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At least 415 records · Page 23Linked to original sources
PubMed2001

[MRI of the temporomandibular joint. Technique, results, indications].

An optimized examination protocol for the MRI evaluation of the temporomandibular joint (TMJ) is presented. The MRI protocol is based on an optimized coil technology, sequence design, and a programmed examination protocol for diagnostics of the TMJ. Depending on the clinical findings, MRI provides an all-in-one diagnostic protocol for the diagnosis of degenerative, inflammatory and tumorous lesions of the TMJ. MRI using an optimized examination protocol should become the primary examination protocol for diseases of the TMJ.

Adult↗
PubMed1994

Optimizing the time course of brachytherapy and other accelerated radiotherapeutic protocols.

PURPOSE: It is likely that early-responding tissues, such as tumors, repair sublethal damage more rapidly than do late-responding tissues. This difference can be exploited to design protocols with a significantly improved therapeutic advantage for accelerated radiotherapeutic regimens, including brachytherapy. METHODS AND MATERIALS: The time course of potential protocols is computer optimized, maximizing the therapeutic difference between tumor-control probability (TCP), and normal-tissue complication probability (NTCP). These quantities are evaluated with the linear-quadratic model, using clinically derived parameters. The optimization is performed by individually adjusting doses in different parts of the treatment, maximizing the therapeutic advantage. In the main calculations, half times for damage repair were T1/2(late) = 4 h, T1/2(early) = 0.5 h. Two component (fast/slow) repair processes were also investigated. RESULTS: Protocols determined by optimization have significantly greater therapeutic advantage than continuous low-dose rate (CLDR) protocols of the same overall dose and time. The optimized protocols are either (a) acute-dose/gap/CLDR/gap/acute-dose; or (b) a series of acute doses separated by 3-4 h. As a typical example, results are given for 60 Gy/120 h CLDR brachytherapy, which is assumed to give NTCP = 0.2 and TCP = 0.8. Under our assumptions, optimized regimes, with the same overall time and dose, produce an NTCP of approximately 0.11 and TCP of approximately 0.83, a significant therapeutic gain over CLDR. CONCLUSION: Difference in repair rates between early- and late-responding tissues can be exploited to produce clinically practical protocols that are significantly superior to current regimens. Such optimized protocols produce slightly better tumor control than CLDR with the same overall dose and time, significantly less late damage, and similar early normal-tissue sequellae. Temporal optimization, thus, promises to be a powerful tool in designing better treatment protocols.

Animals↗
PubMed1999

Improved titers for helper virus-free herpes simplex virus type 1 plasmid vectors by optimization of the packaging protocol and addition of noninfectious herpes simplex virus-related particles (previral DNA replication enveloped particles) to the packaging procedure.

A helper virus-free herpes simplex virus type 1 (HSV-1) plasmid vector system has potential for both gene therapy and physiological studies, but relatively low titers have complicated use of this system. In this article, the packaging efficiency was improved by optimizing the packaging protocol and by adding noninfectious HSV-1-related particles, i.e., previral DNA replication enveloped particles (PREPs), during the packaging procedure. PREPs contain many of the tegument proteins that are thought to enhance an HSV-1 infection. Use of both the optimized packaging protocol and the PREPs resulted in an approximately 50-fold increase in the titer, and five different HSV-1 vectors were packaged using this procedure. A purified vector stock (7.8x10(8) infectious vector particles/ml) was microinjected into the striatum, the rats were sacrificed 4 days after gene transfer, and the brains were found to contain an average of approximately 6740 X-Gal-positive striatal cells. This improved packaging procedure may augment use of this vector system.

Animals↗
PubMed2003

Principles of low temperature cell preservation.

Cell transplantation is becoming an important technique for treatment of heart failure. Preservation is an integral step in any procedure using cells. There are two primary modes of cell preservation at low temperature, hypothermic preservation at temperatures above freezing and cryogenic preservation at temperatures below freezing. Optimal preservation protocols require a fundamental understanding of the principles involved. This review briefly describes the basic mechanisms of damage during hypothermic and cryogenic preservation and the basic principles for developing optimal protocols for preservation of cells.

