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Biomedical subjects

R E Sutton

Publications and source records attributed to R E Sutton.

16 recordsLinked to original sources
PubMed2003

Suicide genes as safety switches in T lymphocytes.

Broader application of adoptive transfer of tumor-specific T-lymphocytes is accompanied by the need for effective suicide genes to ensure the safety of this cell-based therapy. In vivo elimination of T-lymphocytes expressing the herpes simplex virus-derived thymidine kinase gene has demonstrated the feasibility of this suicide gene as safety switch. However, improvements are required to overcome initial problems, such as immunogenicity. Here, newly developed suicide genes, including inducible Fas, inducible caspase and CD20 are discussed. In addition, problems of clinical application of marker genes and gene transfer techniques, which are prerequisites for suicide gene therapy, are addressed.

Antigens, CD20↗
PubMed2001

Systematic determination of the packaging limit of lentiviral vectors.

Because of their ability to transduce nondividing cells, human immunodeficiency virus type 1 (HIV)-based vectors have great potential for the therapeutic delivery of genes to cells. We describe here a systematic study of the packaging limit of HIV-based vectors. Restriction endonuclease-generated bacterial chromosomal DNA fragments of different lengths were cloned at three different positions within a lentiviral vector. Vesicular stomatitis virus G protein (VSV G) pseudotyped lentiviral particles were prepared and the different clones were titered on mammalian cells. We observed that the restriction endonuclease site positions at the 5' and 3' ends of the genome were superior with regard to insertional capacity of foreign DNA. In all cases, viral titers decreased semi-logarithmically with increasing vector length. There appears to be no absolute packaging limit because measurable titers were obtained even when the proviral length was in excess of 18 kb. The reduction in titer appears to occur at the level of viral encapsidation, although we cannot exclude limitations in nuclear export of proviral RNA. These results suggest that HIV-based vectors may have a secondary advantage over oncoretroviral vectors because of their greater packaging limit, although the very low titers of the larger vectors will be of limited utility.

Active Transport, Cell Nucleus↗
PubMed2000

Transduction of human PBMC-derived dendritic cells and macrophages by an HIV-1-based lentiviral vector system.

Professional antigen-presenting cells, such as dendritic cells (DCs) and macrophages, are target cells for gene therapy of infectious disease and cancer. However, transduction of DCs and macrophages has proved difficult by most currently available gene transfer methods. Several recent studies have shown that lentiviral vector systems can efficiently transduce many nondividing and differentiated cell types. In this study, we examined the gene transfer to DCs and macrophages using a lentiviral vector system. Human DCs were propagated from the adherent fraction of peripheral blood mononuclear cells (PBMCs) by culture in medium containing GM-CSF, IL-4, and TNF-alpha. Human macrophages were propagated from adherent PBMCs in medium containing GM-CSF. High titers of a replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced yellow fluorescent protein were produced. In immature DCs (culture days 3 and 5), transduction efficiencies of 25 to 35% were achieved at a multiplicity of infection of 100. However, the transduction efficiency was decreased in more mature DCs (culture day 8 or later). Furthermore, monocyte-derived macrophages were also transduced by the lentiviral vector system. In addition, Alu-LTR PCR demonstrated the integration of the HIV-1 provirus into the cellular genome of the transduced DCs and macrophages. Allogeneic mixed lymphocyte reactions revealed similar antigen-presenting functions of untransduced and lentivirally transduced DCs. Thus, the results of this study demonstrate that both PBMC-derived DCs and macrophages can be transduced by lentiviral vectors.

Alu Elements↗
PubMed1999

Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent.

Human immunodeficiency virus (HIV) type 1 vectors are highly efficient in their ability to transduce human progenitor hematopoietic stem cells (PHSC). Although mitosis was not required for transduction of these cells, transduction rates were much greater once cells had been cultured in the presence of cytokines. Transduction rates, however, rarely exceeded 70%. We demonstrate here that there is a distinct subpopulation that is more easily transduced by HIV vectors. These cells were distinguished by a disproportionate population in the S/G2/M phases of the cell cycle. By sorting them prior to transduction, we found that those cells in either the G1 or S/G2/M fraction were more readily transduced than G0 cells. Maintaining the cells in G0 by omitting cytokines from the medium reduced transduction rates by up to 10-fold. Addition of cytokines to the medium immediately after transduction did not improve the transduction efficiency as measured by expression of the transgene. Analysis of replication intermediates indicated that the block to transduction of G0 cells operated near the time of initiation of reverse transcription. These results suggest that although lentivirus vectors can transduce nondividing PHSC, transduction efficiency is severalfold greater once the cells exit G0 and enter G1. Further characterization of these more transducible cells and identification of the cellular factors responsible may enhance transduction while maintaining the pluripotentiality of the PHSC.

