Ex vivo expansion of human umbilical cord blood hematopoietic stem and progenitor cells

The goal of this thesis research is to establish ex vivo expansion conditions for HSCs derived from UCB. To realize the expansion of HSCs, CD34+ or ACJ33+ UCB cells were cultured in the absence or presence of various cocktails of early acting cytokines including Flt3-L, Tpo, SCF or IL6 under stroma-free or stroma-supported conditions. The HSC and progenitor expansion was assessed using in vitro and in vivo long-term repopulating cells. First, the experiments were designed to test whether HSC expansion would alter the in vivo long-term engraftment potential of CD34+ UCB cells in the presence of EMderived stromal cells during two weeks. Also the cytokines required for expansion of HSCs and progenitors in either the presence or absence of stroma have been evaluated. The experiments described in chapter 3 are closely linked to the work described in the previous chapter. They were designed to investigate whether HSC expansion could be improved when cultured for more than two weeks, and whether the presence of EMderived stromal cells, and combinations of specific cytokines could affect the HSC and progenitor maintenance or expansion. In chapter 4 the effect of a new fusion protein ofiL6 and the soluble IL6R, H-IL6, has been evaluated on the long-term ex vivo expansion ofHCSs derived from AC133+ UCB cells. To do this, we used stroma-free and stroma-supported LTCs and compared several cytokine combinations in the presence or absence of this chimeric protein, or IL6, and estimated the HSC and progenitor output by multiparameter FACS analysis and CAFC assays. Following these experiments, nineteen newly established murine embryonic stromal clones have been investigated for their ability to sustain human HSCs and progenitors in extended LTCs of CD34+ UCB cells in the absence or presence of the cytokines Flt3- L and Tpo for periods as long as twelve weeks. A significant proportion of HSC and progenitor subsets was found in the non-adherent compartment of these co-cultures. With an interest to elucidate the factors that determine the proportion of adherent and non-adherent compartments, we evaluated in chapter 6 the chemoattractive activity of different stromal cells and the effect of exogenously added cytokines herein

Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis

Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers, isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here that the growth and cloning efficiency of embryonic cells (with the exception of the aorta) is increased in the presence of the tsA58 transgene. Furthermore, our results show that the large panel of stromal clones isolated from the different embryonal subregions exhibit heterogeneity in their ability to promote murine and human hematopoietic differentiation. Despite our findings of heterogeneity in hematopoietic growth factor gene expression profiles, high-level expression of some factors may influence hematopoietic differentiation. Interestingly, a few of these stromal clones express a recently described chordin-like protein, which is an inhibitor of bone morphogenic proteins and is preferentially expressed in cells of the mesenchymal lineage

Test-retest reliability of tetrax® static posturography system in young adults with low physical activity level

Purpose/Background: Assessment of postural sway with force plates can be affected by type of measurement and various clinical parameters such as age and activity level of the individual person. For this reason, variability is detected in postural reactions of healthy subjects without balance impairment. Test-retest reliability of postural sway in adolescent athletes has been measured using a force plate and additional test-retest studies have been suggested for subjects of different age groups with different activity levels. Therefore, the purpose of this research was to assess test-retest reliability of Tetrax (R) Static Posturography in young adults with low physical activity level, and examine the relationship between posturography results and low activity level.Methods: Young adults older than 18 years of age were included in the study. Demographic characteristics of the cases were recorded including age, weight, height, body mass index (BMI, kg/m(2)) and dominant extremity. Number of falls in the previous six months, lower body endurance (sit to stand test) and single-leg eyes closed stance test were recorded. Activity level of participants was determined according to the International Physical Activity Questionnaire (IPAQ). Posturographic evaluation of all volunteers was completed using the Tetrax (R) Interactive Postural Balance System (Sunlight Medical Ltd, Israel). Fall risk and general stability index (SI) calculated by the Tetrax (R) were recorded. Following the first test, measurements were repeated 24 to 48 hours later for reliability purposes.Results: Sixty-five subjects (28 male, 37 female; mean age 22.2 +/- 1.1 years, mean BMI 22.6 +/- 3.3 kg/m(2)) were evaluated. All participants were classified as minimally active according to mean IPAQ score (1042.1 +/- 517.7 [231 -2826] MET-minutes per week). ICC scores between the first and second tests for fall index and total stability index were excellent (ICC2,1 = 0.858, 0.850, respectively). Fall risk determined by using the Tetrax (R) device was negatively correlated with lower body endurance (p=0.001, r=-0.446), vigorous activity score (p=0.011, -0.312) and total activity score (p=0.029, r=-0.271), and positively correlated with single leg stance score (p=0.001, r=0.606). There was a weak correlation between fall risk history and the fall risk determined by using Tetrax (R) device (p=0.04, r=0.255). There were no correlations between fall risk and height, weight, and BMI (p>0.05).Conclusions: The results demonstrated the high test-retest reliability of Tetrax (R) interactive balance system in young healthy adults with low physical activity level. Future studies are needed to determine the effectiveness of increasing physical activity level on postural control

