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Results for “transducer selectivity”

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PubMed2001

Human bone marrow stromal cells are efficiently transduced by vesicular stomatitis virus-pseudotyped retrovectors without affecting subsequent osteoblastic differentiation.

This study tested the transduction efficiency of human bone marrow stromal cells (hBMSCs) with vesicular stomatitis virus (VSV)-pseudotyped retrovectors and their subsequent osteogenic differentiation in vitro. Two different retrovectors encoding beta-galactosidase (beta-gal) or enhanced green fluorescent protein (eGFP) as marker genes were examined for transduction of hBMSCs. hBMSCs were obtained from bone marrow filtrates of normal donors (aged 5-35 years), cultured in alpha-minimal essential medium (alpha-MEM) containing 10% fetal calf serum and infected with retrovectors soon after the adherent cells started to form individual colonies. Transduced hBMSCs were observed to express eGFP protein 4-7 days after infection in primary cultures, and the majority of hBMSCs were eGFP-positive. hBMSCs were also stained for beta-gal in the secondary cultures and virtually all hBMSCs expressed beta-gal activity. Transduced hBMSCs were examined for their osteogenic potential. These cells were found to express markers of osteogenic differentiation, including alkaline phosphatase, type I collagen, bone sialoprotein, decorin, and osteocalcin, as strongly as uninfected control cells. Mineralization was also induced by dexamethasone in transduced cells as well as control cells. These results demonstrate that hBMSCs are highly susceptible to infection with VSV-pseudotyped retrovectors with the majority of cultured cells expressing the viral transgenes without antibiotic selection. Transduced cells retain their osteogenic potential in vitro. hBMSCs are a promising cellular vehicle for systemic human gene therapy and VSV-pseudotyped retrovectors should be effective for their in vitro transduction prior to cellular engraftment.

Adolescent↗
PubMed1999

T lymphocyte transduction with herpes simplex virus-thymidine kinase (HSV-tk) gene: comparison of four different infection protocols.

In this study, we assessed the efficiency of T lymphocyte transduction with a retroviral vector carrying the herpes simplex virus thymidine kinase (HSV-tk) and neomycin phosphotransferase (neo) genes by four different protocols: standard supernatant infection, supernatant infection plus centrifugation steps, supernatant infection on fibronectin fragment-coated wells, and cocultivation. After retrovirus-mediated gene transfer of tk-neo in PHA/IL-2-stimulated primary T lymphocytes and G418 selection, T cells retained their proliferative activity, alloresponsiveness, ability to produce and to respond to IL-2, and ganciclovir (gcv)-specific sensitivity. When the four different transduction techniques were compared, no significant differences were seen in terms of cellular viability, proliferation capacity, and immunophenotyping. tk gene expression was the same in all transduced selected populations, as indicated by gcv sensitivity. Transduction efficiency was evaluated by semiquantitative PCR. Using the standard supernatant infection method, the rate of infection was extremely low (<5%). After adding the centrifugation step or performing supernatant infection on fibronectin fragment-coated wells, PCR analysis showed a 30%-40% rate of transduced cells. After infection by cocultivation, the rate of transduced cells was 30%-40%. These results demonstrate that supernatant infection plus centrifugation, supernatant infection on fibronectin fragment-coated wells, and cocultivation methods provide equivalent rates of transduced cells. The lack of reproducibility and safety indicates that cocultivation is not suitable for clinical studies. In our view, supernatant infection plus centrifugation is easier to perform than using fibronectin fragments, and it is currently the optimal method for clinical studies when large quantities of T lymphocytes are being processed.

Cell Division↗
PubMed1992

Immature thymocytes are protected from deletion early in ontogeny.

Self/non-self discrimination occurs within the thymus, where T cells undergo positive selection to produce a repertoire that recognizes foreign antigen in the context of self major histocompatibility complex proteins and negative selection to eliminate from the repertoire those T cells with self-reactive specificities. We have previously shown that two subpopulations of immature antigen receptor-bearing thymocytes exist, one that is susceptible to negative selection induced by ligation of the T-cell receptor for antigen and a second that is resistant to T-cell receptor-mediated negative selection. In the current work, we show that all antigen receptor-bearing thymocytes in early fetal thymuses are resistant to negative selection and that thymocytes susceptible to deletion do not develop until later gestational ages. Thus, deletion is a relatively late event in T-cell ontogeny. In addition, these data suggest that a thymocyte population can be isolated early in ontogeny that is capable of transducing selective signals through the antigen receptor, which do not lead to deletion but may result in positive selection.

