MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3′ untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.     

Family-Based Cluster Randomized Controlled Trial Enhancing Physical Activity and Motor Competence in 4–7-Year-Old Children

Little is known of how to involve families in physical activity (PA) interventions for children. In this cluster randomized controlled trial, we recruited families with four- to seven-year-old children to participate in a year-long study where parents in the intervention group families (n = 46) received tailored counseling to increase children’s PA. Structured PA was not served. Control group families (n = 45) did not receive any counseling. PA in all children (n = 91; mean age 6.16 ± 1.13 years at the baseline) was measured by accelerometers at the baseline and after three, six, nine and 12 months. Motor competence (MC) (n = 89) was measured at the baseline and after six and 12 months by a KTK (KörperkoordinationsTest für Kinder) and throwing and catching a ball (TCB) protocols. The effect of parental counseling on study outcomes was analyzed by a linear mixed-effects model fit by REML and by a Mann-Whitney U test in the case of the TCB. As season was hypothesized to affect counseling effect, an interaction of season on the study outcomes was examined. The results show significant decrease of MVPA in the intervention group when compared to the control group (p < .05). The TCB showed a nearly significant improvement at six months in the intervention group compared to the controls (p = .051), but not at 12 months. The intervention group had a steadier development of the KTK when the interaction of season was taken into account. In conclusion, more knowledge of family constructs associating with the effectiveness of counseling is needed for understanding how to enhance PA in children by parents. However, a hypothesis may be put forward that family-based counseling during an inactive season rather than an active season may provide a more lasting effect on the development of KTK in children.

Trial Registration

Controlled-Trials.com ISRCTN28668090     

A Transgenic Prox1-Cre-tdTomato Reporter Mouse for Lymphatic Vessel Research

The lymphatic vascular system plays an active role in immune cell trafficking, inflammation and cancer spread. In order to provide an in vivo tool to improve our understanding of lymphatic vessel function in physiological and pathological conditions, we generated and characterized a tdTomato reporter mouse and crossed it with a mouse line expressing Cre recombinase under the control of the lymphatic specific promoter Prox1 in an inducible fashion. We found that the tdTomato fluorescent signal recapitulates the expression pattern of Prox1 in lymphatic vessels and other known Prox1-expressing organs. Importantly, tdTomato co-localized with the lymphatic markers Prox1, LYVE-1 and podoplanin as assessed by whole-mount immunofluorescence and FACS analysis. The tdTomato reporter was brighter than a previously established red fluorescent reporter line. We confirmed the applicability of this animal model to intravital microscopy of dendritic cell migration into and within lymphatic vessels, and to fluorescence-activated single cell analysis of lymphatic endothelial cells. Additionally, we were able to describe the early morphological changes of the lymphatic vasculature upon induction of skin inflammation. The Prox1-Cre-tdTomato reporter mouse thus shows great potential for lymphatic research.     

Differential epithelium DNA damage response to ATM and DNA-PK pathway inhibition in human prostate tissue culture

The ability of cells to respond and repair DNA damage is fundamental for the maintenance of genomic integrity. Ex vivo culturing of surgery-derived human tissues has provided a significant advancement to assess DNA damage response (DDR) in the context of normal cytoarchitecture in a non-proliferating tissue. Here, we assess the dependency of prostate epithelium DDR on ATM and DNA-PKcs, the major kinases responsible for damage detection and repair by nonhomologous end-joining (NHEJ), respectively. DNA damage was caused by ionizing radiation (IR) and cytotoxic drugs, cultured tissues were treated with ATM and DNA-PK inhibitors, and DDR was assessed by phosphorylation of ATM and its targets H2AX and KAP1, a heterochromatin binding protein. Phosphorylation of H2AX and KAP1 was fast, transient and fully dependent on ATM, but these responses were moderate in luminal cells. In contrast, DNA-PKcs was phosphorylated in both luminal and basal cells, suggesting that DNA-PK-dependent repair was also activated in the luminal cells despite the diminished H2AX and KAP1 responses. These results indicate that prostate epithelial cell types have constitutively dissimilar responses to DNA damage. We correlate the altered damage response to the differential chromatin state of the cells. These findings are relevant in understanding how the epithelium senses and responds to DNA damage.Key words: DNA damage, prostate, γH2AX, ATM, DNA-PK     

Melanoma cell lines are susceptible to histone deacetylase inhibitor TSA provoked cell cycle arrest and apoptosis

