Impairment of endothelial progenitor cells in women after kidney transplantation.

OBJECTIVE: The long-term survival of kidney transplant patients has substantially improved. However, there is a higher risk for cardiovascular events after transplantation, partly due to immunosuppression. A diminished number of endothelial progenitor cells (EPCs), which play an important role in angiogenesis and the repair of endothelial damage, are associated with an increased cardiovascular risk. The aim of this study was to evaluate whether kidney transplantation affects EPCs in women. METHODS: Twenty-four healthy women and 22 female kidney transplant recipients were recruited. The ratio of angiogenic and non-angiogenic circulating progenitor cells (CPCs) was determined by multicolor flow cytometry and related to clinical parameters. Cord blood-derived endothelial colony-forming cells (ECFCs), a proliferative subgroup of endothelial progenitor cells, were treated with pooled sera from transplant patients or healthy controls and tested for their functional integrity using in vitro models. RESULTS: Kidney transplant recipients displayed a reduced ratio of angiogenic and non-angiogenic CPCs compared to healthy controls. Differences were especially pronounced in premenopausal women. Exposure to sera of transplanted women led to a significant impairment of ECFC proliferation, migration, and angiogenesis ability. CONCLUSIONS: Alterations of EPC populations may contribute to the higher cardiovascular risks after organ transplantation and should be considered in therapeutic strategies.

Downregulation of miR-1270 mediates endothelial progenitor cell function in preeclampsia: Role for ATM in the Src/VE-cadherin axis.

Preeclampsia, a pregnancy-related hypertensive disorder, is associated with endothelial dysfunction and increased cardiovascular risk of the offspring in adulthood. In preeclampsia, endothelial colony-forming cells (ECFC) are reduced in number and function. Recently, we have shown that miR-1270, which is involved in cancer in vitro proliferation, migration, and tumor progression, is downregulated in fetal ECFC from preeclamptic pregnancies. We now hypothesize that miR-1270 dysregulation contributes to vascular endothelial dysfunction occurring after preeclampsia via ATM (ataxia telangiectasia mutated) overexpression, the key kinase of DNA damage repair. Here, we show that miR-1270 silencing in normal ECFC and downregulation in preeclamptic ECFC are accompanied by an increase in the expression levels of ATM. Furthermore, ATM activation correlates with upregulated tyrosine kinase Src leading to phosphorylation and internalization of VE-cadherin (vascular endothelial-cadherin) which subsequently compromises endothelial barrier permeability and morphodynamic cell parameters. Treatment with specific ATM inhibitors reveals a novel role of ATM upstream of tyrosine kinase Src activation. Subsequently, Src phosphorylation and internalization of VE-cadherin compromise endothelial barrier permeability. Our findings suggest that downregulation of miR-1270 contributes to impaired ECFC function via the associated ATM overexpression, which further identifies ATM as a novel and critical factor for ECFC defects in preeclampsia. Our study provides new insights into the understanding of ECFC impairment associated with cardiovascular risk in preeclamptic offspring and identifies potential novel therapeutic targets.

MicroRNA Profiles of Maternal and Neonatal Endothelial Progenitor Cells in Preeclampsia.

Preeclampsia is associated with an increased cardiovascular morbidity of mother and offspring, thus contributing to a substantial burden in women and children's health. It has been proven that endothelial progenitor cell (EPC) numbers and functional characteristics are impaired in cardiovascular disease and preeclampsia, although causative factors for the latter have remained elusive. MicroRNA (miRNA) modifications are a potential mechanism through which exposure to an altered environment translates into the development of chronic disease. In this study, we examined whether development of preeclampsia corresponds to alterations of miRNAs in maternal- and cord-blood-derived EPC. To test this end, we analyzed maternal and neonatal miRNAs via RNA sequencing from endothelial cells of preeclamptic and healthy controls in different cell culture passages. We were able to demonstrate differentially represented miRNAs in all groups. Hsa-miR-1270 showed significantly different levels in cord blood EPC from preeclampsia versus control and was negatively correlated with mRNA levels of its predicted targets ANGPTL7 and TFRC. Transfection with an hsa-miR-1270 inhibitor decreased the tube formation capacity and chemotactic motility but did not change proliferation in vitro. Target predictions and gene set enrichment analyses identified alternative splicing as a significantly enriched pathway for hsa-miR-1270. The top miRNAs in three other groups were predicted to target transcriptional and developmental pathways. Here, we showed for the first time significantly different levels of miRNAs and differently represented mRNA levels of predicted target genes in EPC derived from preeclampsia. Understanding the effects of preeclampsia on the epigenetic mechanisms of EPC will be crucial and may provide initial insights for further evaluation of the benefits of therapies targeting this cell population.

Pravastatin Promotes Endothelial Colony-Forming Cell Function, Angiogenic Signaling and Protein Expression In Vitro.

