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Results for “transducer selectivity”

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At least 127 records · Page 7Linked to original sources
PubMed1995

Down-regulation of interleukin 6 receptor alpha chain in interleukin 6 transduced melanoma cells causes selective resistance to interleukin 6 but not to oncostatin M.

The cytokines interleukin 6 (IL-6) and oncostatin M are able to inhibit the growth of cell lines obtained from early but not advanced melanomas. Resistant cell lines have frequently been found to produce IL-6. Acquisition of IL-6 resistance and the relationship between resistance and endogenous IL-6 production are poorly defined phenomena. We have characterized a panel of melanoma cell lines for susceptibility to IL-6 and oncostatin M and have generated lines that acquired resistance to IL-6 by IL-6 cDNA transduction. These lines retained the previous oncostatin M sensitivity, suggesting that the alpha chain of IL-6 receptor (IL-6R alpha) is involved in the acquisition of resistance. In fact transduced cells lost the ability to bind 125I-IL-6 and to release soluble IL-6R alpha in culture. Moreover, addition of soluble recombinant IL-6R alpha were able to restore IL-6 sensitivity in association with IL-6 production. On the contrary, naturally IL-6 resistant melanoma cell lines were not inhibited by treatment with recombinant soluble IL-6R alpha in association with endogenously produced or recombinant IL-6. These results demonstrate that down-regulation of IL-6 receptor is only one of different mechanisms that are responsible of IL-6 resistance in melanoma cells.

Cell Division↗
PubMed1995

Coexpression of a multidrug-resistance gene (MDR1) and herpes simplex virus thymidine kinase gene as part of a bicistronic messenger RNA in a retrovirus vector allows selective killing of MDR1-transduced cells

A new retroviral vector, pSXLC/pHa, was constructed to coexpress drug-selectable markers with a second gene of interest as a part of a bicistronic mRNA in a retroviral vector using an internal ribosome entry site (IRES) from encephalomyocarditis virus. This system was used to develop a new retroviral vector pHa-MDR-IRES-TK which expresses a single mRNA from which translation of the MDR1 gene is cap dependent and translation of the herpes simplex virus thymidine kinase gene is IRES dependent. The pHa-MDR-IRES-TK transfectants showed high levels of P-glycoprotein expression and multidrug resistance. More than 95% of the vincristine-resistant cells transfected or transduced with pHa-MDR-IRES-TK showed hypersensitivity to ganciclovir, which selects against cells expressing herpes simplex virus thymidine kinase. An amphotropic retrovirus titer of 7.8 x 10(4)/ml was obtained with this vector. This safety-modified vector should be useful for introducing the MDR1 gene into bone marrow cells to protect normal cells from the toxic effects of cancer chemotherapy because this vector allows the elimination of cancer cells that have been unintentionally transduced with the MDR1 vector.

Journal Article↗
PubMed2001

Impact of intraoperative sonography on resection and cryoablation of liver tumors.

PURPOSE: We retrospectively analyzed the impact of intraoperative sonography (IOUS) on the management of patients referred for resection of liver tumors. METHODS: Forty patients underwent IOUS with a 7-MHz curved-array sector transducer; in selected cases, a 5-MHz linear-array transducer attached to a color Doppler unit was also used. The number, size, and location of tumors on IOUS, including tumor proximity to or invasion of major vessels or invasion of the diaphragm, were compared to findings on preoperative imaging studies. The effect of these findings on surgical management was assessed. Unresectable lesions were treated by cryoablation under ultrasound guidance. RESULTS: IOUS detected preoperatively unsuspected lesions in 7 patients (18%). Metastases suspected on CT arterial portography were ruled out in 2 patients (5%), and indeterminate lesions were diagnosed as cysts by IOUS in 2 other patients (5%). Vascular proximity or vascular or diaphragmatic invasion detected by IOUS rendered lesions unresectable in 4 patients (10%). Cryoablation under IOUS guidance and monitoring was attempted in 11 patients (28%) and performed successfully in 10. CONCLUSIONS: IOUS changed the management in 38% of patients and guided cryoablation in 28% of patients. IOUS performed by an experienced sonologist is invaluable for the accurate assessment of liver tumor resectability; the detection of additional, preoperatively unknown lesions; and the guidance of cryoablation of unresectable tumors.

Adolescent↗
PubMed1998

Stable transduction of the interleukin-2 gene into human natural killer cell lines and their phenotypic and functional characterization in vitro and in vivo.

