Transcription profiling of mouse dendritic cells from the spleens of Alymphoplasia or heterozygeous littlermates (wild type) to identify TNIK/NF-kappaB2 regulated gene products.
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ABSTRACT: This study aims at identifying genes that are NIK/NF-kappaB2 responsive in murine dendritic cells matured in vivo. Experiment Overall Design: This study isolated dendritic cells from the spleens of Alymphoplasia or heterozygeous littlermates (wild type). Mice were eiter not treated as a reference or treated I.P. with 25 micrograms of LPS and 100 micrograms of agonistic anti-CD40 antibody. DCs were isolated 6 hours after treatment allowing in vivo maturation via both TLR and CD40 signals. DCs from 3 mice per group were pooled. Experiment was performed in triplicate.
Project description:This study aims at identifying genes that are NIK/NF-kappaB2 responsive in murine dendritic cells matured in vivo. Keywords: NIK/NF-kappaB2 responsive genes in dendritic cells after TLR/CD40 signaling
Project description:In SIV/HIV infection, the gastrointestinal tissue dominates as an important site due to the impact of massive mucosal CD4 depletion and immune activation-induced tissue pathology. Unlike AIDS-susceptible rhesus macaques, natural hosts do not progress to AIDS and resolve immune activation earlier. Here, we examine the role of dendritic cells in mediating immune activation and disease progression. We demonstrate that plasmacytoid dendritic cells (pDC) in the blood upregulate ?7-integrin and are rapidly recruited to the colorectum following a pathogenic SIV infection in rhesus macaques. These pDC were capable of producing proinflammatory cytokines and primed a Tc1 response in vitro. Consistent with the upregulation of ?7-integrin on pDC, in vivo blockade of ?4?7-integrin dampened pDC recruitment to the colorectum and resulted in reduced immune activation. The upregulation of ?7-integrin expression on pDC in the blood was also observed in HIV-infected humans but not in chronically SIV-infected sooty mangabeys that show low levels of immune activation. Our results uncover a new mechanism by which pDC influence immune activation in colorectal tissue following pathogenic immunodeficiency virus infections. SIV negative controls (n=4) and week 12 post SIV infected (n=4) groups of Rhesus macaques and SIV negative controls (n=4) and week 55 post SIV infected (n=4) groups of Sooty mangabeys colorectal tissue biopsies were collected in to RNA later reagent (Qiagen) and were homogenized with syringe and needle method. RNA was extracted with Rneasy mini kit (Qiagen) and was used for microarray experiments. Rhesus GeneChip assays were performed in the Yerkes Microarray Core Facility (www.microarray.emory.edu) , one of the Affymetrix Microarray Core Labs.The 0.5µg of total RNA sample was analyzed on Rhesus Macaque Genome GeneChip that consists of over 52,000 probe sets (Affymetrix, Santa Clara, CA). Target RNA labeling, hybridization and post-hybridization processing were performed following the Affymetrix GeneChip Expression Analysis standard protocols. In brief, The 5 ?g of RNA sample was first reverse-transcribed using T7-Oligo(dT) Promoter Primer and SuperScript II in the first-strand cDNAs synthesis reaction. Following RNase H-mediated second-stranded cDNA synthesis, the double-stranded cDNAs were purified by use of a GeneChip sample clean-up module and served as templates in the generation of biotinylated complementary RNAs (cRNAs) in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix by in vitro transcription (IVT) reaction. The biotinylated cRNAs were cleaned up, fragmented, and hybridized to the rhesus macaque expression arrays at 45°C for 16 h with constant rotation at 60 rpm. The gene chips were then washed and stained with Affymetrix fluidics stations 450 and scanned on Affymetrix scanner 3000. The images are processed to collect raw data with GeneChip Operating Software (GCOS) 1.4. Tissue: Colorectal tissue Time after SIV infection: 12 weeks for SIV infected Rhesus macaques, 55 weeks for Sooty mangabeys Infection: SIVmac251 infection for Rhesus macaques, SIVsm infection for Sooty mangabeys
Project description:The oviduct is a specialized organ playing crucial roles in the success of early reproductive events and it provides an optimal microenvironment for early embryonic development. However, changes in oviductal environment due to estrus synchronization and superovulation hormonal treatments and subsequent influence on embryos transcriptome profile are not yet investigated. For that, the objective of this study was to investigate differences in developmental rate and transcriptome profile of bovine blastocysts cultured under superovulation or synchronization oviductal environment. Influence of oviductal environment on transcriptome abundance of produced blastocysts was examined using the Affymetrix GeneChip Bovine Genome Array. Eighteen Simmental heifers were synchronized, superovulated and artificially inseminated, then nine of them were flushed at day 2 by transvaginal endoscopic means and 2-cell stage embryos were recovered and endoscopicaly transferred to only synchronized recipients. The remaining half of superovulated heifers and the synchronized recipients were flushed at day 7 to collect blastocysts which developed either in superovulated or synchronized oviduct
