High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18–30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.

The transcription factor aryl hydrocarbon receptor (AhR) represents a promising therapeutic target in allergy and autoimmunity. AhR signaling induced by the newly described ligand VAF347 inhibits allergic lung inflammation as well as suppresses pancreatic islet allograft rejection. These effects are likely mediated via alterations in dendritic cell (DC) function. Moreover, VAF347 induces tolerogenic DCs. Langerhans cells (LCs) are immediate targets of exogenous AhR ligands at epithelial surfaces; how they respond to AhR ligands remained undefined. We studied AhR expression and function in human LCs and myelopoietic cell subsets using a lineage differentiation and gene transduction model of human CD34+ hematopoietic progenitors. We found that AhR is highly regulated during myeloid subset differentiation. LCs expressed highest AhR levels followed by monocytes. Conversely, neutrophil granulocytes lacked AhR expression. AhR ligands including VAF347 arrested the differentiation of monocytes and LCs at an early precursor cell stage, whereas progenitor cell expansion or granulopoiesis remained unimpaired. AhR expression was coregulated with the transcription factor PU.1 during myeloid subset differentiation. VAF347 inhibited PU.1 induction during initial monocytic differentiation, and ectopic PU.1 restored monocyte and LC generation in the presence of this compound. AhR ligands failed to interfere with cytokine receptor signaling during LC differentiation and failed to impair LC activation/maturation. VAF347-mediated antiproliferative effect on precursors undergoing LC lineage differentiation occurred in a clinically applicable serum-free culture model and was not accompanied by apoptosis induction. In conclusion, AhR agonist signaling interferes with transcriptional processes leading to monocyte/DC lineage commitment of human myeloid progenitor cells.

Adenoviruses encode a set of highly abundant microRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger noncoding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant multiplicities of infection (MOIs). In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the posttranscriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells, we determined changes in cellular miRNA levels in both total RNA and RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified the direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the two most abundant isoforms in RISCs of lytically infected A549 cells. IMPORTANCE Viral and cellular miRNAs have been recognized as important players in virus-host interactions.This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC targetome during lytic adenovirus infection and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover, this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

Highlights ► SiRNAs inhibit adenovirus multiplication. ► Adenoviral DNA replication is a key target for siRNA-mediated inhibition. ► SiRNAs can delay the death of adenovirus-infected cells.

Highlights► AmiRNAs can be utilized to inhibit adenovirus replication in vitro. ► Adenoviral vectors are an effective expression vehicle for anti-adenoviral amiRNAs. ► A combination of amiRNA and cidofovir results in additive effects.

IntroductionTwo in vitro systems for the generation of human dendritic cells (DCs) are widely used for basic and clinically oriented research: monocyte-derived DCs (moDCs) and hematopoietic progenitor cell (HPC)-derived DCs. In the first system, granulocytemacrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) are added to peripheral blood monocytes to generate moDCs. 1 This system is most widely applied for generating human DCs. In the second model, CD34 ϩ HPCs are stimulated in transforming growth factor ␤1 (TGF-␤1)-supplemented serumfree cultures (containing basic DC cytokines GM-CSF, tumor necrosis factor ␣ [TNF␣], stem cell factor [SCF] with or without Flt3L) for generating Langerhans cells (LCs), a DC subtype that resides within epithelial tissue. 2,3 Exclusion of TGF-␤1 from this latter culture system abrogates LC differentiation and de-represses a program of monocyte generation from CD34 ϩ cells. 3 When these latter HPC cultures are initiated in the presence of serum without TGF-␤1, 2 pathways of CD1a ϩ DCs are simultaneously generated, ie LCs and non-LC interstitial/dermal-type DCs. 4 These populations arise from 2 separate precursor pathways at culture day 5. A CD1a ϩ CD14 Ϫ precursor gives rise to LCs, whereas a CD1a Ϫ CD14 ϩ precursor can give rise to non-LC DCs, which share many features with moDCs generated from peripheral blood monocytes. 4,5 This CD1a Ϫ CD14 ϩ intermediate population can be further subdivided into 2 functional subsets based on CD11b (CD11b ϩ CD14 ϩ versus CD11b Ϫ CD14 ϩ monocytic cells). CD11b ϩ CD14 ϩ cells can be induced by GM-CSF plus IL-4 to develop into intDC/moDCs or by M-CSF to macrophage differentiation. Conversely, CD11b Ϫ CD14 ϩ intermediates, which represent early monocytic cells, retain LC differentiation capacity in the presence of TGF-␤1. 6 Therefore, a CD14 ϩ monocytic precursor can be induced to develop along 2 separate pathways in vitro depending on IL-4 (moDCs) versus TGF-␤1 (LCs) cytokine signals.IL-4 versus TGF-␤1 antagonize each other's function to induce moDCs versus LCs. Neutralizing anti-TGF-␤1 mAb represses LC generation in favor of CD14 ϩ monocyte generation. 7 Similarly, the addition of IL-4 to TGF-␤1-supplemented LC generation cultures of CD34 ϩ cells represses LC differentiation in favor of inducing non-LC DC differentiation. 7 Conversely, the addition of TGF-␤1 to GM-CSF plus IL-4-containing moDC cultures of peripheral blood monocytes polarizes these cells toward an LC-like phenotype. 8 In line with this, TGF-␤1 in the epidermal microenvironment is critical for LC differentiation in vivo. [9][10][11] Functional differences between moDCs versus LCs are increasingly recognized. However, very little is known about the molecular mechanisms controlling the lineage commitment of these cells to develop into different DC subsets (ie, LCs and non-LC moDCs).We recently performed mechanistic studies on the involvement of family members of the nuclear hormone receptor system, ie, retinoid X receptor-alpha (RXR␣) and vitamin D 3 receptor (VDR) i...

We extended the concept of enzyme/prodrug therapy to adenovirus infections by selectively sensitizing adenovirus-infected cells to treatment with GCV.

The transcription factor Ets2 and its transcriptional targets osteopontin (OPN) and osteocalcin (OC) are expressed in tissue-engineered bone constructs in vitro. Up to now little is known about the role of Ets2 in tissue-engineering applications. This study was intended to investigate the hypothesis that protein expression of Ets2 is correlated with the expression of bone-specific proteinsin tissue-engineeredbone constructs. Cell-seeded three-dimensional bone constructs manufactured with osteoblastic cells and poly(lactic-co-glycolic acid) polymer fleeces over a period of 21 days were analyzed by SDS-PAGE and Western blotting. The protein expression of OPN, OC, osteonectin and collagen type I was analyzed. Cellularity, alkaline phosphatase-specific activity and histology confirmed the osteoblastic phenotype of the constructs. Correlations between Ets2 expression and OPN and Ets2 and collagen type I expression could be detected during the phase of late osteoblastic differentiation between days 9 and 21. The correlation between OC and collagen type I was significant in this late stage of osteoblastic differentiation. These results suggest that there is a strong interplay of Ets2 with bone-specific proteins in cell-seeded three-dimensional bone constructs. This study is a crucial step to elucidate the complex interplay of bone-related proteins in the application of bone tissue engineering.