Cell Cycle↗
PubMed1998

HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells.

Recent studies have opened the possibility that quiescent, G0/G1 hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34(+) cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G0/G1. The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G0/G1 HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP+ HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture in vitro for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least in vitro, transgene expression.

Antigens, CD34↗
PubMed1998

Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells.

Lentiviruses are potentially advantageous compared to oncoretroviruses as gene transfer agents because they can infect nondividing cells. We demonstrate here that human immunodeficiency virus type 1 (HIV-1)-based vectors were highly efficient in transducing purified human hematopoietic stem cells. Transduction rates, measured by marker gene expression or by PCR of the integrated provirus, exceeded 50%, and transduction appeared to be independent of mitosis. Derivatives of HIV-1 were constructed to optimize the vector, and a deletion of most of Vif and Vpr was required to ensure the long-term persistence of transduced cells with relatively stable expression of the marker gene product. These results extend the utility of this lentivirus vector system.

Gene Products, vif↗
PubMed1996

Identification of a major co-receptor for primary isolates of HIV-1.

Entry of HIV-1 into target cells requires cell-surface CD4 and additional host cell cofactors. A cofactor required for infection with virus adapted for growth in transformed T-cell lines was recently identified and named fusin. However, fusin does not promote entry of macrophage-tropic viruses, which are believed to be the key pathogenic strains in vivo. The principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-tropic strains of HIV-1 is CC-CKR-5, a receptor for the beta-chemokines RANTES, MIP-1alpha and MIP-1beta.

3T3 Cells↗
PubMed1996

Broad host range of human T-cell leukemia virus type 1 demonstrated with an improved pseudotyping system.

Studies of human T-cell leukemia virus type 1 (HTLV-1) have been hampered by the difficulty of achieving high cell-free and cell-associated infectious titers. Current retroviral pseudotyping systems using the HTLV-1 envelope generate titers of less than 200 infectious particles per ml. We describe here an improved system for pseudotyping using a defective human immunodeficiency virus (HIV) type 1 genome in combination with HTLV-1 env in 293T producer cells. Introduction of additional copies of rev and treatment of cells with sodium butyrate resulted in a cell-associated titer of 10(5)/ml and cell-free titers of greater than 10(4)/ml . By using this system, we found that the host range of HTLV-1 is even greater than previously suspected. Earlier studies which assigned a chromosomal location for the HTLV-1 receptor may therefore reflect cell-to-cell variation in receptor number rather than the absolute presence or absence of a receptor. The generation of higher-titer HIV(HTLV-1) may facilitate identification of the cellular receptor and investigations of the pathophysiology of HTLV-1 infection.

Cell Line↗
PubMed1988

Trypanosome trans-splicing utilizes 2'-5' branches and a corresponding debranching activity.

The 5' ends of trypanosome mRNAs consist of an identical sequence of 39 nucleotides which is derived from a discrete transcript of approximately 140 nucleotides (medRNA). It has been proposed that generation of chimeric mRNAs in trypanosomes occurs by the process of trans-splicing involving medRNA and an acceptor RNA. Part of the basis for this suggestion comes from the ability of HeLa cell extracts (known to contain debranching activity) to catalyze the release of the intron portion of medRNA (minRNA) implying a Y-branched intermediate in the splicing process. Here we provide direct chemical analysis that miniRNA is attached to higher mol. wt RNA molecules by a 2'-5' phosphodiester bond (i.e. as a branched structure). We also demonstrate that trypanosomes have substantial amounts of debranching activity which is similar in nature to that of HeLa cells. These results provide further evidence for trans-splicing in trypanosomes and highlights its similarity to cis-splicing in other eukaryotes.

Animals↗
PubMed1988

The cap of both miniexon-derived RNA and mRNA of trypanosomes is 7-methylguanosine.

Most, if not all, trypanosome mRNAs have the same 35-base sequence at their 5' terminus which is derived from a short RNA (medRNA) probably by the process of trans-splicing. It is of interest, evolutionarily and mechanistically, to determine the chemical structure of the 5' terminus of the precursor (medRNA) and product (mRNA). We demonstrate here that the cap structure of both is most probably 7-methylguanosine in a 5',5' triphosphate linkage, consistent with a precursor/product relationship.

Animals↗
PubMed1986

Evidence for trans splicing in trypanosomes.