Endothelial dysfunction is a primary feature of several cardiovascular diseases. Endothelial colony-forming cells (ECFCs) represent a highly proliferative subtype of endothelial progenitor cells (EPCs), which are involved in neovascularization and vascular repair. Statins are known to improve the outcome of cardiovascular diseases via pleiotropic effects. We hypothesized that treatment with the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor pravastatin increases ECFCs' functional capacities and regulates the expression of proteins which modulate endothelial health in a favourable manner. Umbilical cord blood derived ECFCs were incubated with different concentrations of pravastatin with or without mevalonate, a key intermediate in cholesterol synthesis. Functional capacities such as migration, proliferation and tube formation were addressed in corresponding in vitro assays. mRNA and protein levels or phosphorylation of protein kinase B (AKT), endothelial nitric oxide synthase (eNOS), heme oxygenase-1 (HO-1), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and endoglin (Eng) were analyzed by real time PCR or immunoblot, respectively. Proliferation, migration and tube formation of ECFCs were enhanced after pravastatin treatment, and AKT- and eNOS-phosphorylation were augmented. Further, expression levels of HO-1, VEGF-A and PlGF were increased, whereas expression levels of sFlt-1 and Eng were decreased. Pravastatin induced effects were reversible by the addition of mevalonate. Pravastatin induces beneficial effects on ECFC function, angiogenic signaling and protein expression. These effects may contribute to understand the pleiotropic function of statins as well as to provide a promising option to improve ECFCs' condition in cell therapy in order to ameliorate endothelial dysfunction.

Synthesis of thiolated polysaccharides for formation of polyelectrolyte multilayers with improved cellular adhesion.

Intrinsic cross-linking is not only useful for increasing stability, but also for tailoring mechanical properties of polyelectrolyte multilayers (PEM) on implants and tissue engineering scaffolds. Here, a novel route for synthesizing thiolated chitosan (t-Chi) based on the application of 3,3'-dithiodipropionic acid was applied, while thiolated chondroitin sulfate (t-CS) was conjugated by 3,3'-dithiobis (propanoic hydrazide). Both products were subsequently reduced to obtain the free thiols. The thiol content, structural changes and degree of substitution were studied by UV-vis, FTIR, Raman and 1H NMR spectroscopy, respectively. Chi and CS can be used for PEM formation with the layer-by-layer method, due to the cationic nature of Chi at pH values below 5.0 and the anionic character of CS. Comparative studies on the formation of native Chi/CS versus t-Chi/t-CS PEM with surface plasmon resonance and ellipsometry revealed higher layer mass. We also found that the PEM composed of t-Chi/t-CS had superior cell adhesion properties for human keratinocytes in comparison to the native PEM.

Robotic Companions for Older People: A Case Study in the Wild.

Older people tend to have difficulties using unknown technical devices and are less willing to accept technical shortcomings. Therefore, a robot that is supposed to support older people in managing daily life has to adapt to the users' needs and capabilities that are very heterogeneous within the target group. The aim of the presented case study was to provide in-depth insights on individual usage patterns and acceptance of a mobile service robot in real live environments (i.e. in the users' homes). Results from three cases (users aged 67, 78 and 85 living in their own apartments) are reported. Findings on usability and user experience illustrate that the robot has considerable potential to be accepted to support daily living at home.

Influence of external, intrinsic and individual behaviour variables on serum 25(OH)D in a German survey.

The objective of the present study was to identify external, intrinsic or behavioural factors that significantly influenced serum 25-hydroxyvitamin D (25(OH)D) concentrations in a German survey. Data from 3061 participants in the Cooperative Health Research in the Region of Augsburg, Germany (KORA) F4 survey were used to relate potential determinants to measured mean serum 25(OH)D concentrations using multivariable regression models. The factors significantly associated with hypovitaminosis D (defined as 25(OH)D<25 nmolL(-1)) were season (winter, spring and autumn), urban environment and high body mass index. In contrast, times spent in sunny regions, hours per day spent outdoors in the summer as well as additional oral intake were associated with higher 25(OH)D concentrations. These results suggest that mainly ambient UV exposure but also individual behaviour are the most important determinants for personal 25(OH)D concentrations. The analyses further showed that in winter 43% of subjects were vitamin D deficient and 42% insufficient. Even in summer over half the population has insufficient vitamin D status with 8% deficient and 47% insufficient. Therefore measures to mitigate widespread vitamin D insufficiency such as regular short-term sun exposure and/or improved dietary intake/supplementation recommendations by public health bodies need to be considered.

Differential effect of platelet-rich plasma and fetal calf serum on bone marrow-derived human mesenchymal stromal cells expanded in vitro.

