While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.In eukaryotes, DNA methylation is an epigenetic encryption system that is essential for proper gene regulation (for reviews see references 2, 4, 7, 23, 29, and 30). Defects in methylation lead to diverse disorders from mental retardation to immune deficiencies, and there is particularly strong evidence that methylation defects create a favorable environment for malignant transformation (2, 3). Pharmacologic alterations in methylation of specific genes have also been correlated with tumor response to chemotherapy and patient survival; thus, methylation regulation and the enzymes that catalyze the process represent important areas for treating cancer (2, 30).The enzymatic machinery that mediates methylation involves a number of DNA (cytosine-5) methyltransferase (DNMT) isoforms, including DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b (and a host of DNMT3 splice variants) (4, 29, 30). Dnmt1, Dnmt3a, and Dnmt3b are independent genes and essential; embryos lacking both copies of Dnmt1 or Dnmt3b die before birth, whereas Dnmt3a-nulls survive about 4 weeks (18, 21). Heterozygous mutants appear normal and are fertile (18, 21). The murine DNMT3a and -3b enzymes appear to possess de novo methylation activity (based upon plasmid methylation), and there is evidence that they act on different DNA targets in vivo (12). No transmethylase activity has been found with DNMT2, and biallelic deletions appear to possess normal methylation patterns (8,22). How different DNMTs ar...

BackgroundAdoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells’ ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain.MethodsA second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model.ResultsChimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells.ConclusionsOur study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment.

Background and objectives Cognition in ESRD may be improved by kidney transplantation, but mechanisms are unclear. We explored patterns of resting-state networks with resting-state functional magnetic resonance imaging among patients with ESRD before and after kidney transplantation.Design, setting, participants, & measurements Thirty-seven patients with ESRD scheduled for kidney transplantation and 22 age-, sex-, and education-matched healthy subjects underwent resting-state functional magnetic resonance imaging. Patients were imaged before and 1 and 6 months after kidney transplantation. Functional connectivity of seven resting-state subnetworks was evaluated: default mode network, dorsal attention network, central executive network, self-referential network, sensorimotor network, visual network, and auditory network. Mixed effects models tested associations of ESRD, kidney transplantation, and neuropsychological measurements with functional connectivity.Results Compared with controls, pretransplant patients showed abnormal functional connectivity in six subnetworks. Compared with pretransplant patients, increased functional connectivity was observed in the default mode network, the dorsal attention network, the central executive network, the sensorimotor network, the auditory network, and the visual network 1 and 6 months after kidney transplantation (P=0.01). Six months after kidney transplantation, no significant difference in functional connectivity was observed for the dorsal attention network, the central executive network, the auditory network, or the visual network between patients and controls. Default mode network and sensorimotor network remained significantly different from those in controls when assessed 6 months after kidney transplantation. A relationship between functional connectivity and neuropsychological measurements was found in specific brain regions of some brain networks. ConclusionsThe recovery patterns of resting-state subnetworks vary after kidney transplantation. The dorsal attention network, the central executive network, the auditory network, and the visual network recovered to normal levels, whereas the default mode network and the sensorimotor network did not recover completely 6 months after kidney transplantation. Neural resting-state functional connectivity was lower among patients with ESRD compared with control subjects, but it significantly improved with kidney transplantation. Resting-state subnetworks exhibited variable recovery, in some cases to levels that were no longer significantly different from those of normal controls.

The gut-brain axis in end-stage renal disease (ESRD) is attracting more and more attention. However, the mechanism of gut-brain axis based cognitive disorders in ESRD patients remains unclear. The purpose of this study was to investigate the linkages between the gut microbiota, inflammatory cytokines, brain default mode network (DMN) and cognitive function in ESRD patients.Methods: This prospective study enrolled 28 ESRD patients (13 males and 15 females, mean age of 44 ± 14 years) and 19 healthy controls (HCs) (12 males and 7 females, mean age of 44 ± 10 years). All subjects underwent stool microbiota analysis, blood inflammatory cytokines examination, brain MRI scans and cognitive assessments. Resting state functional MRI (rs-fMRI) data were used to construct DMN and graph theory was applied to characterize network topological properties. Two samples t-test was applied for the comparisons between ESRD and HCs. Correlation analysis and mediation analysis were conducted among factors with significant group differences.Results: ESRD patients displayed gut microbiota alterations, increased systemic inflammation and worse cognitive performance compared to HCs (all p < 0.05). Graph analysis revealed disrupted DMN topological organization, aberrant nodal centralities and functional connectivities (FCs) in ESRD patients relative to HCs (all p < 0.05, FDR corrected). Significant correlations were found between gut microbiota, inflammatory cytokines, DMN network measures and cognitive assessments. Mediation analysis found that gut microbiota alteration impaired DMN connectivity by increasing systemic inflammation.Conclusion: The present study first revealed gut microbiota alterations, systemic inflammation, DMN dissociation and cognitive dysfunction in ESRD patients simultaneously and further illuminated their inner relationship.