Project description:Rhesus macaques (RMs) inoculated with live-attenuated Rev-Independent Nef¯ simian immunodeficiency virus (Rev-Ind Nef¯SIV) as adults or neonates controlled viremia to undetectable levels and showed no signs of immunodeficiency over 6-8 years of follow-up. We tested the capacity of this live-attenuated virus to protect RMs against pathogenic, heterologous SIVsmE660 challenges Blood PBMC Time after SIV infection: 2 weeks post SIV infection Infection:Rev-Ind Nef¯SIV
Project description:The molecular mechanisms underlying the great differences in susceptibility to noise-induced hearing loss (NIHL) exhibited by both humans and laboratory animals are unknown. Using microarray technology, the present study demonstrates that the effects of noise overexposure on the expression of molecules likely to be important to the development of NIHL differ among inbred mice that have distinctive susceptibilities to NIHL including B6.CAST, 129X1/SvJ, and 129S1/SvImJ. The noise-exposure protocol produced, on average, a permanent loss of about 40 dB in sensitivity for auditory brainstem responses in susceptible B6.CAST mice, but no threshold elevations for the two resistant 129S1/SvImJ and 129X1/SvJ substrains. Measurements of noise-induced gene expression changes 6 h after the noise exposure revealed significant alterations in the expression levels of 48 genes in the resistant mice, while by these same criteria, there were seven differentially expressed genes in the susceptible B6.CAST mice. Differentially expressed genes in both groups of mice included subsets of transcription factors. However, only in the resistant mice was there a significant induction of proteins involved in cell-survival pathways such as HSP70, HSP40, p21, GADD45ï¢, Ier3, and Nfï«ï¢iïº. Moreover, increased expression of three of these factors after noise was confirmed at the protein level. Drastically enhanced HSP70, GADD45ï¢, and p21 immunostaining were detected 6 h after the noise exposure in subsets of cells of the lateral wall, spiral limbus, and organ of Corti as well as in cochlear nerve fibers. Upregulation of these proteins after noise exposure likely contributes to the prevalence of survival cellular pathways and thus to the resistance to NIHL that is characteristic of the 129X1/SvJ mice. Experiment Overall Design: Female 10-wk-old mice of the B6.CAST and 129X1/SvJ strains were divided randomly into non-noise control and noise-exposure groups. The non-noise mice served as controls in the gene-profiling experiments to control for the stress induced by experimenter handling and/or confinement of the mice in the noise-exposure chamber that was not directly related to the noise. This mice were in the noise chamber for a sham exposure. In contrast, the ânoiseâ groups were exposed to a 105-dB SPL, 10-kHz octave band of noise for 1 h and sacrificed 6 h after the exposure. Of each of these major groups, eight mice were used for each of three 129X1/SvJ control and three noise-exposed 129X1/SvJ arrays and two B6.CAST control and two noise-exposed B6.CAST arrays. Consequently within each subgroup the arrays are biological replicates.
Project description:Although smoking is the major risk factor for lung cancer, only 7% of female lung cancer patients in Taiwan have a history of cigarette smoking, extremely lower than those in Caucasian females. This report is a comprehensive analysis of the molecular signature of non-smoking female lung cancer in Taiwan. RNA was extracted from paired tumor and normal tissues for gene expression analysis.
Project description:Diffuse Midline Glioma (DMG) is a devastating pediatric brain tumor. The oncolytic adenovirus Delta-24-RGD has shown promising efficacy and safety in DMG patients but is not yet curative. Thus, we hypothesized that activating dendritic cells through the CD40 costimulatory receptor could increase antigen presentation and enhance the anti-tumor effect of the virus, resulting in long-term responses. This study showed that the intratumoral co-administration of Delta-24-RGD and a CD40 agonistic antibody is well tolerated and induced long-term anti-tumor immunity, including complete responses (up to 40%) in DMG preclinical models. Mechanistic studies revealed that this therapy increased tumor proliferating T lymphocytes and proinflammatory myeloid cells, including mature dendritic cells with superior tumor antigen uptake capacity. Moreover, the lack of cross-priming DCs and the prevention of DCs recruitment into the tumor abolish the Delta-24-RGD+anti-CD40 anti-tumor effect. This approach shows potential for combining virotherapy with activating antigen-presenting cells in these challenging tumors.