Animals↗
PubMed1995

[Potential and limitation of myocardial perfusion scintigraphy for detection of viability].

Scintigraphic detection of myocardial viability is required for treatment planning and prognostication in patients with contractile dysfunction. There are four pathophysiological entities of dysfunction in coronary artery disease; one of them, "hibernating" myocardium, cannot be differentiated from scar or necrosis by mere perfusion imaging. Due to the determinants of delayed activity distribution after 201Tl injection, optimized imaging protocols using this tracer allow for adequate differentiation in many instants. Differentiation between "stunned" and "hibernating" myocardium or scar is achieved with all perfusion indicators actually available. Though 201Tl imaging with optimized protocols is almost as efficacious in viability detection as 18F-FDG positron emission tomography, the latter actually remains the reference method particularly in patients with severe left ventricular dysfunction at coronary occlusions.

Coronary Disease↗
PubMed2000

The relationship between exercise work intervals and duration of exercise on lower extremity training induced by electrical stimulation in humans with spinal cord injuries.

A group of 90 male paraplegics were studied to determine the optimal training protocol for isokinetic exercise induced by functional electrical stimulation of the quadriceps muscles. The parameters that were varied were the number of training sessions a week, the length of the training sessions each day, and the work-rest intervals in each training session. Training for 3 days a week for 30 min a day with 6 s of exercise and 6 s of rest proved the optimal protocol. Training for 5 days or for 1 day a week was not as effective in training strength or endurance. A combination of 50% work and 50% rest produced a much greater gain in strength and endurance than work:rest ratios of 66%:33% or 25%:75%. When training was conducted for 5 min, 15 min or 30 min each day, the greatest increase was found when the muscles were exercised for 30 min each day. While more variables need to be examined, this study has provided some initial guidelines for isokinetic training of humans using electrical stimulation.

Adult↗
PubMed1992

Optimization of retroviral vector-mediated gene transfer into endothelial cells in vitro.

Retroviral vector-mediated gene transfer into endothelial cells is relatively inefficient with transduction rates as low as 1-2% in vitro and even lower in vivo. To increase the efficiency of gene transfer into endothelial cells, we used retroviral vectors expressing beta-galactosidase and urokinase and measured endothelial cell transduction efficiencies with quantitative assays for beta-galactosidase and urokinase protein. We evaluated several techniques reported to improve the efficiency of retroviral transduction in vitro, including 1) extended periods of exposure to vector, 2) repeated exposures to vector, 3) maximization of the ratio of vector particles to endothelial cells by increasing the volume and concentration of vector particles or by decreasing the number of endothelial cells exposed, 4) cocultivation of endothelial cells with vector-producing cells, and 5) variation of the type and concentration of polycation used with the retroviral vector. Only the use of more concentrated (higher titer) vector-containing supernatant and the use of the polycation DEAE-dextran improved the efficiency of gene transfer into endothelial cells in vitro. In an optimized transduction protocol, a 60-second exposure to 1 mg/ml DEAE-dextran followed by a single 6-hour exposure to supernatant of a titer of 10(5)-10(6) colony-forming units/ml resulted in transduction efficiencies of 50-90% with both vectors. Decreasing the time of the supernatant exposure to 15 minutes permitted transduction efficiencies of 15-20% while significantly minimizing the duration of the transduction. Therefore, the optimized protocol allows high efficiency in vitro gene transfer into endothelial cells within several hours. The briefer protocol may prove useful for in vivo gene transfer in which the time of exposure to the supernatant is limited.

Cells, Cultured↗
PubMed2000

Immunostaining for cell picking and real-time mRNA quantitation.

Microdissection techniques allow a cell-type or even cell-specific mRNA analysis within complex tissues. Furthermore, valid mRNA quantitation can be performed by real-time reverse transcriptase-polymerase chain reaction from a few isolated cells obtained from cryosections. For a more precise access to many cell types, this technique has to be complemented by a cell-type-specific immunostaining. To evaluate its effect on mRNA quantitation, we analyzed alveolar macrophages (AMs) from control rat lungs and those undergoing stimulation with lipopolysaccharide and interferon-gamma nebulization. Whereas AMs from the left lung were directly harvested for mRNA extraction by bronchoalveolar lavage, tissue sections of the right lung were stained with an optimized immunofluorescence protocol detecting AMs. Fifteen AM profiles per sample were picked by laser-assisted sampling technique. Normalizing to a standard gene, nitric oxide synthase II (NOSII) and tumor necrosis factor (TNF)-alpha mRNA were quantified by real-time reverse transcriptase-polymerase chain reaction. In stimulated lungs, the percentage of picked samples positive for NOSII or TNF-alpha mRNA increased significantly. Moreover, a marked increase in the ratio of target gene mRNA to standard gene mRNA was noted for both NOSII and TNF-alpha in picked AMs from stimulated lungs, which matched very well the increase detected in the lavaged AMs undergoing direct RNA extraction. Thus, when using an optimized protocol for immunofluorescence, this approach may be reliably combined with laser-assisted cell picking and real-time mRNA quantitation in a few immunohistochemically characterized cell profiles within complex tissues.

Animals↗
PubMed1999

Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue.

One of the key end points for understanding the molecular basis of carcinogenesis is the quantitation of gene expression in specific cell populations. Microdissection techniques allow extraction of morphologically distinct cells for molecular analysis. A recent advance in microdissection uses the PixCell laser capture microdissection (LCM) system, which allows for precise removal of pure cell populations from morphologically preserved tissue sections. The objective of this study was to determine the optimal fixation protocol for analyzing RNA from tissue samples using LCM. Optimal fixation must provide acceptable morphology, allow proper laser capture of selected cells, and preserve the integrity of mRNA. We evaluated the effects of both cross-linking and precipitive-type fixatives on frozen and paraffin-embedded mouse liver tissue. For assessment of the quality of the mRNA in LCM samples generated from various fixed tissues, reverse transcription-polymerase chain reaction (RT-PCR)-amplified mouse liver beta2-microglobulin mRNA was detected with ethidium bromide. We also examined mouse glyceraldehyde-3-phosphate-dehydrogenase by using the fluorogenic TaqMan system for real-time quantitative detection of RT-PCR products. Frozen tissues yielded more RT-PCR product than did paraffin-embedded tissues. In both frozen and paraffin-embedded tissues, differences were observed between the fixatives. Precipitive fixatives, such as ethanol and acetone, consistently produced more RT-PCR amplification product than did cross-linking fixatives such as formalin. Optimal fixation protocols for LCM analysis will facilitate the examination of gene expression in specific cell populations, accelerating investigations of the molecular differences responsible for the phenotypic changes observed during carcinogenesis.

Animals↗
PubMedDate not supplied

Optimization of a mouse immunization protocol with Paracoccidioides brasiliensis antigens.

The objectives of the present study were to optimize the protocol of mouse immunization with Paracoccidioides brasiliensis antigens (Rifkind's protocol) and to test the modulation effect of cyclophosphamide (Cy) on the delayed hypersensitivity response (DHR) of immunized animals. Experiments were carried out using one to four immunizing doses of either crude particulate P. brasiliensis antigen or yeast-cell antigen, followed by DHR test four or seven days after the last immunizing dose. The data demonstrated that an immunizing dose already elicited response; higher DHR indices were obtained with two or three immunizing doses; there were no differences between DHR indices of animals challenged four or seven days after the last dose. Overall the inoculation of two or three doses of the yeast-cell antigen, which is easier to prepare, and DHR test at day 4 simplify the original Rifkind's immunization protocol and shorten the duration of the experiments. The modulation effect of Cy on DHR was assayed with administration of 2.5, 20 and 100 mg/kg weight at seven day intervals starting from day 4 prior to the first immunizing dose. Only the treatment with 2.5 mg Cy increased the DHR indices. Treatment with 100 mg Cy inhibited the DHR, whereas 20 mg Cy did not affect the DHR indices. Results suggest an immunostimulating effect of low dose of Cy on the DHR of mice immunized with P. brasiliensis antigens.

Animals↗
PubMed1993

Antitumor response to recombinant murine interferon gamma correlates with enhanced immune function of organ-associated, but not recirculating cytolytic T lymphocytes and macrophages.

The mechanism of therapeutic activity for recombinant murine interferon-gamma (rMu IFN gamma) in the treatment of metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16-BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the tumor-bearing organ) were tested after 1 week and 3 weeks of rMu IFN gamma administration (i.v. three times per week). Natural killer (NK), lymphokine-activated killer (LAK), cytolytic T lymphocyte (CTL) activities against specific and nonspecific targets, and macrophage tumoristatic activity were measured. rMu IFN gamma demonstrated immunomodulatory activity in most assays of immune function. The optimal therapeutic protocol of rMu IFN gamma (2.5 x 10(6) U/kg, three times per week) prolonged survival and decreased the number of pulmonary metastatic foci. This therapeutic activity was correlated with specific CTL activity from pulmonary parenchymal mononuclear cells (PPMC), but not from spleen or blood. Macrophage tumoristatic activity in PPMC also correlated with therapeutic activity, but activity in alveolar macrophages did not. However, therapeutic activity did not correlate with NK or LAK activity at any site. These results demonstrate that the optimal therapeutic protocol is the same as the optimal immunomodulatory dose for pulmonary CTL and macrophage activities. Furthermore, while immunological monitoring may help to optimize treatment protocols, current monitoring procedures that use readily accessible sites, particularly peripheral blood, may not accurately predict the therapeutic efficacy of biological response modifiers in clinical trials.

Animals↗
PubMed2001

Development of an optimized interaction-mating protocol for large-scale yeast two-hybrid analyses.

BACKGROUND: Protein-protein interactions have decisive roles in almost all aspects of the structural and functional organization of cells. But in spite of the increasing amount of complete genome sequence data, the ability to predict protein function from sequences alone is limited. Therefore comprehensive analysis of protein-protein interactions, as derived from the yeast two-hybrid mating system, will yield valuable information for functional biology on a proteomic scale. RESULTS: We have developed an optimized interaction mating protocol for the yeast two-hybrid system, which gives increased mating efficiencies. This significantly reduces the effort and cost of cDNA library screening and allows multiple parallel approaches. Improved preincubation conditions before mating, and optimal cell densities and cell ratios enable almost quantitative mating of the yeast cells carrying the cDNA library. We have proved the applicability of this technology using 20 bait proteins to screen an Arabidopsis thaliana cDNA library, in spite of bait-dependent variations in mating efficiency. CONCLUSIONS: The improved yeast two-hybrid interaction-mating protocol presented here allows the multiple parallel screening of cDNA libraries. It can be carried out without specialized equipment and has the potential to be standardized and automated.

Arabidopsis↗
PubMed2000

[An optimal follow-up protocol for renal cell carcinoma based on the occurrence of recurrences after surgery].

