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Results for “transducer selectivity”

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At least 145 records · Page 8Linked to original sources
PubMed1999

Effects of transducer frequency and output power on the ultrasonographic contrast produced by Optison using fundamental and harmonic imaging techniques.

This in vivo study demonstrated the effects of transducer frequency and output power on the sonographic contrast produced by Optison during continuous ultrasonographic imaging in both fundamental and harmonic modes. During fundamental imaging, Optison produced greater contrast intensity while imaging at higher transducer frequencies. However, in the harmonic mode, lower imaging frequencies caused Optison to produce more intense myocardial enhancement. Regardless of imaging mode, increases in transducer output power caused decreases in both contrast intensity and contrast duration produced by Optison. Proper selection of transducer frequency and output power will help optimize the quality and duration of contrast produced by Optison during continuous ultrasonographic imaging.

Albumins↗
PubMed2003

Increased resistance to nitrogen mustards and antifolates following in vitro selection of murine fibroblasts and primary hematopoietic cells transduced with a bicistronic retroviral vector expressing the rat glutathione S-transferase A3 and a mutant dihydrofolate reductase.

We have constructed a retroviral bicistronic vector, MFG/GID, that transduces the expression of both the A3 isoform of the rat glutathione S-transferase (GST A3), and the tyr-22 variant of the human dihydrofolate reductase (DHFR(L22Y)). Transduction of murine 3T3 fibroblasts with this vector increased their in vitro resistance to chlorambucil (1.8-fold) and trimetrexate (TMTX) (748-fold). TMTX selection of a mixed population of 20% GID-transduced NIH 3T3 cells and 80% control cells resulted in a marked increase in the GST peroxidase activity associated with the GST A3 isoform (17.7-fold). MFG/GID-transduced primary clonogenic murine hematopoietic progenitor cells were likewise more resistant to TMTX and chlorambucil than control beta-gal-transduced cells. Selecting GID-transduced hematopoietic cells with a combination of TMTX and a nucleoside transport inhibitor resulted in a marked increase in resistance upon re-exposure to TMTX (99% survival). Similarly, GID-transduced hematopoietic cells selected with TMTX were more resistant to chlorambucil, with 40% survival at a drug concentration that killed practically all control cells. These results suggest that antifolate-mediated selection of MFG/GID-transduced hematopoietic cells could be used as a mean to enrich the population of transduced cells prior to or following transplantation, thus potentially conferring in vivo chemoprotection to nitrogen mustards and antifolates.

Animals↗
PubMedDate not supplied

Experimental evaluation of indicators of nonlinearity for use in ultrasound transducer characterizations.

Because the number of applications for medical ultrasonic devices continue to increase and, hence, the number of diagnostic ultrasound (US) systems increase, there is a need to reliably characterize the sources in terms of their output pressures. Currently, the transducers are characterized by making pressure measurements in water for every voltage range applied to the source and, then, linearly derate the measured pressure values to estimate the derated acoustic pressure levels. The process is time-consuming and inaccuracies are introduced in the derating process due to nonlinear effects. Therefore, there is a need to find an indicator of nonlinearity that could classify the measured pressure waveform as either linear, where the derating procedure would yield an accurate derated acoustic pressure estimate, or nonlinear, where the derating process would fail. Eight different indicators of nonlinearity were evaluated experimentally using spherically focused US transducers. The transducers were selected to test the indicator sensitivity to frequency (3 to 8 MHz), f-number (1 and 2), and transducer diameter (1.905 and 5.08 cm). Sensitivity to drive voltage conditions was also tested by exciting one of the transducers with pulses of different duration and phase. None of the eight nonlinearity indicators yielded consistent results. The lack of consistency resulted from the competing effects of nonlinear absorption and asymmetrical distortion, which have yet to be combined into a unified theory.

Acoustics↗
PubMed2000

Rapid selection of antigen-specific T lymphocytes by retroviral transduction.

