Untersuchungen zur metabolischen Aktivierung und Toxizität niedrigchlorierter PCBs

Polychlorinated biphenyls (PCBs) are pollutants that can be found everywhere in the environment due to their persistence. Depending on the number and position of chlorine atoms, their chemical and toxicological properties vary. In this thesis, an inhibitory influence of PCB-contaminated plasma samples on telomerase (hTERT) gene expression could be determined ex vivo using two methods. A dose-response relationship could be established between the concentration of lower chlorinated (lc) PCBs in plasma and the inhibition of hTERT expression in hTERT BAC B6B5.1 fibroblasts, which is 1 μg/L. In vitro, qRT-PCR revealed a concentration-dependent inhibitory effect of single chemically synthesized hydroxy (OH)-metabolites of PCB 28 and PCB 101 on hTERT expression in Jurkat T lymphocytes. Lower concentrations of the OH-metabolites led to stronger effects than the parental congeners, which suggests a metabolic activation of lc PCBs as a prerequisite for their mediated toxicity. A combination of the four most reactive metabolites 3-OH-CB28, 3'-OH-CB28, 3-OH-CB101 and 4-OH-CB101 (OH-PCB mix) led to strong inhibitory effects on hTERT expression in concentration ranges which were found to be subtoxic in the MTT assay and approached the content of lc PCBs in the plasma samples. In vitro these concentrations of the OH-PCB mix led to a reduction in ATP production and an increased production of reactive oxygen species (ROS), which could be responsible for inhibition of hTERT expression and PCB-induced senescence. Incubation with the subtoxic OH-PCB mix also led to increased apoptosis of primary and Jurkat T lymphocytes, which could occur via caspase-dependent as well as caspase-independent signaling pathways. In addition, a higher concentration of prostaglandin E2 (PGE2) was detected in PCB-contaminated plasma samples, which inhibited hTERT expression in vitro depending on the concentration. Thus, in addition to mitochondrial dysfunction and energetic deficiency conditions, another inhibition mechanism for hTERT expression of the lc PCBs contained in plasma could exist via PGE2.In the second part of this work, Drosophila melanogaster was established as a model organism for PCB metabolism and PCB-induced neurotoxicity. Wild type Drosophila metabolized PCB 28 into the same OH-metabolites as were detected in human plasma. Contact with PCB 28 in Drosophila led to neurotoxic signs and concentration-dependent lethality. This could be attributed by flow cytometry to a PCB-dependent induction of apoptosis in differentiated neurons. A neuronal knock-down of the cytochrome P450 monooxygenase Cyp18a1 involved in PCB 28 metabolism caused a complete vitality of the flies at high PCB 28 concentration and a strong decrease of apoptosis in differentiated neurons due to the strongly reduced conversion of PCB 28

Telomeres and Telomerase in Hematopoietic Dysfunction : Prognostic Implications and Pharmacological Interventions

Leukocyte telomere length (TL) has been suggested as a marker of biological age in healthy individuals, but can also reflect inherited and acquired hematopoietic dysfunctions or indicate an increased turnover of the hematopoietic stem and progenitor cell compartment. In addition, TL is able to predict the response rate of tyrosine kinase inhibitor therapy in chronic myeloid leukemia (CML), indicates clinical outcomes in chronic lymphocytic leukemia (CLL), and can be used as screening tool for genetic sequencing of selected genes in patients with inherited bone marrow failure syndromes (BMFS). In tumor cells and clonal hematopoietic disorders, telomeres are continuously stabilized by reactivation of telomerase, which can selectively be targeted by telomerase-specific therapy. The use of the telomerase inhibitor Imetelstat in patients with essential thrombocythmia or myelofibrosis as well as the use of dendritic cell-based telomerase vaccination in AML patients with complete remissions are promising examples for anti-telomerase targeted strategies in hematologic malignancies. In contrast, the elevation in telomerase levels through treatment with androgens has become an exciting clinical intervention for patients with BMFS. Here, we review recent developments, which highlight the impact of telomeres and telomerase targeted therapies in hematologic dysfunctions

iPSC-derived mesenchymal stromal cells are less supportive than primary MSCs for co-culture of hematopoietic progenitor cells