A variety of strategies have been attempted in the past to stably transduce natural killer (NK) cells with cytokine or other cellular genes. Here, we demonstrate the successful delivery of the interleukin-2 (IL-2) gene into two human NK cell lines, IL-2-dependent NK-92 and IL-2-independent YT, by retroviral transduction. An MuLV-based retroviral vector expressing human IL-2 and neor markers from a polycistronic message was constructed and transduced into a CRIP packaging cell line. By coincubation of NK cells with monolayers of CRIP cells or by using retrovirus-containing supernatants in a flow-through method, 10% to 20% of NK cells were stably transduced. Upon selection in the presence of increasing G418 concentrations, transduced NK cells were able to proliferate independently of IL-2 for more than 5 months and to secrete up to 5.5 ng/10(6) cells/24 h of IL-2. IL-2 gene-transduced NK-92 cells had an in vitro cytotoxicity against tumor targets that was significantly higher than that of parental cells and secreted interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) in addition to IL-2. Moreover, the in vivo antitumor activity of IL-2 gene-transduced NK-92 cells against established 3-day liver metastases in mice was greater than that of parental nontransduced NK cells. Stable expression of the IL-2 transgene in NK cells improved their therapeutic potential in tumor-bearing hosts. Thus, transduced NK cells secreted sufficient quantities of bioactive IL-2 to proliferate in vitro and mediated the antitumor effects both in vitro and in vivo in the absence of exogenous IL-2. These results suggest that genetic modification of NK cells ex vivo could be useful for clinical cancer therapy in the future.

Animals↗
PubMed1986

Chromatin structure of a P-element-transduced hsp-28 gene in Drosophila melanogaster.

The Drosophila hsp-28 gene was heat inducible when transduced to novel chromosomal sites even when no direct selection for transduced gene expression was imposed. The pattern of DNase I-hypersensitive sites 5' to the wild type and transduced copy of hsp-28 was similar. In addition, DNase I-hypersensitive sites occurred within the P-element sequences flanking transduced loci.

Animals↗
PubMed2005

Gamma-glutamylcysteine synthetase and L-buthionine-(S,R)-sulfoximine: a new selection strategy for gene-transduced neural and hematopoietic stem/progenitor cells.

In most experimental gene therapy protocols involving stem/progenitor cells, only a small fraction of cells, often therapeutically inadequate, can be transduced and made to express the therapeutic gene. A promising strategy for overcoming this problem is the use of a dominant selection marker, such as a drug resistance gene. In this paper, we explore the potential of the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCSh) to act as a selection marker. We found that 3T3 fibroblasts transduced with the bicistronic retroviral vector SF91/GCSh-eGFP, encoding gamma-GCSh and the enhanced green fluorescent protein (eGFP), were highly resistant to L-buthionine-(S,R)-sulfoximine (BSO), a gamma-GCS inhibitor with a low clinical toxicity profile. The level of resistance was not proportional to the increase in intracellular glutathione. In fact, cells overexpressing both heavy and light gamma-GCS subunits had higher intracellular GSH levels, and a lower level of resistance to the cytotoxic activity of BSO, compared with cells overexpressing gamma-GCSh alone. 3T3 fibroblasts overexpressing gamma-GCSh could be selected from cultures containing both naive and gene-modified cells by application of exogenous BSO selection pressure for 4 days. Also, primary neural stem/progenitor cells derived from the lateral ventricles of mouse neonatal brains and primary hematopoietic stem/progenitor cells (HSCs/HPCs) from mouse bone marrow, transduced with the gamma-GCSh-eGFP vector, could be selected by BSO treatment in vitro. On ex vivo BSO selection and reimplantation into a syngeneic myeloablated host, donor HSCs/HPCs repopulated the marrow and continued to express the transgene(s). These results provide proof of principle that somatic stem/progenitor cells, transduced simultaneously with a potentially curative gene and gamma-GCSh, can be selected by treatment with BSO before in vivo transplantation.

3T3 Cells↗
PubMedDate not supplied

Use of bicistronic retroviral vectors encoding the LacZ gene together with a gene of interest: a method to select producer cells and follow transduced target cells.