Infusions of donor peripheral blood T cells can induce durable remissions of Epstein-Barr virus (EBV) lymphomas complicating marrow grafts, but they contain alloreactive T cells capable of inducing graft-versus-host disease. EBV-specific T-cell lines or clones avoid this problem but require 30 to 40 days of culture to establish. To accelerate the generation of EBV-specific T cells, we tested whether retroviral vectors, which only integrate in dividing cells, could be used to transduce and select antigen-reactive T cells early after sensitization to autologous EBV-transformed B cells. T cells were transduced with a dicistronic retroviral vector, NIT, which encodes low-affinity nerve growth factor receptor as an immunoselectable marker and herpes simplex virus thymidine kinase as a suicide gene, at different time points after sensitization. EBV-specific cytotoxic T lymphocyte precursor (CTLp) frequencies in purified NIT(+) T-cell fractions transduced on day 8 of culture were comparable to those of EBV-specific T-cell lines cultured for 30 days or more. Alloreactive CTLp frequencies were markedly reduced in the NIT(+) fraction relative to the untransduced T-cell population. NIT(+) fractions transduced on day 8 possessed more CD4(+) T cells than the cell lines at day 30 and exhibited the same selective pattern of reactivity against immunodominant antigens presented by specific HLA alleles. In contrast, T cells transduced with NIT 5 days after stimulation with mitogen and interleukin-2 were relatively depleted of T cells specific for autologous EBV-transformed cells. Thus, retroviral vectors may be used for rapid selection of viral antigen-reactive T cells depleted of alloreactive T cells.

Cytotoxicity, Immunologic↗
PubMedDate not supplied

Transduction of human renal carcinoma cells with human gamma-interferon gene via retroviral vector.

We used a retroviral vector containing a human gamma-interferon (gamma-IFN) gene to transduce 13 renal carcinoma cell lines. The transduction efficiencies ranged from 0% to 60%, as determined by using an analogous vector containing the LacZ marker gene. In addition, gene-transferred resistance to the antibiotic neomycin was used to select for transduced cells. Nine of 13 lines were successfully transduced. Transduction was associated with the morphologic change of elongation, and there was a marked decrease in cell growth rate. Transduced cells secreted varying amounts (20-1076 pg/10(6) cells/d) of gamma-IFN as measured by enzyme-linked immunosorbent assay for at least 2 to 3 weeks after transduction (including 1 day of transduction, 6-7 days of selection, and an additional 8-12 days before the first passage of the transduced cells). Human leukocyte antigen (HLA) class II expression was markedly increased in six of seven cell lines; HLA class I expression was significantly increased in two of eight lines. Transduced cells that were subjected to cryopreservation after irradiation still produced gamma-IFN and expressed HLA class I and II antigens, although generally at lower levels than before these manipulations. This study confirms that retroviral vector transduction of the human gamma-IFN gene into renal carcinoma cells is feasible and associated with persistent production of gamma-IFN and increased expression of HLA class I and II molecules, and these effects are retained after irradiation and cryopreservation. This suggests that an autologous tumor cell vaccine trial with irradiated gamma-IFN gene-transduced renal carcinoma cell is rationale and feasible.

Carcinoma, Renal Cell↗
PubMed2002

Stable expression of Anthozoa fluorescent proteins in mammalian cells.

