose tissue is dysfunctional, which could lead to increased cardiovascular risk in chronic kidney disease patients

The Role of Governmental Credit in Hemispheric Trade

Ion channels in the plasma membrane are important for the apoptotic process. Different types of voltage-gated ion channels are up-regulated early in the apoptotic process and block of these channels prevents or delays apoptosis. In the present investigation we examined whether ion channels are up-regulated in oocytes from the frog Xenopus laevis during apoptosis. The two-electrode voltage-clamp technique was used to record endogenous ion currents in the oocytes. During staurosporine-induced apoptosis a voltage-dependent Na(+) current increased three-fold. This current was activated at voltages more positive than 0 mV (midpoint of the open-probability curve was +55 mV) and showed almost no sign of inactivation during a 1-s pulse. The current was resistant to the Na(+)-channel blockers tetrodotoxin (1 µM) and amiloride (10 µM), while the Ca(2+)-channel blocker verapamil (50 µM) in the bath solution completely blocked the current. The intracellular Na(+) concentration increased in staurosporine-treated oocytes, but could be prevented by replacing extracellular Na(+) with either K(+) or Choline(+). Prevention of this influx of Na(+) also prevented the STS-induced up-regulation of the caspase-3 activity, suggesting that the intracellular Na(+) increase is required to induce apoptosis. Taken together, we have found that a voltage dependent Na(+) channel is up-regulated during apoptosis and that influx of Na(+) is a crucial step in the apoptotic process in Xenopus oocytes

New strategies for allogeneic hematopoietic stem cell transplantation with umbilical cord

Umbilical cord blood is enriched in hematopoietic stem cells. For this reason, cord blood units may be utilized for allogeneic hematopoietic stem cell transplantations when no adult human leukocyte antigen (HLA)‐matched donor is found. Cord blood units are rapidly available from international cord blood banks and the naivety of cord blood cells allows the transplantation of HLA‐mismatched units without an increase in graft‐versus‐host disease. But cord blood is also beset with some drawbacks compared to other stem cell sources, the most apparent being a slow immune reconstitution after transplantation leading to increased infection related mortality. The overall aim of this thesis work has been to develop new strategies and tools for handling patients transplanted with umbilical cord blood. Donor lymphocyte infusions (DLI), i.e. an additional boost of donor lymphocytes, can be used to treat threatening rejections or malignant relapses in the adult donor setting. However, due to the limited cell dose, this treatment option is currently not available for cord blood transplanted patients. For this reason, we aimed to expand cord blood‐derived T cells for possible use as DLI after transplantation. Starting with an aliquot from the original cord blood graft, we successfully expanded T cells in eight days to adequate numbers for DLI preparation. By studying the cells with multicolor flow cytometry for surface and intracellular markers, functional assays and spectratyping techniques we concluded that the T cells had polyclonal T cell receptor repertoire, were of central and effector memory phenotype and responded in a similar manner towards mitogenic and allogeneic stimulation compared to peripheral blood T cells. The cytokine IL‐7 has previously been shown to protect T cells from apoptosis induced by, e.g. cytokine withdrawal. This feature should be especially important for cord blood T cells due to their sensitivity to activation induced cell death as well as their high expression levels of the IL‐7 receptor. Hence, we aimed to optimize our expansion protocol by adding IL‐7 to a range of IL‐2 concentrations. When IL‐7 was added to low‐dose IL‐2, the resulting T cells presented with a higher degree of polyfunctionality and superior proliferation potential compared with cells expanded without IL‐7. The T cells also had a higher CD4/CD8 ratio and a higher frequency of effector memory cells, which may have positive implications for their use as DLI. The overall one‐year 55% survival after cord blood transplantations at our center highlights the need for predictive risk markers for earlier interventions. We hypothesized that the T cell expansions could be utilized as indirect indicators of graft quality and, thus, as a tool for risk prediction. We correlated phenotypical and functional data from expanded cord blood T cells with clinical features after transplantation. The results indicated that higher frequencies of CD69+ T cells in the expansions were predictive of prolonged patient survival. Since many of the deaths were due to infections, this marker may thus be used as an indicator for e.g. the administration of prophylactic antiviral drugs. To overcome the problem of low cell dose, the strategy of double cord blood transplantations (DCBT) in which two cord blood units are transplanted simultaneously, has been effectively employed. This provides the patient with an increased total nucleated cell dose during the initial critical weeks after transplantation but, in the vast majority of cases, one of the units eventually prevails. However, three out of seven evaluable patients undergoing DCBT at our center presented with a mixed donor chimerism more than two years after transplantation. Since these patients are extremely rare we characterized the phenotype and functionality of their immune systems to gain insight into the significance of mixed donor chimerism. Results indicate that patients with long‐term mixed donor chimerism after double cord blood transplantation have a less functional immune system compared to control patients with one donor immune system. This could be because one of the two immune systems had a more naive T cell profile with poor cytokine production. Moreover, we speculate that the mixed donor chimerism in part may be explained by a graft‐versus‐graft tolerance induced by our use of high‐dose anti‐thymocyte globulin and an inter‐unit match of HLA‐C

