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Human adenovirus infection is life threatening after allogeneic haematopoietic stem cell transplantation (HSCT). Immunotherapy with donor-derived adenovirus-specific T cells is promising; however, 20% of all donors lack adenovirus-specific T cells. To overcome this, we transfected α/β T cells with mRNA encoding a T-cell receptor (TCR) specific for the HLA-A*0101-restricted peptide LTDLGQNLLY from the adenovirus hexon protein. Furthermore, since allo-reactive endogenous TCR of donor T lymphocytes would induce graft-versus-host disease (GvHD) in a mismatched patient, we transferred the TCR into γ/δ T cells, which are not allo-reactive. TCR-transfected γ/δ T cells secreted low quantities of cytokines after antigen-specific stimulation, which were increased dramatically after co-transfection of CD8α-encoding mRNA. In direct comparison with TCR-transfected α/β T cells, TCR-CD8α-co-transfected γ/δ T cells produced more tumor necrosis factor (TNF), and lysed peptide-loaded target cells as efficiently. Most importantly, TCR-transfected α/β T cells and TCR-CD8α-co-transfected γ/δ T cells efficiently lysed adenovirus-infected target cells. We show here, for the first time, that not only α/β T cells but also γ/δ T cells can be equipped with an adenovirus specificity by TCR-RNA electroporation. Thus, our strategy offers a new means for the immunotherapy of adenovirus infection after allogeneic HSCT.

Methylation of cytosines is a prototypic epigenetic modification of the DNA. It has been implicated in various regulatory mechanisms across the animal kingdom and particularly in vertebrates. We mapped DNA methylation in 580 animal species (535 vertebrates, 45 invertebrates), resulting in 2443 genome-scale DNA methylation profiles of multiple organs. Bioinformatic analysis of this large dataset quantified the association of DNA methylation with the underlying genomic DNA sequence throughout vertebrate evolution. We observed a broadly conserved link with two major transitions—once in the first vertebrates and again with the emergence of reptiles. Cross-species comparisons focusing on individual organs supported a deeply conserved association of DNA methylation with tissue type, and cross-mapping analysis of DNA methylation at gene promoters revealed evolutionary changes for orthologous genes. In summary, this study establishes a large resource of vertebrate and invertebrate DNA methylomes, it showcases the power of reference-free epigenome analysis in species for which no reference genomes are available, and it contributes an epigenetic perspective to the study of vertebrate evolution.

Neuroblastomas are the most common extracerebral solid tumors of infancy and childhood. These embryonal tumors of the sympathetic nervous system are unique among pediatric tumors because of their biologic complexity. Neuroblastoma is often lethal, but up to 30 percent of children have favorable outcomes. In this group, neither widespread disease (stage IVS disease) 1 – 3 nor the presence of residual tumor after surgery influences the prognosis. 4 , 5 Mass screening of infants during the first months of life was reported to increase the detection of neuroblastoma in the screened population as compared with an unscreened population. 6 These unusual phenomena can be explained . . .

The EWS-FLI1 transcription factor is consistently expressed in 85% of Ewing tumors (EFT). In heterologous cells, EWS-FLI1 induces p53-dependent cell cycle arrest or apoptosis. It has been speculated that the p53 tumor suppressor pathway may be generally compromised in EFT despite only rare p53 mutations. In order to test for functional integrity of this pathway, we have investigated a series of EFT cell lines that differ from each other with respect to their endogenous p53 and INK4A gene status for their response to ectopic p53 expression and to stimulation of endogenous p53 activity by X-ray treatment. Significant interindividual and intratumoral variations in the apoptotic propensity of EFT cell lines to transient expression of ectopic p53 were observed, which was independent of the level of p53 expression. In cell lines with a low apoptotic incidence, apoptosis was delayed and the surviving fraction showed a prolonged growth arrest. Complete resistance to p53-induced apoptosis in two cell lines established from the same patient was associated with a high BCL2/BAX ratio and low levels of APAF1. Sensitivity to X-rays showed a trend towards a higher apoptotic rate in wild-type (wt) p53 expressing than in p53 mutant cells. However, one wt p53-expressing EFT cell line was completely refractory to irradiation-stimulated cell death despite high apoptotic responsiveness to ectopic p53. No difference in Ser15 phosphorylation and the transcriptional activation of p53 targets was observed in wt p53 EFT cell lines irrespective of the induction of cell death or growth arrest. All together, our results demonstrate that despite significant variability in the outcome, cell death or cell cycle arrest, the p53 downstream pathway and the DNA damage signaling pathway are functionally intact in EFT.