The coordinate expression of a marker gene and a therapeutic gene in one retroviral vector has considerable advantages. High-titer producer lines can potentially be selected on the basis of marker gene expression, and the expression of transduced genes in target cells can readily be followed. Moreover, target cells with stable high expression can be selected before use in therapeutic protocols or research questions. We used internal ribosomal entry site (IRES) sequences to express two genes in the same retroviral vector. We used the LacZ gene as the marker gene and the cytokine interleukin (IL)-7 or dominant negative (dn) forms of the T-cell tyrosine kinases ZAP-70 and lck as genes of interest. Amphotropic packaging cells transfected with MFG-IL-7-IRES-LacZ, MFG-dnZAP-70-IRES-LacZ, or MFG-dnlck-IRES-LacZ were sorted on the basis of beta-galactosidase expression. These LacZ-positive producer cells also expressed the gene of interest, produced high-titer retrovirus, and were capable of efficiently transducing Jurkat T cells and T-cell clones. When MFG-IL-7-IRES-LacZ-transduced Jurkat T cells were sorted on the basis of LacZ expression, a positive correlation with the amount of IL-7 produced by these cells was found. This demonstrates that selection of the LacZ marker gene also selects for cells that express the gene of interest at high levels. Moreover, T cells transduced with the dn tyrosine kinases and selected on the basis of LacZ expression showed functional alterations after T-cell receptor stimulation, demonstrating that retrovirally transduced signaling molecules can alter the function of T cells.

DNA Primers↗
PubMed1999

Generation of aberrant sprouting in the adult rat brain by GAP-43 somatic gene transfer.

The expression of GAP-43 was modulated genetically in the adult rat nigrostriatal or septohippocampal pathway using recombinant adeno-associated virus (rAAV) vectors incorporating the neuron specific enolase (NSE) promoter and either a rat GAP-43 cDNA or the corresponding antisense sequence. Bicistronic expression of green fluorescent protein (GFP) enabled us to evaluate transduced neurons selectively. Single injections of rAAV into the substantia nigra pars compacta (SNc) transduced both dopaminergic and non-dopaminergic neurons stably for the 3-month duration of the study. Transduction with the GAP-43 vector in this region: (1) increased GAP-43 mRNA levels 2-fold compared to controls; (2) led to GAP-43 immunoreactivity in neuronal perikarya, axons, and dendrites that was not observed otherwise; and (3) resulted in GAP-43/ GFP-positive axons that were traced to the striatum where they formed clusters of aberrant nets. The GAP-43 antisense vector, in contrast, decreased neuropil GAP-43 immunoreactivity compared to controls in the SNc. In septum, injections of the GAP-43 expressing vector also caused aberrant clusters of GAP-43 labelled fibers in terminal fields, i.e., fornix and hippocampus, that were not observed in control tissues. It therefore appears that rAAV vectors provide a novel approach for modulating intraneuronal GAP-43 expression in the adult brain.

Afferent Pathways↗
PubMed1997

Reversal of 1,3-bis(2-chloroethyl)-1-nitrosourea-induced severe immunodeficiency by transduction of murine long-lived hemopoietic progenitor cells using O6-methylguanine DNA methyltransferase complementary DNA.

Bone marrow toxicity is a dose-limiting side effect of chloroethylnitrosourea (CNU) chemotherapeutic alkylating agents. A major determinant of CNU cytotoxicity is the methylation of guanine at the O6-position and the subsequent formation of interstrand DNA cross-links. O6-Methylguanine DNA methyltransferase (MGMT) removes alkyl groups from the O6 position of guanine and has been shown to repair CNU-induced DNA damage. We have previously demonstrated that transplantation of murine bone marrow cells transduced with a recombinant retroviral vector expressing MGMT via the human phosphoglycerate kinase promoter (PGK-MGMT) protects animals in vivo from acute myelotoxicity associated with CNU treatment. In the present study, we examined the effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a commonly used CNU, on long term recovery of the lymphoid compartment, including thymus reconstitution, peripheral T and B cell populations, and lymphocyte mitogen responses in mice reconstituted with PGK-MGMT-transduced hemopoietic cells. Mice transplanted with either mock-infected control or PGK-MGMT-transduced stem cells were treated with five weekly doses of BCNU. Analysis of the lymphoid compartment demonstrated significant damage 3 mo after the last BCNU dose in control animals. In contrast, the profound deficiency in CD4+CD8+ double-positive thymocytes and mature lymphocytes observed in control mice surviving BCNU treatment was completely reversed in mice transplanted with PGK-MGMT-transduced bone marrow and was associated with molecular evidence of in vivo selection of transduced cells in the lymphoid compartment. Thus, long term immunodeficiency following CNU therapy may be prevented by genetic modification of murine hemopoietic stem cells with MGMT, leading to significant improvement in post-transplant immune function.