Discovering Genetic Interactions in Large-Scale Association Studies by Stage-wise Likelihood Ratio Tests

Despite the success of genome-wide association studies in medical genetics, the underlying genetics of many complex diseases remains enigmatic. One plausible reason for this could be the failure to account for the presence of genetic interactions in current analyses. Exhaustive investigations of interactions are typically infeasible because the vast number of possible interactions impose hard statistical and computational challenges. There is, therefore, a need for computationally efficient methods that build on models appropriately capturing interaction. We introduce a new methodology where we augment the interaction hypothesis with a set of simpler hypotheses that are tested, in order of their complexity, against a saturated alternative hypothesis representing interaction. This sequential testing provides an efficient way to reduce the number of non-interacting variant pairs before the final interaction test. We devise two different methods, one that relies on a priori estimated numbers of marginally associated variants to correct for multiple tests, and a second that does this adaptively. We show that our methodology in general has an improved statistical power in comparison to seven other methods, and, using the idea of closed testing, that it controls the family-wise error rate. We apply our methodology to genetic data from the PRO-CARDIS coronary artery disease case/control cohort and discover three distinct interactions. While analyses on simulated data suggest that the statistical power may suffice for an exhaustive search of all variant pairs in ideal cases, we explore strategies for a priori selecting subsets of variant pairs to test. Our new methodology facilitates identification of new disease-relevant interactions from existing and future genome-wide association data, which may involve genes with previously unknown association to the disease. Moreover, it enables construction of interaction networks that provide a systems biology view of complex diseases, serving as a basis for more comprehensive understanding of disease pathophysiology and its clinical consequences.</p

Genetic loci on chromosome 5 are associated with circulating levels of interleukin-5 and eosinophil count in a European population with high risk for cardiovascular disease

IL-5 is a Th2 cytokine which activates eosinophils and is suggested to have an atheroprotective role. Genetic variants in the IL5 locus have been associated with increased risk of CAD and ischemic stroke. In this study we aimed to identify genetic variants associated with IL-5 concentrations and apply a Mendelian randomisation approach to assess IL-5 levels for causal effect on intima-media thickness in a European population at high risk of coronary artery disease. We analysed SNPs within robustly associated candidate loci for immune, inflammatory, metabolic and cardiovascular traits. We identified 2 genetic loci for IL-5 levels (chromosome 5, rs56183820, BETA = 0.11, P = 6.73E−5 and chromosome 14, rs4902762, BETA = 0.12, P = 5.76E−6) and one for eosinophil count (rs72797327, BETA = −0.10, P = 1.41E−6). Both chromosome 5 loci were in the vicinity of the IL5 gene, however the association with IL-5 levels failed to replicate in a meta-analysis of 2 independent cohorts (rs56183820, BETA = 0.04, P = 0.2763, I2 = 24, I2 − P = 0.2516). No significant associations were observed between SNPs associated with IL-5 levels or eosinophil count and IMT measures. Expression quantitative trait analyses indicate effects of the IL-5 and eosinophil-associated SNPs on RAD50 mRNA expression levels (rs12652920 (r2 = 0.93 with rs56183820) BETA = −0.10, P = 8.64E−6 and rs11739623 (r2 = 0.96 with rs72797327) BETA = −0.23, P = 1.74E−29, respectively). Our data do not support a role for IL-5 levels and eosinophil count in intima-media thickness, however SNPs associated with IL-5 and eosinophils might influence stability of the atherosclerotic plaque via modulation of RAD50 levels

Early Events in Xenograft Development from the Human Embryonic Stem Cell Line HS181 - Resemblance with an Initial Multiple Epiblast Formation

Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart

Causal relevance of blood lipid fractions in the development of carotid atherosclerosis: Mendelian randomization analysis.