Glioblastoma is the most dangerous brain cancer. One reason for glioblastoma's aggressiveness are glioblastoma stem‐like cells. To target them, a number of markers have been proposed (CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6). A comprehensive study of co‐expression patterns of them has, however, not been performed so far. Here, we mapped the multidimensional co‐expression profile of these stemness‐associated molecules. Gliomaspheres – an established model of glioblastoma stem‐like cells – were used. Seven different gliomasphere systems were subjected to multicolor flow cytometry measuring the nine markers CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6 all simultaneously based on a novel 9‐marker multicolor panel developed for this study. The viSNE dimensionality reduction algorithm was applied for analysis. All gliomaspheres were found to express at least five different glioblastoma stem‐like cell markers. Multi‐dimensional analysis showed that all studied gliomaspheres consistently harbored a cell population positive for the molecular signature CD44+/CD133+/ITGA6+/CD36+. Glioblastoma patients with an enrichment of this combination had a significantly worse survival outcome when analyzing the two largest available The Cancer Genome Atlas datasets (MIT/Harvard Affymetrix: P = 0.0015, University of North Carolina Agilent: P = 0.0322). In sum, we detected a previously unknown marker combination – demonstrating feasibility, usefulness, and importance of high‐dimensional gliomasphere marker combinatorics.

Glioblastoma is the most dangerous brain cancer. One reason for glioblastoma's aggressiveness are glioblastoma stem‐like cells. To target them, a number of markers have been proposed ( CD 133, CD 44, CD 15, A2B5, CD 36, CXCR 4, IL 6R, L1 CAM , and ITGA 6). A comprehensive study of co‐expression patterns of them has, however, not been performed so far. Here, we mapped the multidimensional co‐expression profile of these stemness‐associated molecules. Gliomaspheres – an established model of glioblastoma stem‐like cells – were used. Seven different gliomasphere systems were subjected to multicolor flow cytometry measuring the nine markers CD 133, CD 44, CD 15, A2B5, CD 36, CXCR 4, IL 6R, L1 CAM , and ITGA 6 all simultaneously based on a novel 9‐marker multicolor panel developed for this study. The vi SNE dimensionality reduction algorithm was applied for analysis. All gliomaspheres were found to express at least five different glioblastoma stem‐like cell markers. Multi‐dimensional analysis showed that all studied gliomaspheres consistently harbored a cell population positive for the molecular signature CD 44+/ CD 133+/ ITGA 6+/ CD 36+. Glioblastoma patients with an enrichment of this combination had a significantly worse survival outcome when analyzing the two largest available The Cancer Genome Atlas datasets ( MIT /Harvard Affymetrix: P  = 0.0015, University of North Carolina Agilent: P  = 0.0322). In sum, we detected a previously unknown marker combination – demonstrating feasibility, usefulness, and importance of high‐dimensional gliomasphere marker combinatorics.