BACKGROUND: Fluorescent proteins have become invaluable reporters in many areas of cellular and developmental biology. An enhanced version of the Aequorea victoria green fluorescent protein (AvEGFP) is the most widely used fluorescent protein. For a variety of reasons, it is useful to have alternative fluorescent proteins to AvEGFP. METHODS: The cDNA sequences for enhanced variants of the Anemonia cyan fluorescent protein (AmCyan1), as well as the Zoanthus green (ZsGreen1) and yellow (ZsYellow1) fluorescent proteins, were cloned downstream of a constitutive cytomegalovirus (CMV) promoter within a retroviral expression vector. NIH3T3, HEK293, SW620, and WM35 cells were transduced with recombinant retroviruses at a low multiplicity of infection (MOI) to bias for single-copy integration. Both unselected and stably selected cells transduced with the retroviral expression constructs were characterized. Expression of each fluorescent protein in cells was detected using flow cytometry and fluorescence microscopy with filter sets typically used for AvEGFP/fluorescein isothiocyanate (FITC) detection and was compared with the expression of AvEGFP. In addition, a fluorescence plate reader with several excitation and emission filter sets was used for detection. RESULTS: Expression of each protein was observable by fluorescence microscopy. Under given conditions of flow cytometry, the ZsGreen1 mean fluorescence was approximately 3-fold, 10-fold, and 50-fold greater than that of AvEGFP, ZsYellow1, and AmCyan1, respectively. AmCyan1, ZsGreen1, and AvEGFP were detected by a fluorescence plate reader. CONCLUSION: We determined that fluorescent proteins from Anthozoa species are detectable using a standard flow cytometer and fluorescence microscope. All of the mammalian cell lines tested expressed detectable levels of fluorescent proteins from stable integrated provirus. In cell lines where the AvEGFP protein is toxic or poorly expressed, these Anthozoa fluorescent proteins may serve as alternative fluorescent reporters.

3T3 Cells↗
PubMed1995

Chemotherapy resistance to taxol in clonogenic progenitor cells following transduction of CD34 selected marrow and peripheral blood cells with a retrovirus that contains the MDR-1 chemotherapy resistance gene.

A retrovirus containing the multiple drug resistance (MDR-1) cDNA, was used to transduce cultures of CD34 selected human marrow cells, on stromal monolayers in the presence of hematopoietic growth factors IL-3 and IL-6, following collection from patients recently recovered from chemotherapy-induced myelosuppression. In one experiment, these CD34 selected cells were grown in Dexter cultures for 35 days or more following MDR-1 transduction, and then plated in methylcellulose. Polymerase chain reaction (PCR) analysis of colonies picked after 10-14 days of methylcellulose culture, using a set of primers that are specific for the endogenous or the retrovirally transduced MDR-1, showed that the long-term culture initiating cells (LTCICs) were transduced by the MDR-1 virus. Analysis of the colonies from the CD34 selected MDR-1 transduced cells, with a reverse transcription (RT) PCR assay that could distinguish viral MDR-1 mRNA from endogenous MDR-1 mRNA, showed that the viral MDR-1 mRNA levels were much higher than that of the MDR-1 mRNA from the endogenous MDR-1 gene in the transduced CD34 selected cells. Fluorescence activated cell sorting (FACS) analysis of the CD34 selected transduced marrow cells within 48 h after the transduction, using the C219 and UIC2 monoclonal antibodies for p-glycoprotein, showed that the transduction frequency under these conditions varied from 7 to 20%. Rhodamine efflux studies showed that this additional p-glycoprotein was functional and that the frequency of cells with high p-glycoprotein levels was higher in the transduced cells than in the non-transduced cells. The resistance to taxol of the CD34 selected transduced cells, as judged by the plating efficiency of clonogenic progenitor cells derived from these cells by growth in methylcellulose supplemented with taxol was much higher in the transduced cells than in untransduced cells. In order to test the reproducibility of the transduction frequency of the retroviral supernatants from PA317 MDR-1 viral producer cells on CD34 selected cells, the virus produced from 12 different lots of supernatants from the PA317 MDR-1 producer cell line was used to transduce CD34 selected marrow cells from four different patients, and to transduce the peripheral blood cells of two additional patients collected following chemotherapy-induced myelosuppression. The supernatant lots used for these transduction experiments were tested by Microbiological Associates (Rockville, MD, USA), by the Mus dunni co-cultivation and amplification tests in the S+L-assay and found to be negative for replication-competent retrovirus, and later approved for human use by the Food and Drug Administration.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Protection of CCRF-CEM human lymphoid cells from antifolates by retroviral gene transfer of variants of murine dihydrofolate reductase.