Animals↗
PubMed2005

Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo.

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.

Animals↗
PubMed2002

Selective targeting and inducible destruction of human cancer cells by retroviruses with envelope proteins bearing short peptide ligands.

In the accompanying study, we show how retroviral tropism can be redirected by insertion of short peptide ligands at multiple locations in envelope. Here we use this approach to selectively target and destroy human cancer cells. Many cancer cells overexpress specific cell surface receptors. We have generated Moloney murine leukemia virus (MLV) envelope derivatives bearing short peptide ligands for gastrin-releasing protein (GRP) and human epidermal growth factor receptors. Pseudotyped viruses containing these chimeric envelope derivatives selectively transduce human cancer cell lines that overexpress the cognate receptor. A retrovirus targeting the GRP receptor can deliver the thymidine kinase gene to human melanoma and breast cancer cells, which are killed by the subsequent addition of ganciclovir. Collectively, our results demonstrate that short peptide ligands inserted at appropriate locations in MLV envelope can selectively target retroviruses to human cancer cells and deliver a therapeutically relevant gene.

Amino Acid Sequence↗
PubMed2005

Adaptation of a Pocket PC for use as a wearable voice dosimeter.

This article deals with the adaptation of a commercially available Pocket PC for use as a voice dosimeter, a wearable device that measures the vocal dose of teachers or other individuals on the job, at home, and elsewhere during the course of an entire day. An engineering approach for designing a voice dosimeter is described, and design data are presented. Technical issues include transducer selection, dynamic range, frequency response, memory requirements, power requirements, attachment, cables, connections, and data collection. Advantages and disadvantages of the design are discussed.

Electric Stimulation↗
PubMed1996

Selective transgene expression for detection and elimination of contaminating carcinoma cells in hematopoietic stem cell sources.

Tumor contamination of bone marrow (BM) and peripheral blood (PB) may affect the outcome of patients receiving high dose chemotherapy with autologous transplantation of hematopoietic stem cell products. In this report, we demonstrate that replication defective adenoviral vectors containing the cytomegalovirus (CMV) or DF3/MUC1 carcinoma-selective promoter can be used to selectively transduce contaminating carcinoma cells. Adenoviral-mediated reporter gene expression in breast cancer cells was five orders of magnitude higher than that found in BM, PB, and CD34+ cells. Our results demonstrate that CD34+ cells have low to undetectable levels of integrins responsible for adenoviral internalization. We show that adenoviral-mediated transduction of a reporter gene can detect one breast cancer cell in 5 x 10(5) BM or PB cells with a vector containing the DF3/MUC1 promoter. We also show that transduction of the HSV-tk gene for selective killing by ganciclovir can be exploited for purging cancer cells from hematopoietic stem cell populations. The selective expression of TK followed by ganciclovir treatment resulted in the elimination of 6-logs of contaminating cancer cells. By contrast, there was little effect on CFU-GM and BFU-E formulation or on long term culture initiating cells. These results indicate that adenoviral vectors with a tumor-selective promoter provide a highly efficient and effective approach for the detection and purging of carcinoma cells in hematopoietic stem cell preparations.

Adenoviruses, Human↗
PubMed2004

[Sonography of head and neck].

A brief, schematic summary of a workshop held in Davos 2003 is presented. Sonography of the head and neck has been carried out successfully since the beginning of the sonographical era, as this region is readily accessible to the transducer head and the superficial position of the organ can be very well visualized. The prerequisites are a high-resolution transducer-head (around 7.5 MHz), operator experience and good knowledge of the relevant anatomy. The type of transducer selected--linear or mechanical wobbler--plays a subordinate role, and color Doppler technology is not mandatory. This brief presentation will not address the thyroid gland, the cervical vessels, the paranasal sinuses, the facial soft tissue or the pharynx.

Adenolymphoma↗
PubMed1995

Resistance to human immunodeficiency virus type 1 infection conferred by transduction of human peripheral blood lymphocytes with ribozyme, antisense, or polymeric trans-activation response element constructs.

