Translatome analysis of TRM6/61 overexpressing C6 rat cells.
ABSTRACT: Lysates from control or Trm6/61 overexpressing cells were fractionated on sucrose gradient to isolate polysomes. RNA extracted from heavy polysomal fractions was analyzed by microarray versus total RNA
Project description:We have previously shown that human umbilical cord blood CD34+ progenitor cells undergo in vitro differentiation into functional NK cells and that their co-culture in the presence of HOXB4 transduced stromal MS5 cells resulted in an increase in differentiated NK number. The present study was conducted to compare the stromal effect on NK lytic potential in the presence and absence of HOXB4. Our results provide evidence that HOXB4 transduced MS5 cells as compared to transduced, GFP (+) MS5 cells induced highly differentiated cytotoxic NK cells. Importantly, this difference was not due to the expression of activating NK receptors but was associated with an increased induction of granzyme B degranulation in response to stimulation with NK cell-susceptible targets. DNA microarray-based global transcriptional profiling confirmed the up-regulation of granzyme B. These findings provide further evidence that HOXB4 is a crucial regulator of NK function and that its use in generating functional NK cells with increased lytic potential may be significant for cancer immunotherapy.
Project description:The aim of the present study was to investigate the potential role of HIF-2a in regulating RCC susceptibility to natural killer (NK) cell-mediated killing. We demonstrated that the RCC cell line 786-O with mutated VHL was resistant to NK-mediated lysis as compared to the VHL corrected cell line (WT7). This resistance was independent of immunological synapse formation and NK ligand expression but was found to be reliant on HIF-2a stabilization in 786-0 cells. In order to elucidate the molecular basis of HIF-2a-induced impairment of NK-mediated killing, we performed global gene analysis using DNA microarray. Three candidate genes (ANGPTL4, ADM and ITPR1) were selected on the basis of their fold change and their involvement in cell death and survival. SiRNA targeting of these genes revealed that only ITPR1 silencing resulted in a significant increase of 786-O susceptibility to NK-mediated lysis. Using gene silencing of HIF2-a and chromatin immunoprecipitation assay, we showed for the first time that ITPR1 was a direct novel target of HIF2-a. Notably, a strong and significant correlation was found between ITPR1and HIF2-a staining in RCC patients. Transcriptional profiling of ITPR1silenced RCC cells indicated that several important genes involved in cell survival and apoptosis were differentially regulated. Using ITPR1 silenced Renca cells, we further found that ITPR1 was involved in the control of tumor progression. Moreover ITPR1 targeting combined with NK depletion significantly enhanced tumor growth as compared to ITPR1 knocked down Renca xenografts. Our data provide insights into the link between HIF-2a, ITPR1-related pathway and natural immunity and suggest a role of the HIF2/ITPR1 axis in regulating RCC cell survival.
Project description:A novel avian-origin H7N9 influenza A virus (IAV) emerged in China in early 2013 causing mild to lethal human respiratory infections. H7N9 originated from multiple reassortment events between avian viruses and carries genetic markers of human adaptation. Determining whether H7N9 induces a host-response closer to human or avian IAV is important to better characterize this emerging virus. Here we compared the human lung epithelial cell response to infection with A/Anhui/01/13 (H7N9) or highly pathogenic avian-origin H5N1, H7N7, or human seasonal H3N2 IAV. Here, polarized confluent monolayers of Calu-3 cells were infected apically with the avian-origin IAVs A/Anhui/01/2013 (H7N9) [Anhui01], A/Netherland/219/2003 (H7N7) [NL219], A/Vietnam/1203/2004 (H5N1) [VN1203], or a human seasonal virus A/Panama/2007/1999 (H3N2) [Pan99] at an MOI of 1. Time-matched mocks were also included using the same cell stock as the rest of the samples. Culture medium (same as what the virus stock is in) was used for the mock infections. Quadruplicate wells were infected for each virus/timepoint. Measured timepoints were 3, 7, 12 and 24 hours post-inoculation and the RNA was used for transcriptional analysis via microarray.