Animals↗
PubMed2004

In vivo selection for human and murine hematopoietic cells transduced with a therapeutic MGMT lentiviral vector that inhibits HIV replication.

We have developed an HIV-based lentiviral vector, VRX496, which efficiently transduces human CD34+ progenitors and CD4+ T lymphocytes. VRX496 contains an antisense sequence against the HIV envelope and is currently being evaluated for safety in a clinical trial for treatment of HIV. Selective outgrowth of transduced hematopoietic cells in vivo is anticipated to increase the therapeutic efficacy of this treatment by maximizing the persistence of virus-resistant cells in the body. Although HIV resistance is selective, additional selection may aid in treatment efficacy due to the vast quantity of target cells. Therefore, we engineered VRX496 to express the P140K MGMT gene to drive potent drug-mediated in vivo selection for transduced hematopoietic long-term repopulating cells. Suboptimally transduced T cell cultures treated with O6-benzylguanine and BCNU were selected from 3 to 100%, and after selection cultures did not support HIV replication. Primary CD34+ progenitors derived from G-CSF-mobilized peripheral blood were transduced at 27 to 35% efficiency. Approximate sixfold selection was observed for transduced CD34+ progenitors, colony-forming units, and long-term culture-initiating cells. Multilineage in vivo selection was demonstrated for transduced murine hematopoietic cells in human CD34(+)-derived hematopoietic cells in NOD-SCID mice. These results establish efficient ex vivo and in vivo selection for hematopoietic cells transduced with lentiviral vectors and support the potential therapeutic benefit of this strategy in human gene therapy.

Animals↗
PubMed2001

Selection of drug-resistant transduced cells with cytosine nucleoside analogs using the human cytidine deaminase gene.

Hematopoietic toxicity produced by most anticancer drugs limits their potential for curative therapy. We have shown previously that the human cytidine deaminase (CD) gene can confer drug resistance in murine bone marrow cells (BMCs) to the nucleoside analog, cytosine arabinoside (ARA-C). In the present study, as the first objective we showed that the CD gene can also render drug resistance in BMCs to related analogs, 2',2'-difluorodeoxycytidine (dFdC) and 5-azadeoxycytidine (5-AZA-CdR). As a second objective, we investigated the potential of ex vivo selection with cytosine nucleoside analogs of CD-transduced BMC. The goal of this approach was to enrich the fraction of CD-transduced BMCs so as to increase the transgene expression and level of drug resistance before transplantation. This strategy may have the potential to circumvent the problem in clinical gene therapy of low level of gene transfer and adequate long-term gene expression. Using a bicistronic retroviral vector containing the CD and the green fluorescent protein (CDiGFP), we transduced murine L1210 leukemic cells. All three analogs, ARA-C, dFdC, and 5-AZA-CdR were demonstrated in vitro to enrich (>95%) the population of leukemic cells expressing the GFP transgene. However, with CD-transduced primary murine BMCs cultivated at high cell density we observed that in vitro selection with ARA-C was not possible due to release of CD into the culture medium at amounts that were sufficient to inactivate the analog. The CD-containing medium produced a chemoprotective effect on mock BMCs as shown by lack of significant growth inhibition in the presence of ARA-C. However, at low cell density in a cell mixture containing CD-transduced cells, the mock BMCs showed marked drug sensitivity to ARA-C as determined by clonogenic assay. Selection with ARA-C was shown to significantly increase the CD enzyme activity in transduced BMC. These results suggest that CD gene has the potential to be a good selectable marker and a possible tool for chemoprotection in cancer gene therapy.

Antimetabolites, Antineoplastic↗
PubMed1983

Issues in the selection and use of mechanical transducers to directly measure motor activity in clinical settings.

The application of instrumentation in the social sciences to measure human behavior lags far behind medical practice and the physical sciences. According to Rugh and Schwitzgebel (1977), this primitive state of development and lack of technological sophistication is related to the prevailing belief that many variables of interest to psychologists are not subject to direct measurement. We identified five varieties of maladaptive behaviors commonly displayed by mentally retarded persons which can be measured directly by means of mechanical transducers: hyperactivity, hypoactivity, inadequate postural control, disorders of the sleep/wake cycle, and stereotyped movements. The operating characteristics of several types of motion sensors were described and issues related to the selection and use of these mechanical transducers in clinical settings were considered. Finally, various meanings of the terms reliability and validity were discussed as they pertain to the clinical application of these mechanical measurements of motor activity.

