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

Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a debilitating complication arising in around half of all patients treated with an allogeneic hematopoietic stem cell transplantation. Even though treatment of severe cGVHD has improved during recent years, it remains one of the main causes of morbidity and mortality in affected patients. Biomarkers in blood that could aid in the diagnosis and classification of cGVHD severity are needed for the development of novel treatment strategies that can alleviate symptoms and reduce the need for painful and sometimes complicated tissue biopsies. Methods that comprehensively profile complex biological systems such as the immune system can reveal unanticipated markers when used with the appropriate methods of data analysis. Here, we used mass cytometry, flow cytometry, enzyme-linked immunosorbent assay, and multiplex assays to systematically profile immune cell populations in 68 patients with varying grades of cGVHD. We identified multiple subpopulations across T, B, and NK-cell lineages that distinguished patients with cGVHD from those without cGVHD and which were associated in varying ways with severity of cGVHD. Specifically, initial flow cytometry demonstrated that patients with more severe cGVHD had lower mucosal-associated T cell frequencies, with a concomitant higher level of CD38 expression on T cells. Mass cytometry could identify unique subpopulations specific for cGVHD severity albeit with some seemingly conflicting results. For instance, patients with severe cGVHD had an increased frequency of activated B cells compared to patients with moderate cGVHD while activated B cells were found at a reduced frequency in patients with mild cGVHD compared to patients without cGVHD. Moreover, results indicate it may be possible to validate mass cytometry results with clinically viable, smaller flow cytometry panels. Finally, no differences in levels of blood soluble markers could be identified, with the exception for the semi-soluble combined marker B-cell activating factor/B cell ratio, which was increased in patients with mild cGVHD compared to patients without cGVHD. These findings suggest that interdependencies between such perturbed subpopulations of cells play a role in cGVHD pathogenesis and can serve as future diagnostic and therapeutic targets

Donor Cell Composition and Reactivity Predict Risk of Acute Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Background. Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). We designed a functional assay for assessment of individual risk for acute GVHD. Study Design and Methods. Blood samples were collected from patients and donors before HSCT. Two groups of seven patients each were selected, one in which individuals developed acute GVHD grades II–IV and one in which none showed any clinical signs of GVHD. Peripheral blood mononuclear cells (PBMCs) isolated from donors were incubated in mixed lymphocyte cultures (MLCs) with recipient PBMCs. The cells were characterized by flow cytometry before and after MLC. Results. Samples from donors in the GVHD group contained significantly lower frequencies of naïve γδ T-cells and T-cells expressing NK-cell markers CD56 and CD94. Donor samples in this group also exhibited lower frequencies of naïve CD95+ T-cells compared to controls. After MLC, there were dissimilarities in the CD4/CD8 T-cell ratio and frequency of CD69+ T-cells between the two patient groups, with the non-GVHD group showing higher frequencies of CD8+ and CD69+ T-cells. Conclusion. We conclude that a thorough flow cytometric analysis of donor cells for phenotype and allogeneic reactivity may be of value when assessing pretransplant risk for severe acute GVHD

Voltage-gated ion channel activated during apoptosis.

<p><b>A</b>: Electrophysiological properties of an outward current in control oocytes (black) and staurosporine (1 µM) treated oocytes (red) in <i>Xenopus</i> oocytes at +100 mV. The difference between the current in control and STS-treated oocytes is also plotted (dashed line). <b>B</b>. Mean steady-state current at +100 mV in control (1.8 µA±0.5 µA, <i>n</i> = 17) and apoptotic oocytes treated with 1 µM STS (4.8 µA±0.3 µA, <i>n</i> = 13). Steady state currents at +100 mV are significantly larger in the apoptotic oocytes than in controls (**** P<0.0001). Statistical analyses are mean ± SEM and unpaired t-test. Recordings were done in 100Na solution.</p

Pharmacology of the Na⁺ channel.

<p><b>A</b>. 10 µM amiloride (blue) does not block the STS-induced current at a voltage-clamp step to +100 mV. <b>B</b>. 1 µM Tetrodotoxin (blue) does not block the STS-induced current at a voltage-clamp step to +100 mV. <b>C</b>. 200 µM verapamil (blue) blocks the fast activation current in STS-treated oocytes, leaving a slowly activating current. <b>D</b>. 200 µM verapamil (blue) blocks the fast activating current in control oocytes. <b>E</b>. Difference plots from <b>C</b> and <b>D</b> showing the verapamil sensitive Na⁺ current in STS-treated (red) and control (black) oocytes. <b>F</b>. Dose-response curve for the effects of verapamil (n = 3–5). IC₅₀ = 10.1 µM with the Hill coefficient  = 1.</p