Project description:With 350 million carriers worldwide who may suffer from serious sequelae, including hepatocellular carcinoma, chronic hepatitis B virus (CHB) infection remains an important health issue. Current antiviral therapies hardly eradicate the virus. Therefore, there are still imperative need to develop new therapeutic strategies and predictors for treatment response to cure the disease and avoid futile treatment. By taking the advantage of having the paired liver biopsy samples of IFNalpha responders before/after treatment and pretreatment samples from responders and non-responders, we could characterize the intrahepatic expression profiles associated with necroinflammatory activity and the profiles/signature potentially predictive of response to IFNalpha therapy for CHB in this study. RNA was extracted from pairedliver tissues for gene expression analysis. The gene expression profiles of seven paired liver biopsy samples of IFNalpha responders before/after treatment and other 3 pre- treatment samples were obtained by Affymatrix U133plus2 microarray. We also pooled samples of 11 responders and 11 non-responders to perform affymatrix EXON ST 1.0 array. This dataset is part of the TransQST collection.
Project description:Hyperimmune activation is one of the strong predictors of disease progression during pathogenic immunodeficiency virus infections and is mediated in part by sustained type I interferon (IFN) signaling. Combination antiretroviral therapy suppresses hyperimmune activation only partially in HIV-infected individuals. Here, we show that blockade of Programmed Death-1 (PD-1) during chonic SIV infection significantly reduces the expression of transcripts associated with type I IFN signaling in the blood and colorectal tissue of rhesus macaques (RM). The effect of PD-1 blockade on type I IFN signaling was durable and persisted under high viremia, a condition that is seen in nonprogressive SIV infection in their natural hosts. The reduced type I IFN signaling was associated with enhanced expression of some of the junction-associated genes in the colorectal tissue and a profound decrease in LPS levels in plasma suggesting a possible repair of gut associated junctions and decreased microbial translocation. The reduced type I IFN signaling was also associated with enhanced immunity against gut resident pathogenic bacteria, control of gut associated opportunistic infections and survival of SIV-infected RMs. These results reveal novel mechanisms by which PD-1 blockade enhances survival of SIV-infected RMs and have implications for development of novel therapeutic approaches to control HIV/AIDS. SIV negative controls (n=4); SIV infected and control antibody (SYNAGIS) treated (n=3); and SIV infected Programmed Death 1 antibody (PD-1 Ab) treated (n=3) groups of Rhesus macaques PBMCs were isolated by Ficoll-paque plus medium (Amersham) and were lysed in to RNA later reagent (Qiagen) and samples were homogenized with Qiagen Shredder (Qiagen). RNA was extracted with Rneasy mini kit (Qiagen) and was used for microarray experiments. Rhesus GeneChip assays were performed in the Yerkes Microarray Core Facility (www.microarray.emory.edu), one of the Affymetrix Microarray Core Labs.The 0.5µg of total RNA sample was analyzed on Rhesus Macaque Genome GeneChip that consists of over 52,000 probe sets (Affymetrix, Santa Clara, CA). Target RNA labeling, hybridization and post-hybridization processing were performed following the Affymetrix GeneChip Expression Analysis standard protocols. In brief, RNA sample was first reverse-transcribed using T7-Oligo(dT) Promoter Primer and SuperScript II in the first-strand cDNAs synthesis reaction. Following RNase H-mediated second-stranded cDNA synthesis, the double-stranded cDNAs were purified by use of a GeneChip sample clean-up module and served as templates in the generation of biotinylated complementary RNAs (cRNAs) in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix by in vitro transcription (IVT) reaction. The biotinylated cRNAs were cleaned up, fragmented, and hybridized to the rhesus macaque expression arrays at 45°C for 16 h with constant rotation at 60 rpm. The gene chips were then washed and stained with Affymetrix fluidics stations 450 and scanned on Affymetrix scanner 3000. The images are processed to collect raw data with GeneChip Operating Software (GCOS) 1.4.