Human peripheral blood lymphocytes (PBLs) were transduced with a number of recombinant retroviruses including RRz2, an LNL6-based virus with a ribozyme targeted to the human immunodeficiency virus (HIV) tat gene transcript inserted within the 3' region of the neomycin-resistance gene; RASH5, and LNHL-based virus containing an antisense sequence to the 5' leader region of HIV-1 downstream of the human cytomegalovirus promoter; and R20TAR, an LXSN-based virus with 20 tandem copies of the HIV-1 trans-activation response element sequence driven by the Moloney murine leukemia virus long terminal repeat. After G418 selection, transduced PBLs were challenged with the HIV-1 laboratory strain IIIB and a primary clinical isolate of HIV-1, 82H. Results showed that PBLs from different donors could be transduced and that this conferred resistance to HIV-1 infection. For each of the constructs, a reduction of approximately 70% in p24 antigen level relative to the corresponding control-vector-transduced PBLs was observed. Molecular analyses showed constitutive expression of all the transduced genes from the retroviral long terminal repeat, but no detectable transcript was seen from the internal human cytomegalovirus transcript was seen from the internal human cytomegalovirus promoter for the antisense construct. Transduction of, and consequent transgene expression in, PBLs did not impact on the surface expression of either CD4+/CD8+ (measured by flow cytometry) or on cell doubling time (examined by [3H]thymidine uptake). These results indicate the potential utility of these anti-HIV-1 gene therapeutic agents and show the preclinical value of this PBL assay system.

Animals↗
PubMed1990

Device for the calibration of flow-velocity-measuring Doppler ultrasound equipment.

A new device is described for verifying the calibration accuracy of flow-velocity Doppler ultrasound (US) equipment. A rotating circular disk whose circumferential velocity may be fixed or modulated provides a strong signal source suitable for calibrating clinical Doppler US instruments. Circumferential edge velocity, derived from the disk angular frequency, is used to verify the accuracy of Doppler instrument calibration. A limited survey of routinely used Doppler US instruments demonstrates that calibration accuracy generally is satisfactory for clinical purposes, with minimal variation between instruments. Factors that affect measurements include transducer type, scale range, and display size. All transducers used on a specific piece of equipment need to be tested because the transducer design is an important part of the instrument operation. Variations were observed in reported velocities obtained from different transducers on the same equipment and for the same transducer with different scale ranges. In particular, some scales were more accurate than others, suggesting that individual scales should be tested when performing clinical Doppler US instrument calibration. Other measures of performance including scale display range, sample depth, aliasing, and transducer selection may be studied to identify their effect on measurement accuracy. Temporal modulation of angular velocity produces waveforms that may be used to simulate pulsatile velocity characteristics. The device is simple to construct and use, and it provides a convenient way to verify high-quality instrument performance.

Blood Flow Velocity↗
PubMed1994

Ex vivo expansion and selection of retrovirally transduced bone marrow: an efficient methodology for gene-transfer to murine lympho-haemopoietic stem cells.

An efficient procedure for the insertion of genetic markers into a large proportion of the mouse haemopoietic system was developed, based on the in vitro expansion of retrovirally infected bone marrow and selection of the transduced cells. Bone marrow cells harvested 4 d after 5-FU treatment were incubated under IL-3/SCF stimulation and their growth dynamic, susceptibility to retroviral infection and reconstitution capacity evaluated throughout the incubation period. On the third day of culture a maximum expansion in the CFU-GM and CFU-S12 progenitor pools was observed (130- and 15-fold, respectively), with no apparent impairment in long-term repopulating precursors. This expansion was, however, accompanied by a net decrease in the CFU-GM susceptibility to the infection by supernatants containing a Moloney-derived ecotropic retroviral vector carrying the neor gene. The designed protocol thus involved the infection of freshly harvested 5-FU-treated bone marrow, followed by expansion under IL-3/SCF stimulation and selection for resistance to G418. This procedure allowed us to harvest up to 780 CFU-GM and 50 CFU-S12 per 10(5) bone marrow cells, free from non-genetically marked progenitors. Most of the animals reconstituted with the transduced marrow bore, for at least 5 months, a very high proportion of bone marrow, spleen and thymus cells tagged with the reporter gene. These results, together with the high percentage of haemopoietic precursors bearing the neor gene and expressing resistance to G418 5 months after the transplantation indicates that long-term lympho-haemopoietic repopulating cells were efficiently transduced and selected in vitro under conditions that preserve their self-renewal and differentiation properties. This gene-transfer methodology may improve the development of gene therapy protocols where the purging of non-transduced precursors would guarantee a lasting and uniform expression of exogenous genes.