Behavior↗
PubMed1998

Influence of sensor size on the accuracy of in-vivo ligament and tendon force measurements.

In-vivo tendon forces are commonly measured using transducers, which detect tension in the tendon fibers. A poorly understood source of measurement errors is the difference in stress distribution within the tendon between experimental and transducer calibration conditions. The objective of this study was to investigate this source of error, and to determine whether these errors could be minimized by proper selection of transducer size. The study was conducted using the infrapatellar ligament (patellar tendon) of New Zealand White rabbits. Tendon force was measured with two different size implantable force transducers (IFTs), one Wide and one Narrow, and by a strain gaged load cell in series with the tendon. Tests were conducted at five different loading conditions selected to produce five different stress distributions within the tendon. One loading condition corresponded to a typical post-experiment calibration, and the data from that condition were used to develop a calibration equation for the transducer. The errors that resulted from using this calibration were determined by comparing the tendon force measured by the in-series load cell with the force predicted from the IFT output using the calibration equation. Changes in stress distribution produced measurement errors up to 64 N with the Narrow IFT but only 24 N with the Wide IFT. We found the measurement error was dependent on sensor width. Our results support the hypothesis that measurement errors can be caused by differences in tendon stress distribution between calibration and experimental conditions. We further showed that these errors can be minimized by using an IFT, which samples the tension in a large percentage of the tendon fibers. Information from this study can be used for selection of an appropriately sized implantable force transducer for measuring tendon and ligament force.

Animals↗
PubMed1993

Bretylium differentiates between distinct signal transducing pathways in human lymphocytes.

The selection of signal transducing pathways of T cells depends on the type of triggers. Antigens, antibodies or lectins induce the T cell receptor-CD3 operated pathway, and IL-2 transmits its activation signal via the IL-2 receptor. It has been demonstrated that bretylium, a quaternary ammonium ion, can significantly inhibit the first pathway at the same dose range that stimulates cell activation through the IL-2 receptor system. In the light of the different complexity of the two pathways at the plasma membrane level, and the non-toxic and reversible behavior of the drug, it is suggested that the bretylium induced sustained membrane hyperpolarization is responsible for the observation. This finding may open new possibilities in studying the mechanism of different signal transducing pathways.

Blotting, Northern↗
PubMed2004

Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line.

BACKGROUND: T-cells expressing the HSV1-TK suicide gene can be used for the control of graft-versus-host disease following allogeneic stem cell transplantation. To develop clinical trials based on such a strategy, we have generated under good manufacturing procedures a novel 'split genome' human packaging cell line (1704 cells). METHODS: To minimize the risk of generating replication-competent retroviruses, pol was truncated to remove sequences overlapping with env. To improve retroviral infection and selection of transduced T-cells, high titers of GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector coding for the CD90 GPI-anchored membrane molecule were produced by 1704 cells. RESULTS: Using 1704 cell supernatant and an optimized transduction protocol, approximately 50% of primary T-cells were transduced and could then be purified (approximately 95%) using clinical-grade immunomagnetic beads directed against CD90. Over 96% of these OKT3/IL-2-activated CD90(+)-selected T-cells were killed by ganciclovir. Cell proliferation and cytokine production of transduced T-cells and HLA-restricted cytotoxicity of transduced T-cell clones were identical to those of their non-transduced counterparts cultured under the same conditions. CONCLUSIONS: GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector allow efficient transduction and rapid selection of human T-cells under conditions applicable for clinical trials using the new human 1704 packaging cell line.

Base Sequence↗
PubMed2006

Transduction of human embryonic stem cells by ecotropic retroviral vectors.

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.

Animals↗
PubMed1980

Transducer beam pattern test object.

A new test object has been developed that generates images of transducer beam patterns on B-mode hardcopy. The extent and depth of the transducer focal zone are measured from these images. The predictions made from test object images were verified in the clinical images obtained from two 3.5-MHz focused transducers. Using the test object images, we have found that we can make a more rational selection of transducer for each clinical situation.

Humans↗