Endometrial Mesenchymal Stem/Stromal Cells Modulate the Macrophage Response to Implanted Polyamide/Gelatin Composite Mesh in Immunocompromised and Immunocompetent Mice.

The immunomodulatory properties of human endometrial mesenchymal stem cells (eMSC) have not been well characterised. Initial studies showed that eMSC modulated the chronic inflammatory response to a non-degradable polyamide/gelatin mesh in a xenogeneic rat skin wound repair model, but the mechanism remains unclear. In this study, we investigated the immunomodulatory effect of eMSC on the macrophage response to polyamide/gelatin composite mesh in an abdominal subcutaneous wound repair model in C57BL6 immunocompetent and NSG (NOD-Scid-IL2Rgamma null ) immunocompromised mice to determine whether responses differed in the absence of an adaptive immune system and NK cells. mCherry lentivirus-labelled eMSC persisted longer in NSG mice, inducing longer term paracrine effects. Inclusion of eMSC in the mesh reduced inflammatory cytokine (Il-1ß, Tnfα) secretion, and in C57BL6 mice reduced CCR7+ M1 macrophages surrounding the mesh on day 3 and increased M2 macrophage marker mRNA (Arg1, Mrc1, Il10) expression at days 3 and 7. In NSG mice, these effects were delayed and only observed at days 7 and 30 in comparison with controls implanted with mesh alone. These results show that the differences in the immune status in the two animals directly affect the survival of xenogeneic eMSC which leads to differences in the short-term and long-term macrophage responses to implanted meshes.

Cyclin-dependent kinase 7 is a therapeutic target in high-grade glioma.

High-grade glioma (HGG) is an incurable brain cancer. The transcriptomes of cells within HGG tumors are highly heterogeneous. This renders the tumors unresponsive or able to adapt to therapeutics targeted at single pathways, thereby causing treatment failure. To overcome this, we focused on cyclin-dependent kinase 7 (CDK7), a ubiquitously expressed molecule involved in two major drivers of HGG pathogenesis: cell cycle progression and RNA polymerase-II-based transcription. We tested the activity of THZ1, an irreversible CDK7 inhibitor, on patient-derived primary HGG cell lines and ex vivo HGG patient tissue slices, using proliferation assays, microarray analysis, high-resolution respirometry, cell cycle analysis and in vivo tumor orthografts. The cellular processes affected by CDK7 inhibition were analyzed by reverse transcriptase-quantitative PCR, western blot, flow cytometry and immunofluorescence. THZ1 perturbed the transcriptome and disabled CDK activation, leading to cell cycle arrest at G2 and DNA damage. THZ1 halted transcription of the nuclear-encoded mitochondrial ribosomal genes, reducing mitochondrial translation and oxidative respiration. It also inhibited the expression of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFR-α), reducing signaling flux through the AKT, extracellular-signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) downstream pathways. Finally, THZ1 disrupted nucleolar, Cajal body and nuclear speckle formation, resulting in reduced cytosolic translation and malfunction of the spliceosome and thus leading to aberrant mRNA processing. These findings indicate that CDK7 is crucial for gliomagenesis, validate CDK7 as a therapeutic target and provide new insight into the cellular processes that are affected by THZ1 and induce antitumor activity.

Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny.

Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between de novo DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (POLGA)) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of POLGA in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of POLGA as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of POLGA leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein (GFAP). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of POLGA is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of POLGA and mtDNA copy number in the presence of a DNA demethylation agent that inhibits de novo methyltransferase 1 activity.

Critical role of the transcription factor AP-1 for the constitutive and interferon-induced expression of IFI 16.

IFI 16 is a member of the HIN-200 family of transcriptional regulators that suppress cell growth, modulate the cell cycle and have been linked to cellular differentiation. We hypothesized that the activity of IFI 16 depends on its level of expression and therefore studied the transcriptional activity of the IFI 16 promoter. A discrete sequence within the 5' untranslated region was required for constitutive activity of the promoter and the functional motif within this region was shown to be a consensus AP-1 site. Interestingly, this AP-1 site was also critical for IFN-induced activation of the promoter and consistent with these observations, treatment of cells with IFNgamma resulted in a rapid and robust induction of AP-1 activity that preceded expression of IFI 16. These experiments define the transcriptional mechanisms of IFI 16 gene regulation and provide evidence suggesting that AP-1 activation may be an important event in IFN signaling.

The pathology of non-Rhesus hydrops.

The pathological findings in 50 cases of non-Rhesus hydrops are presented. In 19, pregnancy terminated before the twenty-eighth week of gestation; there were five chromosomally abnormal fetuses and a further six with structural abnormality in this group. Three of 31 infants from the more mature group survived, 16 were stillborn and 12 died in the neonatal period. Nineteen infants had malformations, 14 of these being cardiovascular malformation. Five infants had evidence of intra-uterine infection. Intra-uterine heart failure is the commonest cause of the hydrops.