Animals↗
PubMed2001

Selective transduction of murine myelomonocytic leukemia cells (WEHI-3B) with regular and RGD-adenoviral vectors.

On the basis of the susceptibility of normal myelomonocytic cells to adenoviral vectors, we have studied the possibility of selectively transducing myelomonocytic murine leukemic cells (WEHI-3B) with regular (Reg-Ad) and genetically modified (RGD-Ad) adenoviral vectors. An 8-h incubation of WEHI-3B cells with 100 pfu of Reg-Ad vectors/cell resulted in the whole population becoming positive for transgene expression. Under identical conditions of infection, 20-30% of mouse bone marrow (BM) cells were positive for the transgene. When RGD-Ad vectors were used, a brief exposure (10 min) of WEHI-3B cells to 150 pfu of the virus/cell was enough for 100% of the leukemia cells to become positive for the marker transgene (EGFP). Under these conditions, only 15-20% of BM cells and of primitive hematopoietic progenitors (Lin(-)Sca-1(+) cells) became EGFP(+), indicating an improved selectivity of the vectors for the leukemic cells. The incubation of WEHI-3B but not normal BM cells with soluble fiber protein (FP) inhibited the infection with Reg-Ad. The use of the RGD-Ad bypassed the FP-CAR interaction required for the transduction of WEHI-3B cells with Reg-Ad, suggesting that the abrogation of this requirement accounts for the improved infectivity of these leukemic cells and for the selectivity of RGD-Ad in targeting WEHI-3B leukemia cells.

Adenoviridae↗
PubMed1979

Differential diagnosis of hypoechoic and anechoic masses with gray scale sonography: new observations.

With the technological advances in gray scale sonography that have permitted the use of higher-frequency transducers and expansion of the acoustic dynamic range, increasing problems in differentiating solid masses and fluid-filled masses have become apparent. These difficulties can be overcome by strict adherence to proper scanning techniques, which involve transducer selection, tissue attenuation compensation, and alterations in patient position. The availability of variable-dynamic-range signal processing and the use of real-time scanning can further increase one's confidence in the correct interpretation of these masses. The primary criteria for determining that a mass is fluid-filled have been expanded to include the presence of reverberation echoes, the "lateral shades" sign, and the presence of septations. In the past, hypoechoic masses with low-level internal echoes were termed "complex". Both fluid-filled masses and solid masses may fall into this category. By use of the sonographic criteria, an attempt should be made to determine whether a mass is primarily fluid-filled or solid. Specific anatomic locations and pathologic conditions in which differential diagnosis may be difficult are illustrated; these include abdominal masses, hepatic and renal masses, and pelvic masses.

Abdominal Neoplasms↗
PubMed1999

New insights into positional alcohol nystagmus using three-dimensional eye-movement analysis.

The semicircular canals selectively transduce angular velocity and are normally insensitive to gravity and linear acceleration. In acute alcohol intoxication, however, the cupula becomes lighter than the endolymph, rendering it sensitive to gravity (buoyancy hypothesis). This results in positional alcohol nystagmus (PAN) and rotatory vertigo. We evaluated PAN in 8 normal subjects by means of three-dimensional eye-movement analysis in an attempt to clarify if the buoyancy mechanism is sufficient to explain PAN. Forty minutes after intake of 0.8 g of alcohol/kg of body weight, the subjects were positioned such that the lateral canals were earth vertical. They were then rotated in the plane of the lateral canals about an earth-horizontal axis to either 45 degrees or 90 degrees , right or left ear down, and eye movements were recorded for 40 seconds in each position. The spatial analysis of the responses showed that in addition to the nystagmus induced by the buoyancy of all six cupulae, alcohol intoxication also causes a vertical velocity offset (in all subjects, slow phase down) that is independent of the orientation of the subject in space. The offset may represent a toxic effect on central vestibular pathways, producing a tone imbalance of the vertical vestibulo-ocular reflex.

Adult↗
PubMed1996

Isolating excitatory and inhibitory nonlinear spatial interactions involved in contrast detection.