Vagus nerve stimulation paired with upper limb rehabilitation after chronic stroke

Background and Purpose: We assessed safety, feasibility, and potential effects of vagus nerve stimulation (VNS) paired with rehabilitation for improving arm function after chronic stroke. Methods: We performed a randomized, multisite, double-blinded, sham-controlled pilot study. All participants were implanted with a VNS device and received 6-week in-clinic rehabilitation followed by a home exercise program. Randomization was to active VNS (n=8) or control VNS (n=9) paired with rehabilitation. Outcomes were assessed at days 1, 30, and 90 post-completion of in-clinic therapy. Results: All participants completed the course of therapy. There were 3 serious adverse events related to surgery. Average FMA-UE scores increased 7.6 with active VNS and 5.3 points with control at day 1 post–in-clinic therapy (difference, 2.3 points; CI, −1.8 to 6.4; P=0.20). At day 90, mean scores increased 9.5 points from baseline with active VNS, and the control scores improved by 3.8 (difference, 5.7 points; CI, −1.4 to 11.5; P=0.055). The clinically meaningful response rate of FMA-UE at day 90 was 88% with active VNS and 33% with control VNS (P<0.05). Conclusions: VNS paired with rehabilitation was acceptably safe and feasible in participants with upper limb motor deficit after chronic ischemic stroke. A pivotal study of this therapy is justified

Advances in MASELTOV – Serious Games in a Mobile Ecology of Services for Social Inclusion and Empowerment of Recent Immigrants

Immigration imposes a range of challenges with the risk of social exclusion from the information society (Halfman 1998), such as, getting into communication with the local society and understanding the culture of their host nation. Failure to address these challenges can lead to difficulties in the frame of integrating into the society of the host country, leading to fragmented communities and a range of social issues. As part of a comprehensive suite of services for immigrants, the European project seeks to provide both practical tools and learning services via mobile devices, providing a readily usable resource for immigrants. We introduce recent results, such as the game-based learning aspect of the MASELTOV project is introduced, with the rationale behind its design presented. In doing so, the benefits and implications of mobile platforms and emergent data capture techniques for game-based learning are discussed, as are methods for putting engaging gameplay at the forefront of the experience whilst relying on rich data capture and analysis to provide an effective learning solution

Macrophages mediate colon carcinoma cell adhesion in the rat liver after exposure to lipopolysaccharide

The surgical resection of primary colorectal cancer is associated with an enhanced risk of liver metastases. Moreover, bacterial translocation or anastomic leakage during resection has been shown to correlate with a poor long-term surgical outcome, suggesting that bacterial products may contributeto the formation of metastases. Driven by these premises, we investigated the role of the bacterial product lipopolysaccharide (LPS) in the generation of liver metastases. Intraperitoneal injection of LPS led to enhanced tumor-cell adhesion to the rat liver as early as 1.5 h post-administration. Furthermore, a rapid loss of the expression of the tight junction protein zonula occludens-1 (ZO-1) was observed, suggesting that LPS disrupts the integrity of the microvasculature. LPS addition to endothelial-macrophage co-cultures damaged endothelial monolayers and caused the formation of intercellular gaps, which was accompanied by increased tumor-cell adhesion. These results suggest that macrophages areinvolved in the endothelial damage resulting from exposure to LPS. Interestingly, the expression levels of of ZO-1 were not affected by LPS treatment in rats in which liver macrophages had been depletedas well as in rats that had been treated with a reactive oxygen species (ROS) scavenger. In both settings, decreased tumor-cell adhesion was observed. Taken together, our findings indicate that LPS induces ROS release by macrophages, resulting in the damage of the vascular lining of the liver and henceallowing increased tumorcell adherence. Thus, peri-operative treatments that prevent the activation of macrophages and-as a consequence- limit endothelial damage and tumor-cell adhesion may significantly improve the long-term outcome of cancer patients undergoing surgical tumor resection