Mesenchymal stromal cells (MSCs) derived from various sources have great potential for use in cell-based therapies. Since the proportion of primary MSCs contained in bone marrow or adipose tissue is low, plastic adherence and in vitro expansion are necessary to expand MSCs prior to clinical application. Human platelet-rich plasma has been introduced as an alternative serum source but functional differences have so far not been described. Here we cultured MSCs derived from human bone marrow in medium supplemented with either 10% fetal calf serum (FCS) or 5% and 10% platelet-rich plasma (PRP) until the first or second passage. Parameters under investigation were cell yield, clonogenicity, phenotype as well as migratory and differentiation potential. In addition, the secretion of SDF-1α and the induced migration of CD34(+) haematopoietic stem cells (HSCs) were investigated with regard to the different serum source. The use of PRP resulted in a significantly higher expansion rate and yield at passages 0 and 1. In addition, the level of secreted SDF-1α was significantly increased in the supernatant of MSCs cultured with FCS instead of human PRP. Consistent with this, the migration capacity of MSCs cultured with 10% FCS as well as their capability to induce the migration of CD34(+) haematopoietic progenitors in a transwell assay was higher. Our results demonstrate that human PRP can be seen as an alternative serum source to FCS for MSC cultivation. However, the requirements of the specific clinical application must be carefully considered before the respective serum source is selected.

Maternal nutritional condition and genetic differentiation affect brood size and offspring body size in Nicrophorus.

In most animal species, brood size and body size exhibit some variation within and between populations. This is also true for burying beetles (genus Nicrophorus), a group in which the body size of offspring depends critically on the number of offspring competing for food due to the discrete nature of resource used for larval nutrition (vertebrate carcasses). In one species, brood size and body size are correlated with population density, and appear to be phenotypically plastic. We investigated potential proximate causes of between-population variation in brood size and body size in two species, Nicrophorus vespilloides and Nicrophorus defodiens. Our first experiment supported the notion that brood size is phenotypically plastic, because it was affected by environmental variation in adult nutritional condition. We found that the pre-breeding nutritional status of female N. vespilloides affected the number of eggs they laid, the number of surviving larvae in their broods, and the body size of their offspring. We do not know whether this plasticity is adaptive because greater offspring body size confers an advantage in contests over breeding resources, or whether starved females are constrained to produce smaller clutches because they cannot fully compensate for their poor pre-breeding nutritional status by feeding from the carcass. Our second experiment documents that brood size, specifically the infanticidal brood-size adjustment behavior, has undergone genetic differentiation between two populations of N. defodiens. Even under identical breeding conditions with identical numbers of first-instar larvae, females descended from the two populations produced broods of different size with corresponding differences in offspring body size.

Vaccination of hormone-refractory prostate cancer patients with peptide cocktail-loaded dendritic cells: results of a phase I clinical trial.

BACKGROUND: Immunotherapies might represent promising alternatives for the treatment of patients with hormone-refractory prostate cancer (HRPC). In a Phase I clinical trial, we evaluated a vaccination with dendritic cells (DCs) loaded with a cocktail consisting of HLA-A*0201-restricted peptides derived from five different prostate cancer-associated antigens [prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), survivin, prostein, transient receptor potential p8 (trp-p8)]. METHODS: Eight HRPC patients received a total of four vaccinations every other week. Clinical and immunological responses were monitored by the determination of the serum PSA levels and by enzyme linked immunospot (ELISPOT) analyses, respectively. RESULTS: Apart from local skin reactions no side effects were noted. One patient displayed a partial response (PR; PSA decrease >50%) and three other patients showed stable PSA values or decelerated PSA increases. In ELISPOT analyses, three of four PSA responders also showed antigen-specific CD8+ T-cell activation against prostein, survivin, and PSMA. CONCLUSIONS: The described protocol represents a safe and feasible concept for the induction of clinical and immunological responses. The application of a peptide cocktail-derived from different antigens as a novel treatment modality is supposed to allow for the genetic and biologic heterogeneity of PCa.

Qualitative and quantitative assessment of urinary cytokeratin 8 and 18 fragments compared with voided urine cytology in diagnosis of bladder carcinoma.

OBJECTIVES: To evaluate the value of urine tests based on the detection of cytokeratins 8 and 18 for the diagnosis of bladder cancer compared with urine cytology. METHODS: Samples from 112 patients before transurethral resection (group 1), 40 patients before secondary surgical treatment (group 2), 29 healthy control subjects (group 3, controls), and 10 women with acute urinary tract infection (group 4, controls) were examined with the UBC Rapid and UBC II enzyme-linked immunosorbent assay (ELISA) tests and voided urine cytology. RESULTS: Of the 112 patients in group 1, 90 had transitional cell carcinoma. For the UBC Rapid, UBC ELISA, and cytology, the sensitivity and specificity was 64.4%, 46.6%, and 70.5% and 63.6%, 86.3%, and 79.5%, respectively. The cytology had the greatest accuracy (72.3%) compared with both cytokeratin tests (54.4% and 64.2%). For all three tests, sensitivity increased with tumor grade and stage. In group 2, 16 of 40 patients had residual carcinoma. The sensitivity was similar for all three tests, and the specificity of cytology was lower compared with its specificity in group 1 (47.9% versus 70.5% in group 1). In the controls with or without urinary tract infection, the specificity of cytology was greater than that of both other tests. The combination of the UBC ELISA test with cytology increased the sensitivity to 83% (specificity 68%). CONCLUSIONS: Both cytokeratin tests detected patients with transitional cell carcinoma, but were inferior to voided urine cytology in test quality.