Expression of certain variants of dihydrofolate reductase (DHFR) in mammalian cells protects them from methotrexate. Retroviral transfer of the gene for such a variant DHFR into hematopoietic cells might permit selection of modified cells in vivo by antifolate administration or alleviate antifolate-induced myelosuppression in patients receiving antifolate therapy. We examined protection of cells of the human lymphoblastoid line, CCRF-CEM, transduced with variants of mouse DHFR. In transduced cells selected with G418 but not with antifolate, the variant that had arginine substituted for leucine 22 did not protect against either methotrexate or trimetrexate; however, four other variants did offer protection, with the best having leucine 22 changed to tyrosine. Polyclonal cultures transduced with the different variants express DHFR at about the same level, but clones within each polyclonal population differ in DHFR expression levels and in resistance. These differences in expression were shown to reflect different integration sites for proviral DNA. Exposure to trimetrexate selects highly resistant clones, with high expression due to both high copy number and integration sites that are favorable for expression. Differences in the resistance of cultures expressing different variants at the same level are due to differences in the catalytic activity of the expressed DHFR, its affinity for antifolates, and its stability.

Animals↗
PubMed2000

Extrathymic signals regulate the onset of T cell repertoire selection.

The thymus is the principal site of T cell development, coordinating positive and negative selection of an immunocompetent repertoire. The ability of fetal thymi from C57BL/6 mice to support these selection events in culture is, however, critically dependent on the timing of their excision: whereas thymi from 16 day embryos generate mature CD4+8(-) and CD4(-)8+ thymocytes, 14 day thymi show a profound blockage in positive selection at the CD4+8+ stage. Here we show that this blockage is not due to intrinsic deficiencies in the ability of thymocytes to transduce positive selection signals, suggesting the defect to lie within the thymic microenvironment. Since vascularization of the thymus occurs at day 15 of gestation, we investigated whether the exclusion of plasma proteins from 14 day thymi was responsible. Accordingly, the addition of serum from 16 day embryos to organ cultures of 14 day thymi rescued mature CD4+8(-) and CD4(-)8+ subsets. Activity was found to reside in a low molecular weight fraction of serum, sensitive to proteolysis, which was present only transiently during ontogeny. Our data suggest that repertoire selection is initiated following "priming" of the thymic microenvironment by plasma proteins, thereby ensuring the onset of positive selection to be delayed until the entry of extrathymic proteins to which self tolerance must be established.

Animals↗
PubMed1997

Development of a novel selective amplifier gene for controllable expansion of transduced hematopoietic cells.

To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)-dependent murine cell line, Ba/F3, the cells showed IL-3-independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Delta(5-195)GCRER, whose product lacks the extracellular G-CSF-binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Delta(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

Animals↗
PubMed2004

Retroviral transduction of peptide stimulated t cells can generate dual t cell receptor-expressing (bifunctional) t cells reactive with two defined antigens.

BACKGROUND: Tumors and viruses have developed many mechanisms to evade the immune system, including down-regulation of target antigens and MHC molecules. These immune escape mechanisms may be able to be circumvented by adoptively transferring T cells engineered to express two different T cell receptors, each specific for a different antigen or MHC restriction molecule. METHODS: PBMC from the blood of normal healthy donors were stimulated for three days with an antigenic peptide from cytomegalovirus (CMV) pp65. These CMV reactive cultures were transduced with a encoding the TIL 5 T cell receptor (TCR) that mediates recognition of the dominant epitope of the melanoma antigen MART-1. Following selection for transduced cells, the cultures were evaluated for recognition of CMV pp65 and MART-1 expressing targets. RESULTS: We were able to rapidly create bifunctional T cells capable of recognizing both CMV pp65 and MART-1 using a combination of HLA-A2 tetramer staining and intracellular staining for interferon-gamma. These bifunctional T cells were sensitive to very low levels of antigen, recognize MART-1+ tumor cells, and maintained their bifunctionality for over 40 days in culture. CONCLUSION: Bifunctional T cells can be engineered by transducing short term peptide stimulated T cell cultures. These bifunctional T cells may be more effective in treating patients with cancer or chronic virus infections because they would reduce the possibility of disease progression due to antigen and/or MHC loss variants.

Journal Article↗
PubMed1995

High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.

Animals↗
PubMed1994

Positive selection of CD4+ T cells by TCR ligation without aggregation even in the absence of MHC.

