Characterization of late polyoma mRNA

Polyoma-infected mouse kidney cell cultures were labeled with [(3)H]uridine for 3 h late in the lytic cycle (26 to 29 h after infection) and RNA was extracted from cytoplasm and nuclei and from isolated polyribosomes. Sedimentation velocity analysis in sucrose gradients showed that polyoma-specific "giant" and 26S RNAs are present exclusively in the nucleus. RNA associated with cytoplasmic polyribosomes was analyzed by sedimentation in aqueous sucrose density gradients and dimethylsulfoxide sucrose gradients, as well as by polyacrylamide gel electrophoresis. Polyoma-specific RNA in polyribosomes consists of at least two classes, with sedimentation coefficients of 16 (major fraction) and 19S (minor fraction) in aqueous sucrose gradients and 15 and 17S, respectively, in dimethylsulfoxide gradients. Estimates based on dimethylsulfoxide gradient and analysis suggest a molecular weight of approximately 500,000 for 16S RNA and 700,000 for 19S RNA. These polyoma RNAs seem to undergo reversible conformational changes under the different conditions of analysis. We cannot exclude the possibility that they contain more than one molecular species

Tissue-specific and ubiquitous factors binding next to the glucocorticoid receptor modulate transcription from the mouse mammary tumor virus promoter

Steroid hormones complexed with their receptors play an essential role in the regulation of mouse mammary tumor virus (MMTV) transcription. However, the need for additional tissue-specific regulatory factors is suggested by the lack of virus expression in liver, in which glucocorticoid receptors are highly abundant, and by the tissue-specific transcription of reporter genes linked to an MMTV long terminal repeat in transgenic mice. In this study, we characterized two distal-region regulatory elements, DRa and DRc, which, together with the distal glucocorticoid receptor binding site (DRb), increased transcription from the MMTV promoter in permissive cells. This was demonstrated by transfection of these sequences (DRa, DRb, and DRc) in different combinations with the natural MMTV promoter in mouse fibroblasts and mammary epithelial cells, followed by quantitative S1 nuclease mapping of the transcripts. We further showed by DNase I footprinting, methylation interference, and gel retardation assays with various nuclear extracts from permissive or nonpermissive tissues and cell lines that the factors binding to the DRa site are distinct and tissue-specific whereas those binding to DRc are ubiquitous

Gene transfer into stimulated and unstimulated T lymphocytes by HIV-1-derived lentiviral vectors

Genetic modification of T lymphocytes holds great potential for treatments of cancer, T cell disorders and AIDS. While in the past recombinant murine retroviruses were the vectors of choice for gene delivery to T cells, vectors based on lentiviruses can provide additional benefits. Here, we show that VSV-G pseudotyped HIV 1 vector particles delivering the enhanced green fluorescent protein (EGFP) efficiently transduce human T lymphocytes. Transduction efficiency was optimal when infection included centrifugation of cells with concentrated vector supernatant in the presence of Polybrene. In contrast to previous reports describing murine retrovirus-mediated gene transfer to T lymphocytes, fibronectin did not improve the transduction efficiency of the VSVG-pseudotyped HIV-1 particles. Similar gene transfer efficiencies were observed following stimulation of cells with PHA/IL-2 or anti-CD3i/CD28i antibodies, although greater transgene expression was observed in the latter case. Interestingly, production of vectors in the absence of the accessory proteins Vif, Vpr, Vpu and Nef was accompanied by a 50% decrease in transduction efficiency in activated T cells. Transduction of T cells that were not stimulated before infection was achieved. No transduction of non-prestimulated cells was observed with a GAL V-pseudotyped murine retroviral vector. The requirement for accessory proteins in non-prestimulated cells was more pronounced. Our results have implications for lentiviral vector targeting of other cells of the hematopoietic system including stem cells

Stable transduction with lentiviral vectors and amplification of immature hematopoietic progenitors from cord blood of preterm human fetuses