The 5' ends of trypanosome mRNAs consist of an identical sequence of 35 nucleotides. This "mini-exon" sequence is derived from the 5' end of a 137 nucleotide RNA (medRNA). The remainder of each mRNA is derived from a protein-coding exon that is not linked to the mini-exon. We propose that medRNA is spliced in trans to de-novo-initiated transcripts of protein-coding genes. This trans splicing model predicts that the downstream portion of medRNA will be part of a branched structure and then be released as a free product (minRNA). We demonstrate that significant levels of minRNA exist in trypanosome RNA. Furthermore, minRNA can be released from high molecular weight RNA by a HeLa cell S100 "debranching" extract. We conclude that trans splicing is the physiological process by which mature mRNA molecules are synthesized in trypanosomes.

Animals↗
PubMed1984

Corticotropin-releasing factor regulates proopiomelanocortin messenger ribonucleic acid levels in vivo.

Ovine corticotropin-releasing factor (oCRF) was administered to male rats for 3-15 days. Continuous intravenous infusion, repeated subcutaneous, and intracerebroventricular injections of oCRF resulted in a 50-100% increase in messenger ribonucleic acid (mRNA) coding for proopiomelanocortin (POMC) in the anterior pituitaries (AP) of these animals. The synthetic glucocorticoid, dexamethasone, reversed the actions of oCRF. None of the above treatments altered POMC mRNA content in the neurointermediate lobe. Bilateral adrenalectomy enhanced POMC mRNA levels 4.8-fold. To determine whether this effect was dependent on increased hypothalamic production and release of CRF, a bilateral ablation of the paraventricular nucleus (PVN), known to contain CRF neurons directly involved in ACTH secretion, was performed. 6 days after the PVN destruction a parallel 75% reduction of both stalk-median eminence CRF-like immunoreactivity and plasma ACTH had occurred. The AP content of POMC mRNA decreased by 53%, while the ACTH content increased by 56%. These observations suggest that CRF regulates the biosynthesis of POMC-derived polypeptide products in the AP, while the peptide does not seem to be directly involved in intermediate lobe POMC biosynthesis. The reduction of POMC mRNA levels following PVN destruction demonstrates that CRF and other substances located in the nucleus may cause the increased POMC gene transcription following adrenalectomy.

Adrenalectomy↗
PubMed1982

Effects of neurotransmitters and cyclic AMP on somatostatin release from cultured cerebral cortical cells.

The influence of cortical neurotransmitters and cyclic AMP on the release of immunoreactive somatostatin (IRS)from cultured cortical cells was examined. Cells were obtained by mechanoenzymatic dispersal of telencephalons of 17-day-old rat embryos and were maintained as monolayers in minimum essential medium with 10% heat-inactivated horse serum. After the cultures had stabilized morphologically and in cellular IRS content they were subjected to rapid sequential changes of a buffered salt solution with or without test substances added. The amount of somatostatin released was measured by a specific radioimmunoassay. Acetylcholine and the GABA antagonist, picrotoxin, both stimulated IRS release. The cholinergic stimulation was predominantly muscarinic. GABA and histamine, to a lesser extent, were inhibitory and norepinephrine and serotonin produced no net change in IRS release. Both cAMP and theophylline (DMX) stimulated IRS release. These results confirm the potential of intrinsic cortical somatostatinergic neurons to respond to endogenous neurotransmitters and further establishes somatostatin as a cortical neuromodulator.

Acetylcholine↗
PubMed1982

Sodium- and calcium-dependent somatostatin release from dissociated cerebral cortical cells in culture.

The influence of membrane depolarization on somatostatin release from cerebral cortical neurons was examined. Fetal rat telencephalic cells, obtained by mechanoenzymatic dispersal, were maintained as organotypic monolayer cultures for 12 days before experimental studies. The immunoreactive somatostatin (IRS) released into the medium during a treatment epoch was compared to the amount released from the same cells during an immediately preceding control period. Potassium (60 mM) induced an increase in IRS secretion which was dependent on extracellular calcium concentration and could be prevented by the addition of the calcium channel blockers, cobalt or verapamil. Depolarization by veratridine, a sodium ionophore, also stimulated IRS release. The effect of veratridine was reversed by simultaneous exposure of the cells to either tetrodotoxin, a sodium channel blocker, or verapamil, a calcium channel blocker. These findings indicate that IRS release by cerebral cortical cells is stimulated by membrane depolarization and is dependent on both Na+ and Ca++ entry into the cells.

Animals↗