Melanoma is the most aggressive of skin cancers because of its high resistance to currently available therapy. Although melanoma cells often retain wild-type p53 tumour suppressor protein and express it at high levels, the p53 mediated apoptosis pathway is suppressed. Histone deacetylase (HDAC) inhibitors are a promising group of compounds inducing differentiation, growth arrest and apoptosis in tumour cells in preclinical studies. We have studied the cellular effects of trichostatin A (TSA), a HDAC inhibitor, in a panel of melanoma cell lines and its mechanism of action in relation to p53. TSA stabilized wild-type p53, but p53 protein accumulation was overridden by simultaneous downregulation of p53 mRNA leading to a decrease in p53 protein. While growth arrest was induced in all cell lines studied and apoptosis in most (6/7), these cellular effects were independent of the p53 status of the cells. Inhibiting p53 function by a dominant negative p53 (p53(175His)) confirmed that the HDAC inhibitor induced apoptosis was independent of wild-type p53, even though TSA slightly activated p53 in a reporter assay. The results indicate that while the action of TSA is independent of p53, the activation of the apoptosis pathway by the HDAC inhibitors may provide therapeutic approaches for melanoma treatment.     

Smooth muscle-endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels--smooth muscle and endothelial cells--and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.     

Genes involved in systemic and arterial bed dependent atherosclerosis--Tampere Vascular study

Background

Atherosclerosis is a complex disease with hundreds of genes influencing its progression. In addition, the phenotype of the disease varies significantly depending on the arterial bed.

Methodology/Principal Findings

We characterized the genes generally involved in human advanced atherosclerotic (AHA type V–VI) plaques in carotid and femoral arteries as well as aortas from 24 subjects of Tampere Vascular study and compared the results to non-atherosclerotic internal thoracic arteries (n=6) using genome-wide expression array and QRT-PCR. In addition we determined genes that were typical for each arterial plaque studied. To gain a comprehensive insight into the pathologic processes in the plaques we also analyzed pathways and gene sets dysregulated in this disease using gene set enrichment analysis (GSEA). According to the selection criteria used (>3.0 fold change and p-value <0.05), 235 genes were up-regulated and 68 genes down-regulated in the carotid plaques, 242 genes up-regulated and 116 down-regulated in the femoral plaques and 256 genes up-regulated and 49 genes down-regulated in the aortic plaques. Nine genes were found to be specifically induced predominantly in aortic plaques, e.g., lactoferrin, and three genes in femoral plaques, e.g., chondroadherin, whereas no gene was found to be specific for carotid plaques. In pathway analysis, a total of 28 pathways or gene sets were found to be significantly dysregulated in atherosclerotic plaques (false discovery rate [FDR] <0.25).

Conclusions

This study describes comprehensively the gene expression changes that generally prevail in human atherosclerotic plaques. In addition, site specific genes induced only in femoral or aortic plaques were found, reflecting that atherosclerotic process has unique features in different vascular beds.     

Short-latency stretch reflex modulation in response to varying soleus muscle activities.

The current investigation examined the effect of various types of background muscle contractions on the short-latency stretch reflex (SLR) elicited from the soleus muscle while subjects were in a sitting position. A stretch was applied to the calf muscles while they performed an isometric (pre-ISO), shortening (pre-SHO) and lengthening contraction (pre-LEN) with several pre-contraction levels. The ankle was at a 90 degrees tibio-tarsal joint angle when the perturbation was applied. Subjects developed and maintained a given pre-load level, which was maintained at various percentages of the maximum voluntary isometric plantar flexion torque. This was performed at 80 degrees in pre-SHO, 90 degrees in pre-ISO and 100 degrees in pre-LEN for about 2s before the contractions. The SLRs in trials with 0, 35 and 50% of the maximum voluntary contraction torque level were compared among the three conditions. The main results were as follows. (1) Pre-ISO and pre-SHO showed an equal SLR area and a different SLR waveform in the active muscle. (2) Pre-LEN showed the smallest SLR area of three conditions in the active muscle. (3) Pre-LEN showed shorter SLR latencies than the other conditions. (4) Pre-SHO showed a longer SLR latency in the relaxed muscle than in the active muscle. (5) The SLR area was larger in the active muscle than in the relaxed muscle. These findings demonstrate that the muscle contraction type and the pre-contraction level before a stretch perturbation have a considerable influence on the latency, the area and the waveform of the SLR. In particular, the equal area and the different waveforms of the SLR between pre-ISO and pre-SHO were a unique finding in the present study. They might result from differences in muscle spindle sensitivity and afferent input from various receptors induced by the present motor task.