The developmental fate of immature thymocytes is determined by the specificity of their T-cell antigen receptors (TCRs). Immature CD4+8+ thymocytes are positively selected to differentiate into mature T cells by recognition of peptides associated with major histocompatibility complex (MHC) encoded molecules on thymic epithelial cells. But neither the identity of molecules transducing positive selection signals nor the nature of the signals themselves is fully known. Here we report that direct ligation of TCR molecules by monoclonal antibodies specific for either clonotypic or CD3 chains can signal immature thymocytes to differentiate into mature CD4+8- T cells, even in the absence of MHC expression and MHC-dependent CD4 co-receptor signalling. Moreover, we show that TCR engagement induces positive selection signals only in the absence of TCR aggregation and that TCR aggregation is inhibitory for positive selection. Thus, low valency of TCR crosslinking is a critical parameter, distinguishing positive selection from other TCR-mediated signalling events.

Animals↗
PubMed2000

A comparative evaluation of gene transfer into blood cells using the same retroviral backbone for independent expression of the EGFP and deltaLNGFR marker genes.

BACKGROUND AND OBJECTIVES: Retroviral vectors are widely used to deliver foreign genes to hematopoietic stem cells (HSC). Improvement of marking protocols needs reporter genes to allow rapid detection and efficient selection of transduced cells. The great potential of EGFP and LNGFR as reporter systems prompted us to compare them simultaneously, using the same retroviral backbone and the same gene transfer procedures. DESIGN AND METHODS: The EGFP and LNGFR coding sequences were separately cloned into the MFG retroviral backbone. A cloning strategy assuring that both genes utilize the same ATG as the start codon was adopted. Marker gene expression, viral titers, transduction efficiency, and vector stability were evaluated in expanded amphotropic packaging clones and human hematopoietic cell lines by flow cytometry and PCR analysis. Vectors were also tested for their ability to transduce CD34+ peripheral blood cells. RESULTS: A significantly larger number of MFG- LNGFR packaging clones were obtained that produced high viral titers. A direct correlation between viral titer and marker gene expression in packaging clones was demonstrated for both constructs. Similar expression kinetics and absence of in vitro toxicity in transduced cells were also observed for both constructs. Successful infection of CD34+ cells was achieved even after a short time of exposure to recombinant viruses. INTERPRETATION AND CONCLUSIONS: Our results demonstrate that EGFP and LNGFR marker genes are equally useful for a rapid, specific and non-toxic detection of transduced cells. The MFG-EGFP construct appears useful to optimize gene transfer protocols in vitro. On the other hand, the MFG-LNGFR construct, for making possible a more efficient selection of high titer producer clones, as well as for safety and adaptability to the in vivo use, is more suitable for clinical applications.

Cell Line↗
PubMed2003

Real-time determination of picomolar free Cu(II) in seawater using a fluorescence-based fiber optic biosensor.

We report real-time, in situ determination of free copper ion at picomolar levels in seawater using a fluorescence-based fiber optic biosensor. The sensor transducer is a protein molecule, site-specifically labeled with a fluorophore that is attached to the distal end of an optical fiber, which binds free Cu(II) with high affinity and selectivity. The transducer reports the metal's concentration as a change in fluorescence intensity or lifetime, using a frequency domain approach. The transducer's response time is diffusion-limited, with a typical measurement requiring 30 s. The sensor demonstrates a detection limit of 0.1 pM free Cu(II) in a seawater model. Accuracy and precision of the sensor were at least comparable to cathodic ligand exchange/adsorptive cathodic stripping voltammetry. Measurements of tidal flushing of a copper-contaminated inlet are shown.

Biosensing Techniques↗
PubMed1995

A new reporter system for detection of retroviral infection.

