Genome-wide DNA methylation patterns are established and maintained by the coordinated action of three DNA methyltransferases (DNMTs), DNMT1, DNMT3A and DNMT3B. DNMT3B hypomorphic germline mutations are responsible for two-thirds of immunodeficiency, centromere instability, facial anomalies (ICF) syndrome cases, a rare recessive disease characterized by immune defects, instability of pericentromeric satellite 2-containing heterochromatin, facial abnormalities and mental retardation. The molecular defects in transcription, DNA methylation and chromatin structure in ICF cells remain relatively uncharacterized. In the present study, we used global expression profiling to elucidate the role of DNMT3B in these processes using cell lines derived from ICF syndrome and normal individuals. We show that there are significant changes in the expression of genes critical for immune function, development and neurogenesis that are highly relevant to the ICF phenotype. Approximately half the upregulated genes we analyzed were marked with low-level DNA methylation in normal cells that was lost in ICF cells, concomitant with loss of repressive histone modifications, particularly H3K27 trimethylation, and gains in transcriptionally active H3K9 acetylation and H3K4 trimethylation marks. In addition, we consistently observed loss of binding of the SUZ12 component of the PRC2 polycomb repression complex and DNMT3B to derepressed genes, including a number of homeobox genes critical for immune system, brain and craniofacial development. We also observed altered global levels of certain histone modifications in ICF cells, particularly ubiquitinated H2AK119. Therefore, this study provides important new insights into the role of DNMT3B in modulating gene expression and chromatin structure and reveals new connections between DNMT3B and polycomb-mediated repression.

Survivin is an anti-apoptotic protein that is highly expressed in many cancers, including malignant gliomas. Preclinical studies established that the conjugated survivin peptide mimic SurVaxM (SVN53-67/M57-KLH) could stimulate an anti-tumor immune response against murine glioma in vivo, as well as human glioma cells ex vivo. The current clinical study was conducted to test safety, immunogenicity and clinical effects of the vaccine. Recurrent malignant glioma patients whose tumors were survivin-positive, and who had either HLA-A*02 or HLA-A*03 MHC class I allele-positivity, were given subcutaneous injections of SurVaxM (500 μg) in Montanide ISA 51 with sargramostim (100 μg) at 2-week intervals. SurVaxM was well tolerated with mostly grade one adverse events (AE) and no serious adverse events (SAE) attributable to the study drug. Six patients experienced local injection site reactions; three patients reported fatigue (grades 1 and 2), and 2 patients experienced myalgia (grade 1). Six of eight immunologically evaluable patients developed both cellular and humoral immune responses to vaccine. The vaccine also stimulated HLA-A*02, HLA-A*03 and HLA-A*24 restricted T cell responses. Three patients maintained a partial clinical response or stable disease for more than 6 months. Median progression-free survival was 17.6 weeks, and median overall survival was 86.6 weeks from study entry with seven of nine patients surviving more than 12 months.

Perinatal hypoxia/ischemia (HI) is a common cause of neurological deficits in children. Interleukin-1 (IL-1) activity has been implicated in HI-induced brain damage. However, the mechanisms underlying its action in HI have not been characterized. We used a 7-day-old rat model to elucidate the role of nuclear factor-jB (NF-jB) activation in HI stimulation of IL-1 signaling. HI was induced by permanent ligation of the left carotid artery followed by 90 min of hypoxia (7.8% O 2 ). Using ELISA assays, we observed increased cell death and caspase 3 activity in hippocampus and cortex 3, 6, 12, 24 and 48 h post-HI. IL-1b protein expression increased, beginning at 3 h after HI and lasting until 24 h post-HI in hippocampus and 12 h post-HI in cortex. Intracerebroventricular injection of 2lg IL-1 receptor antagonist (IL-1Ra) 2 h after HI significantly reduced cell death and caspase 3 activity. Electrophoretic mobility shift assay analyses of hippocampus and cortex after HI for NF-jB activity showed increased p65/p50 DNA-binding activity at 24 h post-HI. Western blot analyses showed significant nuclear translocation of p65. Protein expression levels of two known inflammatory agents, inducible nitric oxide synthase and cycloxygenase 2, known to be transcriptionally regulated by NF-jB, also increased at 24 h after HI. All these HI-induced changes were reversed by IL-1Ra blockade of IL-1 signaling, consistent with IL-1 triggering of inflammatory apoptotic outcomes via NF-jB transcriptional activation. The observed increase in cytoplasmic phosphorylated inhibitor jBa (IjBa) and nuclear translocation of Bcl-3 24 h after HI was also significantly attenuated by IL-1Ra blockade, suggesting that HI-induced IL-1 activation of NF-jB is via both the degradation of IjBa and the nuclear translocation of Bcl-3. Keywords: Bcl-3, hypoxia/ischemia, inhibitor jBa, interleukin-1, nuclear factor-jB. Perinatal hypoxia/ischemia (HI) during gestation/delivery or the accidental asphyxia of infants is a common cause of neurological deficits and delayed cognitive and behavioral deficits (Kaltschmidt et al. 1994). A rapidly expanding body of evidence indicates that inflammatory mediators, including inflammatory cytokines, contribute substantially to the pathogenesis associated with perinatal HI brain injury (Hagberg et al. 1996;Bona et al. 1999;Hedtjarn et al. 2002). The best-characterized early response inflammatory cytokine is interleukin-1 (IL-1) (Szaflarski et al. 1995). The IL-1 family includes the agonists IL-1a and IL-1b, the endogenous receptor antagonist IL-1 receptor antagonist (IL-1Ra), and a newly discovered member, IL-18/IL-1c (Shapiro et al. 1998). IL-1b has consistently been detected in central nervous system after injury to the brain or spinal cord (Rothwell et al. 1997;Nesic et al. 2001). There is also evidence showing a correlation between increased IL-1b levels and subsequent neurodegeneration. Thus, administration of exogenous IL-1b markedly exacerbates neuronal/glial damage in rodents exposed to focal cerebral ischemia or...