Umbilical cord blood (CB) from the early gestational human fetus is recognized as a rich source of hematopoietic stem cells. To examine the value of fetal CB for gene therapy of inborn immunohematopoietic disorders, we tested the feasibility of genetic modification of CD34(+) cells from CB at weeks 24 to 34 of pregnancy, using lentiviral vector-mediated transfer of the green fluorescent protein (GFP) gene. The transduction rate of CD34(+) cells was 42 +/- 9%, resulting in GFP expression in 23 +/- 4% of colonies derived from colony-forming units (CFUs) and 11 +/- 1% from primitive long-term culture-initiating cells (LTC-ICs). Cell cycle analysis demonstrated transduction and GFP expression in cells in the G(0) phase, which contains immature hematopoietic progenitors. Transduced fetal CD34(+) cells could be expanded 1000-fold in long-term cultures supplemented with megakaryocyte growth and development factor along with Flt-3 ligand. At week 10, expression of GFP was observed in 40.5 +/- 11.7% of CFU-derived colonies. While prestimulation of CD34(+) cells with cytokines prior to transduction increased the efficiency of GFP transfer 2- to 3-fold, long-term maintenance of GFP-expressing CFUs occurred only in the absence of prestimulation. The GFP gene was found integrated into the genomic DNA of 35% of LTC-IC-derived colonies initiated at week 10, but GFP expression was not detectable, suggesting downregulation of transgene activity during the extended culture period. These results indicate that human fetal CB progenitors are amenable to genetic modification by lentiviral vectors and may serve as a target for gene therapy of hematopoietic disorders by prenatal autologous transplantation

Lower risk of peripheral venous catheter-related bloodstream infection by hand insertion

INTRODUCTION Little is known about the bloodstream infection (BSI) risk associated with short-term peripheral venous catheters (PVCs) and no large study investigated the insertion site-related risk for PVC-BSI. METHODS We performed a cohort study at the University of Geneva Hospitals using the prospective hospital-wide BSI surveillance database. We analyzed the association between insertion site and risk of PVC-BSI on the upper extremity using univariable and multivariable marginal Cox models. RESULTS Between 2016 and 2020, utilization of 403'206 peripheral venous catheters were prospectively recorded in a 2000-bed hospital consortium with ten sites. Twenty-seven percent of PVC (n = 109'686) were inserted in the hand. After adjustment for confounding factors, hand insertion was associated with a decreased PVC-BSI risk (adjusted hazard ratio [HR] 0.42, 95% CI 0.18-0.98, p = 0.046) compared to more proximal insertion sites. In a sensitivity analysis for PVCs with ≥ 3 days of dwell time, we confirmed a decreased PVC-BSI risk after hand insertion (HR 0.37, 95% CI 0.15-0.93, p = 0.035). CONCLUSION Hand insertion should be considered for reducing PVC infections, especially for catheters with an expected dwell time of more than 2 days

Human and murine APOBEC3s restrict replication of koala retrovirus by different mechanisms

Background: Koala retrovirus (KoRV) is an endogenous and exogenous retrovirus of koalas that may cause lymphoma. As for many other gammaretroviruses, the KoRV genome can potentially encode an alternate form of Gag protein, glyco-gag. Results: In this study, a convenient assay for assessing KoRV infectivity in vitro was employed: the use of DERSE cells (initially developed to search for infectious xenotropic murine leukemia-like viruses). Using infection of DERSE and other human cell lines (HEK293T), no evidence for expression of glyco-gag by KoRV was found, either in expression of glyco-gag protein or changes in infectivity when the putative glyco-gag reading frame was mutated. Since glyco-gag mediates resistance of Moloney murine leukemia virus to the restriction factor APOBEC3, the sensitivity of KoRV (wt or putatively mutant for glyco-gag) to restriction by murine (mA3) or human APOBEC3s was investigated. Both mA3 and hA3G potently inhibited KoRV infectivity. Interestingly, hA3G restriction was accompanied by extensive G → A hypermutation during reverse transcription while mA3 restriction was not. Glyco-gag status did not affect the results. Conclusions: These results indicate that the mechanisms of APOBEC3 restriction of KoRV by hA3G and mA3 differ (deamination dependent vs. independent) and glyco-gag does not play a role in the restriction

Loss of amphiregulin reduces myoepithelial cell coverage of mammary ducts and alters breast tumor growth

Abstract Background Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth. Methods We crossed AREG-null (AREG−/−) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG−/− PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors. Results Intriguingly, PyMT-induced lesions progress more rapidly in AREG−/− mice than in AREG+/+ mice. Quantification of K8+ luminal and K14+ myoepithelial cells in non-PyMT AREG−/− mammary glands showed fewer K14+ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG−/− PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas. Conclusions Our study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer

Synergistic action of GA-binding protein and glucocorticoid receptor in transcription from the mouse mammary tumor virus promoter.

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position -139 to -146 from the cap site) and fp2 (at -157 to -164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways