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PubMed1999

Development of a new catheter-tip pressure transducer.

Two major disadvantages of the conventional catheter-tip transducer have been incapability to calibrate baseline pressure and to adjust sensitivity after insertion. A vast majority of conventional catheter-tip transducers are classified as gauge type. Due to their structure they are inherently incapable of recalibrating after insertion. We have overcome this problem by developing a new differential type catheter-tip transducer equipped with a lumen that connects a small chamber at the backside of the transducer to another external port. This lumen is capable of pressure passage. The output of this type depends on the difference between the two imposed pressures. This passage makes a baseline standard possible, when the end of the lumen is exposed to atmospheric pressure. When pneumatic pressure is imposed to the end of the lumen using a syringe, for example, the transducer output shifts up and down in accordance to that pressure, enabling baseline pressure recalibration and verifying the degree of sensitivity after insertion. By obtaining the following data, we confirmed the stability and availability of this transducer: Baseline drift less than 0.04 mmHg/8 hour, frequency characteristics flat up to 60 Hz, and common mode rejection ratio more than 46 dB.

Catheterization, Peripheral↗

PubMed2001

Growth inhibition of interleukin-2 receptor gene-transduced peripheral blood lymphocytes on human ovarian cancer cells.

OBJECTIVE: To investigate the growth inhibition of interleukin-2 receptor (IL-2R) gene-transduced peripheral blood lymphocytes (PBLs) on human ovarian cancer cells. METHODS: Interleukin-2 (IL-2) and IL-2R genes were transfected into human ovarian cancer cell line 3AO and PBLs, respectively, using the same Fugene vector. Twenty-four hours later transfected and nontransfected PBLs were cocultured with transfected and nontransfected 3AO for 48 hours. cytotoxicity of PBLs on 3AO was detected by the MTT assay. RESULTS: The morphology of IL-2-transduced 3AO and IL-2R-transduced PBLs remained unchanged. 3AO cells could be transfected with the IL-2 gene and expressed IL-2 mRNA, and PBLs could be transfected with the IL-2R gene and expressed IL-2R mRNA. IL-2 transduced 3AO cells enhanced their response to the cytotoxicity of PBLs. Furthermore, growth inhibition of PBLs to 3AO cells increased significantly when the IL-2R was transfected into PBLs and when the IL-2 gene was transfected into 3AO cells and the two were combined. CONCLUSIONS: IL-2R gene transduced PBLs are able to enhance their cytotoxicity on IL-2 gene transduced ovarian cancer cells. This method may be a new way to investigate IL-2 gene therapy for ovarian cancer.

Cell Count↗

PubMed1984

Minimal size plasmids containing an M13 origin for production of single-strand transducing particles.

We have studied the requirements for efficient production of single-strand transducing particles from minimal size plasmids containing the phage M13 origin of replication. The most favorable origin fragment for production of transducing particles was found to extend from nucleotides 5372 to 5943 of the phage sequence, which includes a segment of the phage gene IV coding region and eliminates part of the gene II promoter. Minimizing vector size for M13 origin plasmids appears to be beneficial since transducing particle titer decreases with increasing plasmid size. A mutant helper phage was isolated and shown to improve the production of transducing particles from small plasmids, but to outcompete plasmids bearing large inserts. Several plasmid vectors which produce high yields of single-strand transducing particles and good ratios of transducing particles to helper phage have been constructed.

Coliphages↗

PubMed1994

Transplantation of retrovirus-transduced canine keratinocytes expressing the beta-galactosidase gene.

We studied transplantation of retrovirus vector transduced canine keratinocytes to determine whether keratinocytes could persist and express the transferred gene after superficial transplantation to full-thickness wounds of dogs, a large random-bred model for gene transfer studies. Canine keratinocytes were transduced by co-cultivation with PA317 retrovirus packaging cells which produced helper-free amphotropic retroviral vectors (LZSN and LNPOZ) encoding the genes for beta-galactosidase (beta-gal) and neomycin phosphotransferase (neo). Efficient transfer and expression of the two genes could be demonstrated in confluent keratinocyte cultures for both vectors. When transduced keratinocytes were grown in organotypic cultures on a collagen matrix containing autologous dermal fibroblasts at the air-liquid interface, the cultures showed well-organized and defined epidermal cell layers and several markers of terminal differentiation, including the presence of keratohyalin granules and a multilayered stratum corneum. To determine whether the transferred beta-gal gene was also expressed in vivo, we performed autologous transplantation of transduced keratinocytes onto full-thickness wounds of dogs. beta-Gal expressing keratinocytes could be demonstrated in situ in the regenerating epidermis 2 weeks after transplantation. We conclude that keratinocytes can be efficiently transduced by retroviral vectors, that retroviral transduction does not interfere with proliferation or differentiation, and that transduced keratinocytes express the transferred gene after transplantation to full-thickness skin wounds of dogs. Keratinocytes thus seem to be good target cells for gene therapy.

Animals↗

PubMed1996

Urokinase expression in transduced endothelial cells.

Increased thromboresistance through the release of lytic agents by endothelial cells may improve the patency of endothelial lined prosthetic grafts. We have evaluated the expression of urokinase from cells transduced with a retrovirus containing the gene for a human preprourokinase. Endothelial cells were enzymatically harvested from canine external jugular vein in nine animals and grown to confluence in culture. One-third of these cells served as controls, and the remaining two-thirds were transduced via incubation with an LXSN-type retroviral vector carrying the urokinase gene and a neomycin resistance gene. Successfully transduced cells were selected by incubation with 400 micrograms/mL G418 and pure cultures grown to confluence. Supernatants from confluent control and experimental cell cultures after 48 hours in defined, serum-free medium were assayed for human urokinase concentration and overall enzyme activity. ELISA quantitation of concentration using mouse antihuman urokinase antibody showed 0.15 +/- 0.11 ng/mL/hr/10(6) cells in the transduced cell supernatant; no measurable concentration was found in the control cells. (P < 0.01) Overall (human plus canine) enzyme activity of urokinase was determined using an indirect spectrophotometric assay based on plasminogen activation (ploug U/mL). Transduced cells showed activities of 0.12 at 10 days and 0.45 at confluence; control cell activity was 0.0 and 0.15, respectively. (P < 0.05) These data show that endothelial cells can be transduced with a urokinase expressing gene that increases the release of this thrombolytic agent. Lining small diameter prosthetic grafts with these cells may improve their thromboresistance and long-term patency.

Animals↗

PubMedDate not supplied

beta-galactosidase transduced T lymphocytes: a comparison between stimulation by either PHA and IL-2 or a mixed lymphocyte reaction.

BACKGROUND: Retroviral-mediated gene transfer stably introduces exogenous genes into normal and neoplastic cells of the hematopoietic system. METHODS: We used two retroviral vectors [the first, FLac, expresses a chimeric protein (Sh-ble::LacZ) between the product of the phleomycin resistance gene (Sh-ble) and the bacterial beta-galactosidase encoded by the LacZ gene; the second, NuNL vector, contains a fusion sequence (LacZ::Neo) that expresses the LacZ and the neomycin resistance genes] to transduce T lymphocytes derived from the peripheral blood of healthy human donors. Two lymphocyte activation procedures were employed: a) phytohemagglutinin/interleukin-2 (PHA/IL-2) polyclonal stimulation; b) allogeneic stimulation in a mixed irradiated or non irradiated lymphocyte reaction, both supplemented with IL-2 (MLR/IL-2). Infection was achieved by co-cultivating activated T cells with the producing amphotropic cell line pretreated with mitomycin C for 96 hours. Infection and transduction efficiency were assayed by LacZ gene expression, which is detected as indigo blue staining with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X..Gal). RESULTS: The highest percentage of transduced T cells was reached on the 3rd PHA/IL-2 and on 9th MLR/IL-2 activation days. In these conditions with FLac vector we obtained up to 80% X-Gal+ cells after PHA/IL-2 activation and 66% and 44%, respectively, with non irradiated and irradiated MLR/IL-2, respectively. Up to 40% X-Gal+ cells were obtained with NuNL vector after PHA/IL-2 stimulation, 40% with irradiated and 48% with non irradiated MLR/IL-2 activated cells. In term of transduction efficiency, large variability was observed among patients. There were no immunophenotypical differences between FLac or NuNL vector-transduced cells activated by either of the two techniques and the control cells. CONCLUSIONS: Our results indicate that: a) the use of FLac or NuNL vector retroviral-mediated gene transfer into T-lymphocytes derived from peripheral blood and stimulated by either PHA/IL-2 or a MLR produces a high percentage of transduced T cells; b) MLR is a good system for generating a transduced alloreactive lymphocyte population. The combination of high transduction efficiency and the capacity to obtain alloreactive transduced lymphocytes should open up the possibility of generating new in vitro and in vivo studies with selectable genes for in vivo therapeutic use.

Drug Resistance, Microbial↗

PubMed1997

Protection of herpes simplex virus thymidine kinase-transduced cells from ganciclovir-mediated cytotoxicity by bystander cells: the Good Samaritan effect.

Although considerable attention has been directed in the field of gene therapy toward elucidating the mechanism by which a transduced cell could kill a bystander cell, little is known about how bystander cells may affect transduced cells. We hypothesized that bystander cells, particularly if they were capable of gap junctional communication, could protect cells transduced with the herpes simplex virus thymidine kinase (HSV-TK) from ganciclovir (GCV)-induced cytotoxicity. To test this hypothesis, we used a rat hepatocyte cell line (WB) that can carry out efficient gap junctional communication, a WB clone transduced with HSV-TK (WB-TK), and a communication-incompetent subclone of WB cells (aB1). We cocultured WB-TK cells with either WB or aB1 cells, treated them with GCV, and then plated the cells into selective media that permitted us to quantify independently the surviving fraction of WB-TK cells or bystander cells. We found that WB bystander cells conferred up to a 1000-fold protection on WB-TK cells treated with GCV. aB1 cells conferred detectable, but significantly less, protection. These findings demonstrate that herpes simplex virus thymidine kinase-transduced cells can be significantly protected by bystander cells, particularly those that can carry out gap junctional communication. Whether this "Good Samaritan" effect improves the overall efficacy of gene therapy, by prolonging the survival of the source of toxic metabolites, or decreases effectiveness by increasing the survival of transduced cells will need to be determined in vivo.

Animals↗

PubMedDate not supplied

Vaccination for experimental gliomas using GM-CSF-transduced glioma cells.

Brain tumors have an immunoprivileged status which contributes to their refractoriness to treatment. In this study, immune rejection of GL261 glioma tumors in the mouse brain was achieved by subcutaneous vaccination with GM-CSF-transduced glioma cells. Cultured GL261 cells were transduced to secrete murine GM-CSF using a retrovirus vector, then irradiated, and injected subcutaneously into H-2 matched C57BL/6 mice. In prevaccination studies, the median survival time (MST) of animals vaccinated with 5 x 10(4) or 5 x 10(5) GM-CSF-transduced cells 7 days prior to intracranial injection of 10(6) nontransduced, nonirradiated GL261 cells was significantly prolonged by 45-50% compared with animals vaccinated in parallel with nontransduced, irradiated glioma cells. In treatment of established gliomas, the MST of animals, which were treated subcutaneously with 5 X 10(6) irradiated GM-CSF-transduced cells 3 days after intracranial injection of 2 x 10(4) nontransduced cells, was prolonged significantly by 36% compared with animals treated with the same number of nontransduced, irradiated cells or to sham-treated animals. In prevaccination studies, histology of brain tumors 4 days after intracranial tumor cell injection revealed infiltrates of CD8+ lymphocytes and eosinophils, the latter exclusively in animals vaccinated with GM-CSF-transduced cells, Thus, subcutaneous injection of irradiated GM-CSF-transduced glioma cells can induce a potent immune response to intracranial gliomas both as a vaccination against subsequent intracranial glioma cell implantation and for treatment of established intracranial glioma.

Animals↗

PubMed1998

Delayed targeting of cytokine-nonresponsive human bone marrow CD34(+) cells with retrovirus-mediated gene transfer enhances transduction efficiency and long-term expression of transduced genes.

Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34(+) cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol using neoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34(+) cells. BM CD34(+) cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers of neoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transduced neoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

Adult↗

PubMed1985

Transducer system for the noninvasive recording of arterial pressure contours.

A transducer system is described that permits noninvasive recordings of the pressure-vs-time arterial profile at any palpable site on the body. A thin (30 microns) piezoelectric polymer film of polyvinylidene fluoride serves as the active element and as the mechanical coupler to the skin. The system has high frequency response and a low frequency cut-off of 0.16 Hz. The active element and its support provides a good mechanical impedance match with the skin. The transducer housing resembles a thick wrist watch and is strapped in place. The complete system is designed to record as many as four simultaneous profiles on a subject. Comparisons of the noninvasive transducer records with records taken with an indwelling catheter indicate congruence of the two signals and the first and second derivatives of the two signals, both when the transducer is applied directly to the skin and when a layer of fat is interposed between the skin and the transducer. The system provides a simple atraumatic means of periodically screening and/or monitoring cardiovascular changes in large populations of subjects and would provide a means of tracking changes induced by therapeutic programs.

Arteriosclerosis↗

PubMed1986

Extended use of disposable pressure transducers. A bacteriologic evaluation.

In a prospective randomized study, contamination rates of disposable pressure transducers changed every two days (n = 81) were compared with those changed at four (n = 26) or eight days (n = 50); the mean daily incidence of contamination was 3% for each group. After four days of use, the cumulative prevalences of contamination were similar. However, after eight days, the cumulative prevalence was significantly higher in transducers used without change (6.9%) than in those changed every two days (2.9%). Gram-negative bacilli were present in 63% of contaminated transducers; over half were from the patients' own flora. The only definite transducer-related bacteremia occurred on a day of initial contamination and should have been unaffected by the interval of change. Routine use of disposable transducers can be safely extended to four days, even in a busy intensive care unit.

Bacteria↗

PubMed1991

Recording fetal breathing movements with a passive transducer based on an inductive principle.

A new transducer has been developed, based on an inductive principle, for recording fetal movements and sounds. The compliance of this transducer, the INductive PHOno-sensor (INPHO), can be matched to that of the maternal abdomen to provide an optimal transfer of displacement between maternal abdomen and transducer. In this manner, it is possible to detect fetal breathing movements by digital filtering of the INPHO signal in a frequency band between 0.5 and 2.0 Hz. Singular breathing movements can be detected and this was verified by real time ultrasound imaging. The INPHO transducer shows a flat (+/- 1.5 dB) frequency response between 0.2 and 200 Hz. The signal-to-noise ratio of the transducer system is better than 95 dB, and enables very weak movements and sounds to be detected. Spectral analysis of the processed signal shows that modulation of fetal breathing by maternal breathing takes place. The measuring setup allows for the quantitative assessment of fetal respiratory sinus arrhythmia.

Fetal Heart↗

PubMed1997

ICP monitoring with a re-usable transducer: experimental and clinical evaluation of the Gaeltec ICT/b pressure probe.

Intracranial pressure monitoring requires reliable transducers at a justifiable price. At present, transducers for single or repeated use are available. We examined the Gaeltec model ITC/b solid state miniature transducer experimentally and clinically. Measurement accuracy was assessed in vitro at increasing steps of 5 mmHg from 0 to 80 mmHg. While new and recently serviced probes revealed minimal deviations from the preset values, frequently used transducers differed up to 7 mmHg. This occurred especially in the high pressure range above 50 mmHg. Additionally the drift was investigated at different pressure levels. After 24 hours we already found drifts of 2 mmHg with new and serviced probes and up to 4 mmHg with used ones. In clinical practice we implanted 150 transducers in 121 patients from 1983 until 1995. The probes were re-used up to twelve times, the average time being 7 times. 32.7% of all measurements were regarded as not reliable. Dislocation (16.7%), inability to calibrate (3.3%) and defect pressure probes (3.3%) were the most common complications. Repeated use of the Gaeltec ICT/b probe also seemed to result in an additional decay of measurement quality. The strain of frequent cleaning and sterilizing may have caused changes of the physical properties of the probes with time. Whether these results also apply to other types of ICP probes for repeated use needs further evaluation.

Adolescent↗

PubMed1997

[Adjusting the geometry of implantable hearing aid components to human temporal bone. I: Electromechanical transducer].

Prerequisite to implantation of a piezoelectrical transducer of an implantable hearing aid is a shape allowing its implantation into human mastoid and middle ear. To approach this problem, a consecutive series of six transducer prototypes was created in an iterative process. Their functional geometry was evaluated in 50 human temporal bones. A shape for a functioning transducer was found which will enable implantation in 78% of the cases examined (confidence interval: 61.5%-89.2%). It will allow simultaneous implantation of the transducer into the mastoid and microphone, which is situated transmastoidal in the posterior wall of the ear canal. Furthermore, the transducer may be coupled to the ossicular chain or the perilymph.

Cochlear Implantation↗

PubMed1997

[Intraoperative evaluation of signal transduction of prototypes of implantable hearing aid transducers in the human].

During routine middle ear surgery prototypes of implantable piezoelectric hearing aid transducers were coupled to the ossicular chain of 28 patients. Pure tone thresholds in a broad frequency range (250 Hz-8 kHz) and perception of music were the basis of psychoacoustic evaluation of the transfer properties of the prototypes in this human study. The piezoelectric transducer reached an equivalent transducer sound-pressure level of up to 145 dB SPL and frequencies of 10 kHz. The dynamics for music reached 32 dB, which was identical with the results of the preoperative investigations using hi-fi headsets (33 dB). The low nonlinear distortions of around 0.1% and the frequency range up to 10 kHz are reflected in the positive evaluation of sound quality by 84% of the patients involved. The study is the basis for realizing a piezoelectric transducer designed for routine clinical implantations. The transducer is thought to be part of a totally implantable future hearing aid for rehabilitation of moderate and severe sensory neural hearing loss predominantly in the middle and high frequency range.