BackgroundGlioma stem cells (GSCs) are a subpopulation of stem-like cells that contribute to glioblastoma (GBM) aggressiveness, recurrence, and resistance to radiation and chemotherapy. Therapeutically targeting the GSC population may improve patient survival, but unique vulnerabilities need to be identified.ResultsWe isolate GSCs from well-characterized GBM patient-derived xenografts (PDX), characterize their stemness properties using immunofluorescence staining, profile their epigenome including 5mC, 5hmC, 5fC/5caC, and two enhancer marks, and define their transcriptome. Fetal brain-derived neural stem/progenitor cells are used as a comparison to define potential unique and common molecular features between these different brain-derived cells with stem properties. Our integrative study reveals that abnormal expression of ten-eleven-translocation (TET) family members correlates with global levels of 5mC and 5fC/5caC and may be responsible for the distinct levels of these marks between glioma and neural stem cells. Heterogenous transcriptome and epigenome signatures among GSCs converge on several genes and pathways, including DNA damage response and cell proliferation, which are highly correlated with TET expression. Distinct enhancer landscapes are also strongly associated with differential gene regulation between glioma and neural stem cells; they exhibit unique co-localization patterns with DNA epigenetic mark switching events. Upon differentiation, glioma and neural stem cells exhibit distinct responses with regard to TET expression and DNA mark changes in the genome and GSCs fail to properly remodel their epigenome.ConclusionsOur integrative epigenomic and transcriptomic characterization reveals fundamentally distinct yet potentially targetable biologic features of GSCs that result from their distinct epigenomic landscapes.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1420-6) contains supplementary material, which is available to authorized users.

Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation (alloHCT), a potentially curative therapy for hematologic diseases. It has long been thought that murine bone marrow (BM) derived T cells do not mediate severe GVHD because of their quantity and/or phenotype. During the course of experiments testing the impact of housing temperatures on GVHD, we discovered that this apparent resistance is a function of the relatively cool ambient housing temperature. Murine BM-derived T cells have the ability to mediate severe GVHD in mice housed at a thermoneutral temperature. Specifically, mice housed at IACUC mandated, cool standard temperatures (~22°C) are more resistant to developing GVHD than mice housed at thermoneutral temperatures (~30°C). We learned that the mechanism underlying this housing-dependent immunosuppression is associated with increased norepinephrine production and excessive signaling through β-adrenergic receptor (β-AR) signaling which is increased when mice are cold stressed. Treatment of mice housed at 22°C with a β2-adrenergic antagonist reverses the norepinephrine driven suppression of GVHD and yields similar disease to mice housed at 30°C. Conversely, administering a β2-adrenergic agonist decreases GVHD in mice housed at 30°C. In further mechanistic studies using β2-adrenergic receptor deficient (β2-AR−/−) mice, we found that it is host cell β2-AR signaling that is essential for decreasing GVHD. These data reveal how baseline levels of β-AR signaling can influence murine GVHD and point to the feasibility of manipulation of β2-AR signaling to ameliorate GVHD in the clinical setting.