Abstract In vitro culture of hematopoietic stem and progenitor cells (HPCs) is supported by a suitable cellular microenvironment, such as mesenchymal stromal cells (MSCs)—but MSCs are heterogeneous and poorly defined. In this study, we analyzed whether MSCs derived from induced pluripotent stem cells (iPS-MSCs) provide a suitable cellular feeder layer too. iPS-MSCs clearly supported proliferation of HPCs, maintenance of a primitive immunophenotype (CD34+, CD133+, CD38-), and colony-forming unit (CFU) potential of CD34+ HPCs. However, particularly long-term culture-initiating cell (LTC-IC) frequency was lower with iPS-MSCs as compared to primary MSCs. Relevant genes for cell-cell interaction were overall expressed at similar level in MSCs and iPS-MSCs, whereas VCAM1 was less expressed in the latter. In conclusion, our iPS-MSCs support in vitro culture of HPCs; however, under the current differentiation and culture conditions, they are less suitable than primary MSCs from bone marrow

Additional file 2: of iPSC-derived mesenchymal stromal cells are less supportive than primary MSCs for co-culture of hematopoietic progenitor cells

DNA methylation is in line with differential expression of VCAM1, CDH2, and LAMB1. DNA methylation levels of CpG dinucleotides in the genes VCAM1, CDH2, and LAMB1 were analyzed for bone marrow-derived MSCs, iPS-MSCs, and iPSCs using the Illumina 450 k BeadChip data (GSE17448 and GSE54767) as described in detail in our previous work [1]. DNA methylation level is given as β-value ranging from 0 (no methylation) to 1 (100 % methylation). Genomic location of the respective CpG sites and statistical significance of MSCs vs. iPS-MSCs are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, TSS1500 = 1500 bp upstream of transcription start site; TSS200 = 200 bp upstream of TSS; UTR = untranslated region). DNA methylation of VCAM1 was higher in iPS-MSCs than MSCs. In contrast, close to the transcription start site of LAMB1 and CDH2 several CpGs revealed significantly lower DNA methylation in iPS-MSCs than primary MSCs. These epigenetic differences may therefore be relevant for the observed differences in gene expression. (PDF 362 kb

Additional file 1: of iPSC-derived mesenchymal stromal cells are less supportive than primary MSCs for co-culture of hematopoietic progenitor cells

Functional characterization of iPS-MSCs. (A) Phase contrast images of MSCs and iPS-MSCs: iPS-MSCs revealed similar fibroblastoid morphology as MSCs. (B) iPS-MSCs displayed similar immunophenotypic characteristics as primary MSCs (MFI = mean fluorescence intensity; mean ± S.D. of three biological replicates is presented; *P < 0.05, **P < 0.01). (C) MSCs and iPS-MSCs were differentiated for three weeks towards adipogenic, osteogenic, and chondrogenic lineages and subsequently stained with BODIPY/DAPI, Alizarin Red, or Alcian Blue/PAS, respectively (in analogy to our previous work [1]). Controls were simultaneously cultured in normal growth medium (DMEM supplemented with 10 % human platelet lysate). Representative images are shown. (PDF 153 kb

Data standards for heart failure : the European Unified Registries for Heart Care Evaluation and Randomized Trials (EuroHeart)

Standardized data definitions are essential for assessing the quality of care and patient outcomes in observational studies and randomized controlled trials. The European Unified Registries for Heart Care Evaluation and Randomized Trials (EuroHeart) project of the European Society of Cardiology (ESC) aims to create contemporary pan-European data standards for cardiovascular diseases, including heart failure (HF). We followed the EuroHeart methodology for cardiovascular data standard development. A Working Group including experts in HF registries, representatives from the Heart Failure Association of the ESC, and the EuroHeart was formed. Using Embase and Medline (2016–21), we conducted a systematic review of the literature on data standards, registries, and trials to identify variables pertinent to HF. A modified Delphi method was used to reach a consensus on the final set of variables. For each variable, the Working Group developed data definitions and agreed on whether it was mandatory (Level 1) or additional (Level 2). In total, 84 Level 1 and 79 Level 2 variables were selected for nine domains of HF care. These variables were reviewed by an international Reference Group with the Level 1 variables providing the dataset for registration of patients with HF on the EuroHeart IT platform. By means of a structured process and interaction with international stakeholders, harmonized data standards for HF have been developed. In the context of the EuroHeart, this will facilitate quality improvement, international observational research, registry-based randomized trials, and post-marketing surveillance of devices and pharmacotherapies across Europe