Project description:BJAB cells were infected with pNL-SIN-CMV-AcGFP (see accession number GSE8867 for data) or pNL-SIN-CMV-AcGFP expressing KSHV miR-K1, miR-K12-11 or miR-K4-3p and sorted 48 hours after infection. 12 or 16 days after transduction, cytoplasmic RNA was harvested and gene expression analysis of independent BJAB cell pools was performed using Human Operon v3.0.2 arrays. Each sample was run against Universal Human Reference RNA, Stratagene. The samples listed here were processed in parallel to those with accession number GSE8867, which includes all matched control cell lines. 6 independent B cell pools each expressing KSHV miR-K1, miRK12-11, or miR-K4-3p are included. Matched controls include unmodified BJAB (3 arrays) and cells transducted with parental vector pNL-SIN-CMV-AcGFP (6 replicates).
Project description:A series of conditional mouse models of embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma and spindle cell sarcoma were generated and validated for relavence to corresponding human cancers. Conditional mouse models of embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma and spindle cell sarcoma were created by activation or deletion of Pax3:Fkhr, p53, Ptch1 or Rb1 genes.
Project description:NYVAC, a highly attenuated, replication-restricted poxvirus, is a safe and immunogenic vaccine vector. Deletion of immune evasion genes from the poxvirus genome is an attractive strategy for improving the immunogenic properties of poxviruses. Using systems biology approaches, we describe herein the enhanced immunological profile of NYVAC vectors expressing the HIV-1 clade C env, gag, pol, and nef genes (NYVAC-C) with single or double deletions of genes encoding type I (B19R) or type II (B8R) interferon (IFN)-binding proteins. Transcriptomic analyses of human monocytes infected with NYVAC-C, NYVAC-C with the B19R deletion (NYVAC-C- B19R), or NYVAC-C with B8R and B19R deletions (NYVAC-C- B8RB19R) revealed a concerted upregulation of innate immune pathways (IFN-stimulated genes [ISGs]) of increasing magnitude with NYVAC-C- B19R and NYVAC-C- B8RB19R than with NYVAC-C. Deletion of B8R and B19R resulted in an enhanced activation of IRF3, IRF7, and STAT1 and the robust production of type I IFNs and of ISGs, whose expression was inhibited by anti-type I IFN antibodies. Interestingly, NYVAC-C- B8RB19R induced the production of much higher levels of proinflammatory cytokines (tumor necrosis factor [TNF-], interleukin-6 [IL-6], and IL-8) than NYVAC-C or NYVAC-C- B19R as well as a strong inflammasome response (caspase-1 and IL-1 ) in infected monocytes. Top network analyses showed that this broad response mediated by the deletion of B8R and B19R was organized around two upregulated gene expression nodes (TNF and IRF7). Consistent with these findings, monocytes infected with NYVAC-C- B8RB19R induced a stronger type I IFN-dependent and IL-1-dependent allogeneic CD4 T cell response than monocytes infected with NYVAC-C or NYVAC-C- B19R. Dual deletion of type I and type II IFN immune evasion genes in NYVAC markedly enhanced its immunogenic properties via its induction of the increased expression of type I IFNs and IL-1 and make it an attractive candidate HIV vaccine vector. Primary Human monocytes from 5 donors were incubated with each following vectors nyvacdb, nyvadb19r, nyvaccdb8rb19r_6h, untreated control and collected at 6h.