Interactions between filters tuned to different orientations and spatial locations were investigated with a masking paradigm. Targets were masked by pairs of Gabor signals presented either at a different orientation (+/- delta theta) or at a different spatial location (+/- delta y). The two mask components were either of equal phase or of opposite phase to each other. Detection thresholds of the target were measured as a function of mask contrast. Typically, the curves obtained showed the following behavior: for increasing mask contrast the threshold first decreased, then reached a minimum and then increased linearly on a log-log scale reflecting a power-law behavior. Mask pairs of equal phase as well as pairs of opposite phase were shown to facilitate detection. Facilitation by mask pairs of equal phase was larger (up to 0.4 log units) and decreased for increasing delta theta and delta y. The facilitation for mask pairs of opposite phase (approximately 0.1 log units) was observed only for larger delta theta and delta y. Phase independent suppression was observed with higher mask contrasts at smaller delta theta and delta y. The strength of this suppression was shown to decrease with practice. We account for the observed facilitation with an accelerating transducer function applied on a second-stage filter. Suppression is modeled with an additional inhibitory second stage filter that divides the output of this transducer. Selective reduction of the inhibitory gain accounts for the practice effects.

Contrast Sensitivity↗
PubMed1996

Particular nuclear transcription factors responsive to systemic administration of kainic acid in murine brain.

Gel retardation electrophoresis revealed that binding of a radiolabeled double stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 (AP1) was markedly potentiated 2 h after the intraperitoneal injection of kainic acid (KA) at a dose range of 10-40 mg/kg in a dose-dependent manner in the murine hippocampus. The potentiation was seen in a manner independent of the crisis of convulsive seizures following the administration of KA at different doses. At the highest dose employed, the systemic KA significantly potentiated the AP1 binding in most central discrete structures examined except the cerebellum. In contrast, KA significantly potentiated binding of a radiolabeled probe for cyclic AMP response element binding protein (CREB) in a dose-dependent fashion in the hippocampus, without altering that in other parts of murine brain. No significant alteration was detected in binding of a probe for c-Myc in any brain regions examined 2 h after the administration of KA at different doses. However, immunoblotting analysis demonstrated that KA was ineffective in altering endogenous levels of both CREB and CREB phosphorylated at serine133 in the hippocampus and cerebellum. These results suggest that in vivo systemic KA signals may be selectively transduced to nuclear AP1 in the hippocampus through a mechanism different from phosphorylation of CREB at serine133 in murine brain.

Animals↗
PubMed1992

Temporomandibular joint sounds and condyle/disk relations on magnetic resonance images.

This study compared the condyle/disk relationships on magnetic resonance images (MRIs) in a group of subjects with completely silent temporomandibular joints (TMJ) when tested clinically with those in subjects with readily discernible TMJ sounds. The sounds were recorded with an accelerometer as the transducer. Selected degrees of jaw separation were electronically determined and recorded with interocclusal wafers for use with the imaging process. Of the "silent joints" 89% were found to have sounds when tested with the accelerometer. These "subclinical" sounds tended to be of shorter duration and occurred at a greater degree of vertical opening than the clinically discernable sounds. The MRIs of the group with clinically discernable sounds tended to show a change in the relationship between the head of the condyle and the intermediate zone of the disk, at the degree of jaw separation of the sound occurrence, whereas no condyle/disk change occurred in the group with "clinically silent joints." It is likely that all joints create sound during function. The different characteristics of the subclinical sounds versus the clinical sounds may indicate differing sound origins.

Adult↗
PubMed2005

Altering AAV tropism with mosaic viral capsids.

Over the past decade, AAV-based vectors have emerged as promising candidates for gene therapeutic applications. Despite the broad tropism of the first eight serotypes identified, certain cell types are refractory to transduction with AAV-based vectors. Furthermore, for certain applications the targeting of specific cell types is desirable. To improve on present methods to alter AAV2 tropism, we take advantage of AAV2 mosaics. Here, we show that AAV2 mosaics have improved infectivity compared with all-mutant virions. Using an AAV2 mutant that contains the immunoglobulin-binding Z34C fragment of protein A, we demonstrate the utility of AAV2 mosaics to alter AAV2 tropism. This system allows us to transduce selectively and efficiently MO7e and Jurkat cells. The use of AAV2 mosaics with a protein A fragment inserted into their capsid, together with targeting antibodies, is a versatile method that allows the specific transduction of a wide array of cell types.

Capsid↗