INTRODUCTION AND OBJECTIVE: The tumor biology of renal cell carcinoma (RCC) remains poorly understood and the prognosis of which is unpredictable. To define an optimal follow-up protocol in nonmetastatic RCC, tumor recurrences and the clinical course were assessed in the patients who had undergone nephrectomy. PATIENTS AND METHODS: Between March 1972 to July 1999, 265 patients with PT1-3N0 (Nx) M 0 RCC underwent radical or partial nephrectomy. Clinicopathological variables were compares with the time of the first recurrence, site of metastasis and reason for diagnosis. RESULTS: In July 1999, the median (range) follow-up for the surviving patients was 50 (1-244) months. Metastases were diagnosed in 45 of the 265 patients (17%). Sixty percent of the patients had their metastases diagnosed within 3 years and 87% by 6 years. The incidence and the mean time to first diagnosis of recurrence were 9.0% and 43.6 months for pT1, 32.4% and 39.6 months for pT2, and 32.7% and 25.0 months for pT3. Metastatic sites and the mean time of diagnosis were 40.6 months in lung (46.7%), 26.1 months in bone (17.8%), 18.6 months in retroperitoneal organs (11.1%), 38.0 months in liver (6.7%), 51.0 months in thyroid gland (4.4%) and 31.0 months in brain (2.2%). Most recurrence in lung, retroperitoneal organs and liver were incidentally diagnosed, whereas recurrences in bone, thyroid gland and brain had some symptoms associated with metastases. Incidence of lung metastasis in pT1 (9 patients, 56%) and pT2 (6, 55%) was greater than that in PT3 (6, 33%). In contrast, incidence of abdominal metastasis (retroperitoneal organs and the liver) in pT3 (4 patients, 33%) was grater than pT1 (2, 12%) and PT2 (1, 16%). CONCLUSIONS: The results indicate that the risk of tumor progression and site of metastasis seemed to depend on original TNM stage. Therefore stage specific postoperative follow-up protocol should be considered. The yearly follow-up may suffice for pT1a patients after surgery. For pT1b and pT2 patients, the follow-up should be intense during the first 3 years by a routine chest X-ray. For pT3 patients, the follow-up during the first 2 years may be important. Particularly, considering the high incidence of abdominal metastasis in these patients, examination by CT scan is recommended every 6 months in the first 2 years. No need for bone scintigraphy and brain CT was indicated for asymptomatic patients.

Carcinoma, Renal Cell↗
PubMed1986

Dissociation of therapeutic and toxic effects of polyinosinic-polycytidylic acid admixed with poly-L-lysine and solubilized with carboxymethyl cellulose in tumor-bearing mice.

In this paper, we describe a study of the therapeutic parameters (dose and schedule) and immunomodulatory activity (macrophage, natural killer cell, and T-cell number and function) of polyinosinic-polycytidylic acid admixed with poly-L-lysine and solubilized with carboxymethyl cellulose [poly(I,C)-LC] in the treatment of MBL-2 tumor ascites. Tumor-bearing mice received an optimal therapeutic protocol [100 micrograms poly(I,C)-LC administered twice a wk], a maximum tolerated dose [50 micrograms poly(I,C)-LC administered daily], or the optimal immunomodulatory protocol for normal mice [10 micrograms poly(I,C)-LC administered daily]. The percentage of tumor-associated macrophages and their cytotoxic activity correlated with host survival. In addition, splenic T-cell activity correlated with host survival, and splenic natural killer cell function had a near significant correlation with host survival. These results indicate that the optimal dose and schedule of poly(I,C)-LC for immunomodulation in tumor-bearing animals are also the optimal therapeutic protocol but have less toxicity than the maximum tolerated dose.

Animals↗
PubMed1991

Weaning of three hybridoma cell lines to serum free low protein medium.

A general weaning procedure is described which allowed a range of hybridomas to be weaned readily off serum without loss of antibody production. Initial work was carried out with one cell line only (SPO1 cells) and one serum substitute containing a final protein concentration of 40 mg l-1. The SPO1 cells were first adapted to a range of readily available basal media and then weaned off serum by a range of protocols. From this work an optimal weaning protocol and basal medium for weaning were determined. These were then used to wean the SPO1 cells and two other cell lines off serum with a second, protein free, serum substitute with varying concentrations of defined proteins added. All three cell lines investigated were readily weaned off serum by this protocol at protein concentrations as low as 1 mg l-1. No loss of antibody production was observed with any of the cell lines. The weaning procedure outlined in both simple and rapid and has been successfully adopted in our laboratory by relatively inexperienced cell culture technicians.

Animals↗
PubMed2003

Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research.