Adult↗

PubMed2006

A comparative study of water perfusion catheters and microtip transducer catheters for urethral pressure measurements.

The aim of this study was to compare the maximum urethral closure pressure (MUCP) measures with two different techniques: water perfused catheter and microtip transducer catheters with respect to reproducibility and comparability for urethral pressure measurements. Eighteen women with stress urinary incontinence had repeat static urethral pressure profilometry on a different day using a dual microtip transducer and water perfused catheter (Brown and Wickham). The investigators were blinded to the results of the other. The microtip measurements were taken in the 45 degrees upright sitting position with the patient at rest at a bladder capacity of 250 ml using an 8 Fr Gaeltec double microtip transducer withdrawn at 1 mm/s, and the transducer was orientated in the three o'clock position. Three different measures were taken for each patient. Three water perfusion measurements were performed with the patient at rest in the 45 degrees upright position at a bladder capacity of 250 ml using an 8 Fr BARD dual lumen catheter withdrawn at 1 mm/s. The mean water perfusion MUCP measure was 26.1 cm H2O, significantly lower than the mean microtip measure of 35.7 cm H2O. The correlation coefficient comparing each water perfusion measurement with the other water perfusion measures in the same patient was excellent, at 0.95 (p = 0.01). Correlation coefficient comparing each microtip measure with the other microtip measure in the same patient was also good, ranging from 0.70 to 0.80. This study confirms that both water perfusion catheters and microtip transducers have excellent or very good reproducibility with an acceptable intraindividual variation for both methods.

Adult↗

PubMed1992

A miniature piezoelectric polymer transducer for in vitro measurement of the dynamic contact stress distribution.

Previous studies of contact pressure measurement between articular surfaces have been mostly limited to static techniques. The purpose of our study was to develop a new dynamic technique for a direct measurement of the local contact stresses, and to apply the new method to an in vitro cadaver study of the patellofemoral joint pressures. The miniature transducer consists of a 2 mm diameter and 28 microns thick piece of piezoelectric polymer film sandwiched between two stainless steel electrodes of similar diameter. A water-resistant capsule consisting of Teflon film and Hysol epoxy was applied around the transducer. The transducer was 3 mm in diameter and 0.7 mm in thickness. A 3 mm well was made at six locations in the patella, corresponding to superior, middle, and inferior regions of both facets. Six transducers were cemented within each well, flush with the articular cartilage. The transducers were calibrated in situ before and after the experiment. The femur was rigidly fixed to the loading apparatus and the tibia was allowed to flex and extend through a 90 degrees range of motion using an Instron and a pulley system connected to the quadriceps tendon. Q angles of 0, 5, 10 and 15 degrees were established by adjusting the direction of the quadriceps tendon. Stresses ranging from 0.1-1.3 MPa were recorded at various locations. These values varied in flexion and extension. An overall decrease in these stresses was noted after tuberosity elevation up to 1.5 cm, following which increased values up to 1.8 MPa were recorded mostly in the superior section.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomechanical Phenomena↗

PubMed1988

Measurement of discrete vertical in-shoe stress with piezoelectric transducers.

The purpose of this investigation was to design and validate a system suitable for non-invasive measurement of discrete in-shoe vertical plantar stress during dynamic activities. Eight transducers were constructed, with small piezoelectric ceramic squares (4.83 x 4.83 x 1.3 mm) used to generate a charge output proportional to vertical plantar stress. The mechanical properties of the transducers included 2.3% linearity and 3.7% hysteresis for stresses up to 2000 kPa and loading times up to 200 ms. System design efficacy was analysed by means of a multiple day, multiple trial data collection. With the transducers placed beneath plantar landmarks, the footstrike of one subject was recorded ten times on each of five days while running at 3.58 m/s on a treadmill. Within-day and between-day proportional error (PE) was used to estimate the error contained in the mean peak stress during foot contact. Within-day PE focused on trial to trial variability associated with the subject and equipment, and averaged 3.1% (range 2.5-4.0%) across transducer location. Between-day PE provided a cumulative estimate of subject, transducer placement, and random equipment variability, but excluded trial to trial variability. It ranged from 4.9 to 15.8%, with a mean of 9.9%. Peak stress, impulse, and sequence of loading data were examined to identify discrete foot function patterns and highlight the value of discrete stress analysis.

Foot↗