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Cai F.F.,University of Basel | Kohler C.,University of Basel | Zhang B.,University of Basel | Chen W.J.,University of Basel | And 6 more authors.
Anticancer Research | Year: 2011

Background: Alterations of mitochondrial DNA (mtDNA) have been found in cancer patients, therefore informative mtDNA mutations could serve as biomarkers for the disease. Materials and Methods: The two hypervariable regions HVR1 and HVR2 in the D-Loop region were sequenced in ten paired tissue and plasma samples from breast cancer patients. Results: MtDNA mutations were found in all patients' samples, suggesting a 100% detection rate. Examining germline mtDNA mutations, a total of 85 mutations in the D-loop region were found; 31 of these mutations were detected in both tissues and matched plasma samples, the other 54 germline mtDNA mutations were found only in the plasma samples. Regarding somatic mtDNA mutations, a total of 42 mutations in the D-loop region were found in breast cancer tissues. Conclusion: Somatic mtDNA mutations in the D-loop region were detected in breast cancer tissues but not in the matched plasma samples, suggesting that more sensitive methods will be needed for such detection to be of clinical utility.

Tatura R.,University of Duisburg - Essen | Zeschnigk M.,University of Duisburg - Essen | Adamzik M.,University of Duisburg - Essen | Probst-Kepper M.,Institute for Clinical Transfusion Medicine | And 2 more authors.
PLoS ONE | Year: 2012

During sepsis, a relative increase of regulatory T (Treg) cells has been reported. Its persistence is associated with lymphocyte anergy, immunoparalysis and a poor prognosis. Currently, an exact quantification of human Treg cells based on protein expression of marker molecules is ambiguous, as these molecules are expressed also by activated non-regulatory T cells. Furthermore, no firm criteria for flow cytometer gate settings exist so far. Recently, a specific DNA methylation pattern within FOXP3-TSDR has been reported that allows distinguishing Treg and non-regulatory T cells, independent of their activation status. Using this epigenetic marker, we established a single-tube real-time PCR based methylation assay (QAMA) for relative quantification of Treg cells. Validation was performed on defined ratios of methylated and unmethylated target sequence and on mixtures of Treg and non-regulatory T cells. DNA-methylation was measured in CD4+ T cells isolated from blood samples of 30 septic patients and 30 healthy subjects and compared with results of Treg cell quantification by flow cytometry based on CD4+ CD25hiCD127low measurement. In septic patients both methods showed an increased ratio of Treg cells to all CD4+ T cells. In healthy individuals, the results obtained by both methods were clearly positively correlated. However, the correlation between both methods in septic patients was only weak. We showed that quantification of Treg cells by QAMA detects CD4+ T cells with unmethylated FOXP3-TSDR, hidden in the CD25med/low fraction of flow cytometry. Given that unmethylated FOXP3-TSDR is the most specific feature of Treg cells to date, our assay precisely quantifies Treg cells, as it additionally detects those committed Treg cells, hidden in the CD25med/low fraction of CD4+ cells. Furthermore, QAMA is a reliable method, which is easier to standardize among laboratories and can thus improve reproducibility of Treg cell quantification. © 2012 Tatura et al.

Kehrmann J.,University of Duisburg - Essen | Tatura R.,University of Duisburg - Essen | Zeschnigk M.,University of Duisburg - Essen | Probst-Kepper M.,Institute for Clinical Transfusion Medicine | And 3 more authors.
Immunology | Year: 2014

Summary: The epigenetic regulation of transcription factor genes is critical for T-cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate on human CD4+ CD25- T cells for generating demethylation within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage-specifying transcription factors of the major T-cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. The FOXP3-TSDR methylation pattern was determined by using deep amplicon bisulphite sequencing. 5-aza-2'-deoxycytidine induced FOXP3-TSDR hypomethylation and expression of the Treg-cell-specific genes FOXP3 and LRRC32. Proliferation of 5-aza-2'-deoxycytidine-treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin-3-gallate induced global DNA hypomethylation to a lesser extent than 5-aza-2'-deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg-cell-specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5-aza-2'-deoxycitidine-treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells. © 2014 John Wiley & Sons Ltd.

Hoerning A.,University of Duisburg - Essen | Kalkavan H.,University of Duisburg - Essen | Rehme C.,University of Duisburg - Essen | Menke J.,Johannes Gutenberg University Mainz | And 4 more authors.
Pediatric Transplantation | Year: 2011

The presence of microchimerism in peripheral blood of solid organ transplant recipients has been postulated to be beneficial for allograft acceptance. Kinetics of donor cell trafficking and accumulation in pediatric allograft recipients are largely unknown. In this study, we implemented SNPs of the HVRs I and II of mitochondrial DNA to serve as molecular genetic markers to detect donor-specific cell chimerism after pediatric renal transplantation. Serial dilution of artificial chimeric DNA samples showed a linear correlation coefficient of R > 0.98 and a detection sensitivity of 0.01% with high reproducibility. Longitudinal semiquantitative analysis of donor-specific SNPs was then performed in peripheral blood mononuclear cells samples up to two yr post-transplant. Quantity of donor-specific cell chimerism in peripheral blood was highest in the early post-transplant period reaching values of ∼10% after liver-kidney and 2.8% after renal transplantation. From one wk after transplantation, renal transplant patients exhibited an amount of donor-specific mtDNA ranging from 0.01% to 0.1%. We developed a highly accurate, sensitive, and rapid real-time quantitative PCR method using sequence-specific primers and fluorescent hydrolysis probes for the detection of at least 0.01% donor-specific cells in the recipient's peripheral blood after renal transplantation. © 2011 John Wiley & Sons A/S.

Scheiter M.,Helmholtz Center for Infection Research | Lau U.,Helmholtz Center for Infection Research | Van Ham M.,Helmholtz Center for Infection Research | Bulitta B.,Helmholtz Center for Infection Research | And 6 more authors.
Molecular and Cellular Proteomics | Year: 2013

The recent Natural Killer (NK) cell maturation model postulates that CD34+ hematopoietic stem cells (HSC) first develop into CD56 bright NK cells, then into CD56dimCD57- and finally into terminally maturated CD56dimCD57+. The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56 bright NK cells versus CD56dim NK cells and CD56 dimCD57- NK cells versus CD56dimCD57 + NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions. Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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