Electroporation designates the use of short high-voltage pulses to overcome the barrier of the cell membrane. By applying an external electric field, which just surpasses the capacitance of the cell membrane, transient and reversible breakdown of the membrane can be induced. This transient, permeabilized state can be used to load cells with a variety of different molecules, either through simple diffusion in the case of small molecules, or through electrophoretically driven processes allowing passage through the destabilized membrane--as is the case for DNA transfer. Initially developed for gene transfer, electroporation is now in use for delivery of a large variety of molecules: From ions to drugs, dyes, tracers, antibodies, and oligonucleotides to RNA and DNA. Electroporation has proven useful both in vitro, in vivo and in patients, where drug delivery to malignant tumours has been performed. Whereas initial electroporation procedures caused considerable cell damage, developments over the past decades have led to sophistication of equipment and optimization of protocols. The electroporation procedures used in many laboratories could be optimized with limited effort. This review (i) outlines the theory of electroporation, (ii) discusses factors of importance for optimization of electroporation protocols for mammalian cells, (iii) addresses particular concerns when using electroporation in vivo, e.g. effects on blood flow and considerations regarding choice of electrodes, (iv) describes DNA electrotransfer with emphasis on use in the in vivo setting, and (v) sums up data on safety and efficacy of electroporation used to enhance delivery of chemotherapy to tumours in cancer patients.

Animals↗
PubMed1999

Protocols and future directions in imaging of renal artery stenosis: CT angiography.

Computed tomographic angiography (CTA) has become an established technique for minimally invasive imaging of renal artery stenosis. This article describes in detail the current scan protocols suggested for renal artery stenosis and explains the rationale behind the choice of such parameters as collimation, pitch, reconstruction interval, scan duration and scan length. It discusses the optimized use of contrast agents including bolus triggering and a saline flush to improve contrast agent utilization. Examples of optimized scan protocols are given for various scanner types including subsecond scanners and multislice computed tomography. With standard 1-s scanners, a slice collimation of 2 mm is recommended. A pitch factor of 1.5-2 is important for optimum volume coverage. Smoothing reconstruction kernels improve signal-to-noise ratio and allow for dose reduction. For optimum results, the plateau phase of contrast enhancement has to be matched as precisely as possible. Thus, a test bolus injection or an automated bolus triggering technique should be employed. Axial CT images remain the basis for making the diagnosis but require interactive viewing on a monitor display. Image processing (multiplanar reformats; maximum intensity projections; shaded surface displays) is employed to display the renal arteries. Given optimized examination protocols, high sensitivity and specificity can be achieved (90-98%) with negative predictive values of greater than 95%. Thus, a normal CT angiogram virtually rules out renal artery stenosis.

Angiography↗
PubMed1996

Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol.

Reliable flow cytometric analysis of normal and diseased skin requires pure epidermal single-cell suspensions. Several methods to separate the dermis from the epidermis are available. The proteolytic enzyme thermolysin separates the epidermis from the dermis at the lamina lucida and therefore permits reliable dermoepidermal separation. In the present study an optimized cell isolation procedure using thermolysin and trypsin is described, which is particularly suitable for punch biopsies. A 16-20-h (overnight) incubation of biopsies taken from normal and hyperproliferative skin with thermolysin (0.5 mg/ml) at 4 degrees C produced a selective separation of the dermis and epidermis. After a 30-min trypsin incubation (0.25 mg/ml) at 37 degrees C a cell suspension was produced which was characterized by minimal cell damage (cellular debris and clumps), a high recovery of basal cells and high quality DNA histograms. Furthermore, dermal contamination was very low. The thermolysin-trypsin separation methodology followed by triple-labelling flow cytometry provided a precise quantification of the percentage of keratin 10-positive cells, vimentin-positive cells and cells in S and G2M phases. Proliferative activity was selectively measured in the basal, the suprabasal and the non-keratinocyte compartment at various time intervals during epidermal regeneration after adhesive tape stripping. In contrast to the non-keratinocytes, the percentage of cells in S and G2M phases in the basal keratinocytes and in the suprabasal compartment increased 44-48 h after stripping. The increased proliferation following tape stripping was paralleled by an increased invasion of vimentin-positive cells into the epidermis and preceded by a decreased number of keratin 10-positive cells. Thermolysin-trypsin separation followed by three-colour flow cytometry permits a highly selective characterization of normal and hyperproliferative epidermis.

Adolescent↗