We describe a novel reporter molecule, the murine surface antigen Thy-1, useful for immunoselection and detection of retrovirus-mediated transduction by flow cytometry. A cDNA encoding the murine thy-1 gene was isolated, and cell surface expression of its gene product was demonstrated. The Thy-1 glycoprotein was tested as a cell surface reporter molecule in the context of replication-defective and -competent retroviruses. Cells transduced via murine retroviral vectors carrying the thy-1 and the neomycin phosphotransferase genes express Thy-1 glycoprotein on their surfaces. The Thy-1 marker is potentially useful in gene transfer protocols because selection of transduced cells can be achieved by immunoselection with anti-Thy-1 antibodies shortly after infection with the retroviral vector. In addition, a human immunodeficiency virus type 1 (HIV-1) recombinant expressing Thy-1 is described, which is replication-competent and syncytium-inducing in human peripheral blood mononuclear cells (PBMCs) and immortalized CD4-positive cell lines. Cells infected with this HIV-1 recombinant express Thy-1 on their surfaces and can be detected and purified by fluorescence-activated cell sorting (FACS). Because of these properties, retroviruses expressing this genetic marker can be useful for studies in gene therapy and of the retroviral life-cycle.

Animals↗
PubMed1994

Helper T cell-independent proliferation of CD8+ cytotoxic T lymphocytes transduced with an IL-1 receptor retrovirus.

Although the proliferation of CD8+ CTL typically requires cytokine support provided by helper T cells, a subset of naturally occurring CD8+ CTL are capable of proliferating independently of T cell help. Such helper-independent CTL have previously been shown to possess IL-1 receptors (IL-1R) and to proliferate in response to IL-1 through endogenous production of IL-2. In this study, we have transduced conventional helper-dependent CTL clones with a retroviral vector encoding the murine type I IL-1R. Transduced CTL selected in G418 expressed vector-derived transcripts encoding IL-1R and displayed approximately 1000 cell surface receptors with an IL-1 affinity typical for the type I IL-1R. In contrast to parental cells, transduced CTL proliferated in response to IL-1 in the presence of Ag, without a requirement for helper T cells, IL-2, or other cytokine support. Stimulation with both IL-1 and Ag was necessary for the proliferative response. No endogenous synthesis of IL-2 could be detected in the IL-1R transduced cells in response to IL-1 stimulation, in the presence or absence of Ag. The IL-1R-induced phenotype was demonstrated in two independent T cell clones, both of which retained Ag-specific cytolytic activity. No such conversion to a helper-independent phenotype was induced by a retroviral vector encoding only the neo gene. The behavior of the IL-1R-transduced CTL in proliferation assays thus resembled that of the naturally occurring helper-independent CTL.

Animals↗
PubMed1995

In situ interleukin-4 gene expression in cancer patients treated with genetically modified tumor vaccine.

Patients with advanced malignancies, participating in our ongoing phase I interleukin-4 (IL-4) gene therapy protocol at the Pittsburgh Cancer Institute, were vaccinated with irradiated autologous tumor cells together with IL-4 gene-transduced irradiated autologous fibroblasts. The level of expression of the IL-4 gene in cultured transduced and selected fibroblasts and in biopsies obtained from vaccination sites was evaluated using quantitative reverse transcription-polymerase chain reaction (RT-PCR). The number of copies of IL-4 mRNA/ng of total cellular RNA was determined in the transduced fibroblasts. Good agreement was observed between IL-4 message expression, as determined by RT-PCR, and IL-4 production, as determined by enzyme-linked immunosorbent assay (ELISA) in the fibroblast supernatants. Tissue biopsies of multiple vaccination sites were obtained from the patients to determine the level of gene expression in situ for IL-4 and Neo-r. The Neo-r gene was used as a marker for transduced fibroblasts. Two weeks after the first vaccination, mRNA for the IL-4 gene was still detectable in all tissue biopsies. The Neo-r gene was also detectable, indicating the presence of transduced fibroblasts in the biopsy. After the second vaccination, expression of the IL-4 and Neo-r genes was generally the highest on day 1 after vaccine administration and was considerably lower but still detectable on day 14 in all biopsies tested. These data indicate that autologous dermal fibroblasts transduced with the IL-4 and Neo-r genes and used as a source of IL-4 in tumor vaccine are able to express the IL-4 gene in vivo.

Base Sequence↗