Project description:Myeloid dendritic cells from WT, Irf3-/-xIrf7-/-, Irf3-/-xIrf5-/-xIrf7-/-, Mavs-/- (IPS1-/-)and Ifnar-/- mice were infected with West Nile virus to assess the contributions of specific signaling and transcription factors in initiating the antiviral response RPMI + rmGM-CSF purified bone marrow derived myeloid DC (mDC), mock and WNV infected with insect derived virus and RNA isolated at 24 hours post infection . Each genotype has matched mock and infected samples. n=6 for WT mock, WT infected, IFNAR mock, IFNAR infected, IRF3x7 infected; n=5 IRF3x7 infected; n=3 IRF3x5x7 mock, IRF3x5x7 infected, MAVS mock, MAVS infected. RNA was prepared using the Illumina bead station assay and hybridized to Illumina RefSeq-8 V2 BeadChips. Note: MAVS=IPS1
Project description:Progressive fibrosis of the skin and internal organs accounts for the intractable nature and the high mortality of scleroderma. As the principal effector cells responsible for fibrosis, stromal fibroblasts and myofibroblasts contribute to excessive deposition of collagens and other extracellular matrix proteins. Transforming growth factor β (TGF-β), which stimulates collagen synthesis, myofibroblast differentiation and epithelial-mesenchymal transition (EMT), is implicated as a key initiating factor in both physiological and pathological tissue remodeling. However, the mechanism responsible for the persistence of the fibrotic process associated with pathological repair remains poorly understood. In this study, we analyzed the gene expression in dermal fibroblasts using different treatments (Poly I:C, IFN-beta, Egr-3 overexpression and other conditions). Primary fibroblast cultures were established by explantation from neonatal foreskin. Biopsy protocols were approved by the Institutional Review Board at Northwestern University. Fibroblasts were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Lonza, Basel, Switzerland), 50 µg/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C, and studied between passages 2-8. At confluence, serum-free media supplemented with 0.1% bovine serum albumin (BSA) were added to the cultures for 24 h prior to the treatment of Poly I:C for 2 hours or 24 hours (10 ug/ml), or IFN-β (100 U/ml) or other reagents for 24 hours, or infected with Ad-Egr-3 or Ad-GFP at 50 MOI for 48 hours. Total RNA was isolated using RNeasy Mini Plus Kits (Qiagen, Valencia, CA). The integrity of RNA was determined using Agilent Bioanalyzer (Santa Clara, CA). Fluorescently labeled cDNA was prepared (Ambion, Austin, TX), and was hybridized to Illumina Human HT-12 Version 4 microarray chips containing 44,000 probes (Illumina, San Diego, CA). Raw signal intensities for each probe were obtained using Illumina BeadStudio data analysis software and imported to the Bioconductor lumi package for transformation and normalization. The data were preprocessed using a variance stabilization transformation method followed by quantile normalization. Data from probes that produced signals near or below background levels (estimated based on Illumina negative control probes) with all samples were discarded.
Project description:The generation of CD8+ T-cell memory is an important aim of immunization. While several distinct subsets of CD8+ T-cell memory have been described, the lineage relationships between effector (EFF), effector memory (EM) and central memory (CM) T cells remain contentious. Specifically, there is contradictory experimental evidence to support both the linear (Naive>EFF>EM>CM) and progressive differentiation (Naive>CM>EM>EFF) models. In this study, we applied a systems biology approach to examine global transcriptional relationships between the three major CD8+ T cell subsets arising endogenously as a result of vaccination with three different prime-boost vaccine regimens. Differential gene expression analysis and principle component analysis revealed that central memory cells were more closely related to naive T cells than both effector memory and effector cells. When the transcriptional relationships between subsets were enriched in an unbiased fashion with known global transcriptional changes that result when T-cells repeatedly encounter antigen, our analysis favored a model whereby cumulative antigenic stimulation drives differentiation specifically from Naive > CM > EM > EFF. These findings provide an insight into the lineage relationship between mature CD8+ T-cell subsets and will help in the rational design of vaccines aimed at generating effective immune responses against infections and cancer. Effector (EFF), effector memory (EM), central memory (CM) and naive CD8+ T cells from mice spleen. Memory subset arise endogenously as a result of vaccination with three different prime-boost vaccine regimens: DNA-rAd5, rAd5-rAd5 and rAd5-rLCMV.