BACKGROUND: Carotid intima-media thickness (CIMT), a subclinical measure of atherosclerosis, is associated with risk of coronary heart disease events. Statins reduce progression of CIMT and coronary heart disease risk in proportion to the reduction in low-density lipoprotein cholesterol. However, interventions targeting triglycerides (TGs) or high-density lipoprotein cholesterol (HDL-C) have produced inconsistent effects on CIMT and coronary heart disease risk, making it uncertain whether such agents are ineffective for coronary heart disease prevention or whether CIMT is an inadequate marker of HDL-C or TG-mediated effects. We aimed to determine the causal association among the 3 major blood lipid fractions and common CIMT using mendelian randomization analysis. METHODS AND RESULTS: Genetic scores specific for low-density lipoprotein cholesterol, HDL-C, and TGs were derived based on single nucleotide polymorphisms from a gene-centric array in ≈5000 individuals (Cardiochip scores) and from a genome-wide association meta-analysis in >100 000 individuals (Global Lipids Genetic Consortium scores). These were used as instruments in a mendelian randomization analysis in 2 prospective cohort studies. A genetically predicted 1 mmol/L higher low-density lipoprotein cholesterol concentration was associated with a higher common CIMT by 0.03 mm (95% confidence interval, 0.01-0.04) and 0.04 mm (95% confidence interval, 0.02-0.06) based on the Cardiochip and Global Lipids Genetic Consortium scores, respectively. HDL-C and TGs were not causally associated with CIMT. CONCLUSIONS: Our findings confirm a causal relationship between low-density lipoprotein cholesterol and CIMT but not with HDL-C and TGs. At present, the suitability of CIMT as a surrogate marker in trials of cardiovascular therapies targeting HDL-C and TGs is questionable and requires further study

Tissue factor A-603G genotype associates with carotid intima-media thickness in subjects undergoing cardiovascular risk prevention

Tissue factor (TF), key initiator of coagulation, is also ascribed a non-haemostatic function in inflammation, cell migration and proliferation, suggesting a role of TF not only in thrombosis but also in atherosclerosis development. Polymorphisms in the TF gene promoter have been shown to modulate the expression of TF, and thereby potentially also its involvement in atherosclerosis and individual predisposition to atherosclerotic disease. Hence, this study was aimed at investigating associations between TF promoter polymorphisms and carotid intima-media thickness (IMT), a well-established surrogate marker of atherosclerotic disease. To this end, the TF A-603G polymorphism was analyzed in 316 subjects enrolled in a primary and secondary cardiovascular risk prevention programme, with measurements of carotid IMT by B-mode ultrasound. Also, the TF Ins-1208Del polymorphism was investigated in a limited number of subjects, which confirmed the previously reported complete concordance between the -603A and -1208Del alleles. The subjects were aged 60.2\ub18.4 years, 80% were male, and 78% were undergoing secondary prevention with a history of coronary, cerebrovascular, or peripheral atherosclerotic disease. Both mean and maximum carotid IMT (measured at the common carotid artery, carotid bifurcation, and internal carotid artery) differed significantly according to A-603G genotype, being highest in -603N A (n=93), intermediate in NG (n=161) and lowest in G/G (n=62) (mean IMT: A/A 1.31\ub10.36 mm, NG 1.27\ub10.33 mm, G/G 1.19\ub10.32 mm; max IMT: A/A 2.36\ub10.88 mm, NG 2.26\ub10.85 mm, 2.05\ub10.88 mm; both p<0.05; adjusted for age, gender, and statin treatment). In summary, a significant association between TF promoter genotype and carotid IMT was observed, perhaps mediated via alterations of TF expression levels in the circulation or within the carotid vessel wall. These findings support the hypothesis that TF plays a role in the atherosclerotic process, beyond its well-known role in haemostasis and thrombosis, thus further implicating TF not only in thrombotic complications of atherosclerotic disease, but also in plaque progression

In Vitro Evaluation of Graft-versus-Graft Alloreactivity as a Tool to Identify the Predominant Cord Blood Unit before Double Cord Blood Transplantation

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Fatty acid binding protein 4 in circulating leucocytes reflects atherosclerotic lesion progression in Apoe(-/-) mice

Biopharmaceutic