Human adenovirus infection is life threatening after allogeneic haematopoietic stem cell transplantation (HSCT). Immunotherapy with donor-derived adenovirus-specific T cells is promising; however, 20% of all donors lack adenovirus-specific T cells. To overcome this, we transfected [alpha]/[beta] T cells with mRNA encoding a T-cell receptor (TCR) specific for the HLA-A*0101-restricted peptide LTDLGQNLLY from the adenovirus hexon protein. Furthermore, since allo-reactive endogenous TCR of donor T lymphocytes would induce graft-versus-host disease (GvHD) in a mismatched patient, we transferred the TCR into [gamma]/[delta] T cells, which are not allo-reactive. TCR-transfected [gamma]/[delta] T cells secreted low quantities of cytokines after antigen-specific stimulation, which were increased dramatically after co-transfection of CD8[alpha]-encoding mRNA. In direct comparison with TCR-transfected [alpha]/[beta] T cells, TCR-CD8[alpha]-co-transfected [gamma]/[delta] T cells produced more tumor necrosis factor (TNF), and lysed peptide-loaded target cells as efficiently. Most importantly, TCR-transfected [alpha]/[beta] T cells and TCR-CD8[alpha]-co-transfected [gamma]/[delta] T cells efficiently lysed adenovirus-infected target cells. We show here, for the first time, that not only [alpha]/[beta] T cells but also [gamma]/[delta] T cells can be equipped with an adenovirus specificity by TCR-RNA electroporation. Thus, our strategy offers a new means for the immunotherapy of adenovirus infection after allogeneic HSCT.

Human adenovirus infection is life threatening after allogeneic haematopoietic stem cell transplantation (HSCT). Immunotherapy with donor-derived adenovirus-specific T cells is promising; however, 20% of all donors lack adenovirus-specific T cells. To overcome this, we transfected alpha / beta T cells with mRNA encoding a T-cell receptor (TCR) specific for the HLA-A*0101-restricted peptide LTDLGQNLLY from the adenovirus hexon protein. Furthermore, since allo-reactive endogenous TCR of donor T lymphocytes would induce graft-versus-host disease (GvHD) in a mismatched patient, we transferred the TCR into gamma / delta T cells, which are not allo-reactive. TCR-transfected gamma / delta T cells secreted low quantities of cytokines after antigen-specific stimulation, which were increased dramatically after co-transfection of CD8 alpha -encoding mRNA. In direct comparison with TCR-transfected alpha / beta T cells, TCR-CD8 alpha -co-transfected gamma / delta T cells produced more tumor necrosis factor (TNF), and lysed peptide-loaded target cells as efficiently. Most importantly, TCR-transfected alpha / beta T cells and TCR-CD8 alpha -co-transfected gamma / delta T cells efficiently lysed adenovirus-infected target cells. We show here, for the first time, that not only alpha / beta T cells but also gamma / delta T cells can be equipped with an adenovirus specificity by TCR-RNA electroporation. Thus, our strategy offers a new means for the immunotherapy of adenovirus infection after allogeneic HSCT.

We analyzed the p53 mutational status, mRNA and protein expression in 24 human breast carcinoma cell lines. Following measurement of their DNA content with flow cytometry, we ascertained the copy numbers of the centromere of chromosome 17 (cen17) and p53 with fluorescence in situ hybridization (FISH). A functional yeast assay (FASAY) was used to screen for inactivating mutations. Positive results were subsequently verified by DNA sequencing. Finally, we assessed the mRNA expression with a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay and the protein expression with immunocytochemical staining, western blot, and quantitative flow cytometry. The DNA content of the cell lines ranged from 0.85 to 2.58. Nine cell lines had concordant copy numbers (between two and four) of p53 and cen17, whereas 12 had more, and three less cen17 than p53 copies. The FASAY was successful in all but one cell line and revealed the presence of mutated alleles in 16 of them, 13 cell lines expressed only the mutated, and three both the mutated and the wild-type alleles. The mutations were comprised of 11 missense, two nonsense, and three frameshift mutations. Immunocytochemical staining, western blot and quantitative flow cytometry yielded comparable p53 protein expression results. However, both the mRNA and the protein expression levels varied considerably in the different cell lines and no consistent pattern with regard to the respective p53 mutational status became evident. The results obtained in these breast carcinoma cell lines indicate that no clear-cut linear relationship exists between the p53 mutational status and the extent of its respective mRNA and protein expression. Therefore, direct DNA analyses and functional assays remain the only methods for the reliable detection of p53 mutations.