The standard form of cell adhesion glycoprotein CD44 is a metastasis suppressor in prostate cancer. However, we previously showed by RT-PCR and Western blotting that cancer overexpresses unique CD44 variant v7-v10 isoforms. Muc18 is another cell adhesion marker reportedly overexpressed by prostate cancer. Matched frozen section-confirmed tumor and benign tissues were harvested from 10 prostatectomy specimens and tumor was microdissected from two lymph node metastases. Tissues were homogenized for RNA preparations, and RT-PCR was performed for the CD44v7-v10 sequence. In cultured prostate cancer cells, we caused RNA interference against CD44v9 and/or Muc18. We used PC3M cells and a derivative cell line called G s a, that constitutively expresses this G-protein and is more invasive. Lipofection was performed for a green fluorescent protein plasmid and for two 22-mer DNA fragments, cloned into a plasmid expression vector to generate hairpin, interfering dsRNA. Assays for invasion into Matrigel, a basement membrane matrix, were performed in 4-5 experiments. RT-PCR demonstrated expression of a 608 bp band representing CD44v7-v10 or a 638 bp band of Prostate cancer remains a public health problem of enormous magnitude; it is the second most common cause of cancer fatality among North American men and the most common in Great Britain. Only a minority of all cases invade locally and metastasize, thus requiring altered expression of cell adhesion molecules to allow tumor cell detachment, migration through a stromal matrix, and lymphovascular invasion.CD44, a family of transmembrane glycoproteins involved in homotypic cell, cell-matrix, and cellcytoskeletal interaction, holds promise as a prostate tumor marker. The extracellular domain of CD44 binds numerous matrix substituents: hyaluronic acid, heparin-affinity growth factors, vascular endothelial growth factor, p185 HER2 , epidermal growth factor, and hepatocyte growth factor. Its intracellular domain binds to ezrin, radixin, moesin and merlin, which interact with the cytoskeleton; notably, neutral endopeptidase 24.11 competes with CD44 for this interaction. 1 CD44 also binds directly to the cytoskeletal substituent ankyrin, thus determining cell and tissue architectural form. 2 CD44 is an oncodevelopmental protein, with roles in

It has not been possible to generate naïve human pluripotent stem cells (hPSCs) that substantially contribute to mouse embryos. We found that a brief inhibition of mTOR with Torin1 converted hPSCs from primed to naïve pluripotency. The naïve hPSCs were maintained in the same condition as mouse embryonic stem cells and exhibited high clonogenicity, rapid proliferation, mitochondrial respiration, X chromosome reactivation, DNA hypomethylation, and transcriptomes sharing similarities to those of human blastocysts. When transferred to mouse blastocysts, naïve hPSCs generated 0.1 to 4% human cells, of all three germ layers, including large amounts of enucleated red blood cells, suggesting a marked acceleration of hPSC development in mouse embryos. Torin1 induced nuclear translocation of TFE3; TFE3 with mutated nuclear localization signal blocked the primed-to-naïve conversion. The generation of chimera-competent naïve hPSCs unifies some common features of naïve pluripotency in mammals and may enable applications such as human organ generation in animals.

Glioma cells release exosomes in culture and into the extracellular matrix in vivo. These nanobodies transport an array of biomolecules and are capable of mediating cell-cell communication. Circulating exosomes in cancer patients may be indicative of disease status and response to therapy. The inhibitor of apoptosis protein (IAP) survivin (SVN) promotes cancer cell proliferation, local immune suppression and resistance to chemotherapy and it is a potential cancer biomarker. We used imaging flow cytometry to perform quantitative measurements of circulating SVN+ exosomes in the serum of malignant glioma patients undergoing investigational treatment with an anti-survivin vaccine (SurVaxM). Serum from glioma patients contained abundant CD9+ exosomes with both SVN and glial fibrillary acidic protein (GFAP) on their surface. Survivin and GFAP were evaluated both independently and together as possible tumor markers on CD9+ exosomes. Patients with longer time to tumor progression generally exhibited a decrease in circulating CD9+/SVN+ and CD9+/GFAP+/SVN+ exosomes immediately following survivin vaccination; whereas, those with early tumor progression had an increase in exosomes, despite anti-survivin immunotherapy. Serum from non-cancer healthy control individuals had very few detectable CD9+/GFAP+/SVN+ exosomes, although CD9+/GFAP+ exosomes were detectable in small numbers. This study demonstrates that patients with malignant gliomas have CD9+/GFAP+/SVN+ and CD9+/SVN+ exosomes that are released into the circulation and that early reductions in their numbers following anti-survivin immunotherapy might be associated with longer progression-free survival.