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Results for “protocol optimization”

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At least 73 records · Page 4Linked to original sources
PubMed1987

T cell depletion of bone marrow for clinical marrow allografting: optimalization of conditions for depletion by anti-CD2 and anti-CD8 monoclonal antibodies with rabbit complement, and for detection of residual T cell content.

Normal bone marrow obtained at harvest for bone marrow transplantation was passed over a Ficoll density gradient to obtain the mononuclear cell fraction. These cells were incubated with anti-T cell monoclonal antibodies (MAb) anti-HuLym-1 and anti-HuLym-8 plus rabbit complement, washed, and the degree of T cell depletion was assessed by culture in phytohemagglutinin, by flow cytometry and by limiting dilution analysis. The optimal protocol for T cell depletion was found to be a 2-stage incubation with 2 cycles of complement treatment, using a bone marrow cell concentration as low as possible. The precise conditions of complement dilution and incubation temperature depended on the batch of complement used, and had to be assessed for each batch. Of the 3 methods used for assessing T cell contamination, culture in PHA was found to be inappropriate due to large variation in background proliferation of treated and untreated bone marrow cells. Flow cytometry was accurate for residual T cell content of greater than 1% (corresponding to a 90-95% depletion of T cells) but could not detect lower levels of T cell contamination. The limit dilution assay was found to be by far the most sensitive, being capable of detecting a T cell contamination of 1 in 10,000 cells. When combined with flow cytometry on untreated marrow for calculation of the cloning efficiency, it gave very accurate determinations of residual T cell content of marrow.

Animals↗
PubMedDate not supplied

Artificial intelligence techniques for cancer treatment planning.

An artificial intelligence system, NEWCHEM, for the development of new oncology therapies is described. This system takes into account the most recent advances in molecular and cellular biology and in cell-drug interaction, and aims to guide experimentation in the design of new optimal protocols. Further work is being carried out, aimed to embody in the system all the basic knowledge of biology, physiopathology and pharmacology, to reason qualitatively from first principles so as to be able to suggest cancer therapies.

Animals↗
PubMed1989

Efficacy of fertilization in artificially inseminated turkey hens.

Research was conducted to develop an artificial insemination protocol optimizing the use of spermatozoa by turkey breeder hens. Large White turkey hens were inseminated on Days 14 and 17 postphotostimulation with 200 million spermatozoa from one male phenotype to fill the oviductal storage sites. Artificial inseminations were then performed weekly for 20 wk with different spermatozoa numbers of another male phenotype. Fertility and phenotype of each poult were determined at hatch to ascertain which insemination, initial or subsequent, was responsible for fertility. Inseminating weekly with 200 million viable spermatozoa cells resulted in better fertility but did not optimize the hen's utilization of spermatozoa from the initial inseminations. When fewer spermatozoa were inseminated weekly (50 million cells), more progeny were fertilized by spermatozoa already residing in the oviduct than would be expected. When the number of spermatozoa inseminated weekly was increased at intervals during a laying cycle, spermatozoa from the initial inseminations were utilized more efficiently, but fertility was depressed at times during the laying cycle. Gradually increasing weekly inseminated numbers of spermatozoa from 50 to 200 million viable cells/hen as the hens age results in nearly equivalent fertility to that resulting from insemination by 200 million cells each week. This represents a savings of 1.4 billion spermatozoa/hen over a 20-wk laying period.

Animals↗
PubMed1992

Drug delivery optimization through Bayesian networks.

This paper describes how Bayesian Networks can be used in combination with compartmental models to plan Recombinant Human Erythropoietin (r-HuEPO) delivery in the treatment of anemia of chronic uremic patients. Past measurements of hematocrit or hemoglobin concentration in a patient during the therapy can be exploited to adjust the parameters of a compartmental model of the erythropoiesis. This adaptive process allows more accurate patient-specific predictions, and hence a more rational dosage planning. We describe a drug delivery optimization protocol, based on our approach. Some results obtained on real data are presented.

Anemia↗
PubMed1991

Psoralen plus ultraviolet A in the prophylactic treatment of benign summer light eruption.

We report the results of a French multicentre study to evaluate the efficiency of psoralen plus ultraviolet A (PUVA) therapy in the prophylactic treatment of benign summer light eruption (BSLE) and to establish the optimal protocol of radiation. Nine photobiology centres took part in this study; 83 patients (76 of them women) were evaluated. The radiation protocols were as follows: oral psoralen (8-methoxypsoralen; 0.6 mg/kg) was taken at each session; the starting dose of UVA radiation was determined according to skin type, with increments of 0.5 J/cm2 every 2 sessions. The subjects were randomized to receive 10-20 sessions 3 times per week. PUVA therapy was very effective: 68 patients (82%) reported total protection from BSLE. Four patients (5%) showed progress. Only 13% showed no improvement. The satisfactory results were not correlated with either the number of sessions or the J/cm2 of UVA. The intensity of tanning after the PUVA sessions did not appear to predict cure. Thirty-six percent of the patients had adverse reactions to treatment, including erythema, pruritus and triggering of BSLE. However, these effects only required the treatment to be stopped in 2% of the cases (for severe pruritus). The results in the various centres were similar.

Adolescent↗
PubMed1991

Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays.

The ability to transfer new genetic material into human hematopoietic cells provides the foundation for characterizing the organization and developmental program of human hematopoietic stem cells. It also provides a valuable model in which to test gene transfer and long-term expression in human hematopoietic cells as a prelude to human gene therapy. At the present time such studies are limited by the absence of in vivo assays for human stem cells, although recent descriptions of the engraftment of human hematopoietic cells in immune-deficient mice may provide the basis for such an assay. This study focuses on the establishment of conditions required for high efficiency retrovirus-mediated gene transfer into human hematopoietic progenitors that can be assayed in vitro in short-term colony assays and in vivo in immune-deficient mice. Here we report that a 24-hour preincubation of human bone marrow in 5637-conditioned medium, before infection, increases gene transfer efficiency into in vitro colony-forming cells by sixfold; interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) provide the same magnitude increase as 5637-conditioned medium. In contrast, incubation in recombinant growth factors IL-1, IL-3, and granulocyte-macrophage colony-stimulating factor increases gene transfer efficiency by 1.5- to 3-fold. Furthermore, preselection in high concentrations of G418 results in a population of cells significantly enriched for G418-resistant progenitors (up to 100%). These results, obtained using detailed survival curves based on colony formation in G418, have been substantiated by directly detecting the neo gene in individual colonies using the polymerase chain reaction. Using these optimized protocols, human bone marrow cells were genetically manipulated with a neo retrovirus vector and transplanted into immune-deficient bg/nu/xid mice. At 1 month and 4 months after the transplant, the hematopoietic tissues of these animals remained engrafted with genetically manipulated human cells. More importantly, G418-resistant progenitors that contained the neo gene were recovered from the bone marrow and spleen of engrafted animals after 4 months. These experiments establish the feasibility of characterizing human stem cells using the unique retrovirus integration site as a clonal marker, similar to techniques developed to elucidate the murine stem cell hierarchy.

Adult↗
PubMedDate not supplied

The value of multiple preparations in the diagnosis of malignant pleural effusions. A cost-benefit analysis.

A cost-benefit analysis of five techniques employed in processing 108 malignant pleural effusions for cytopathologic examination was performed. Ethanol-fixed, Papanicolaou-stained smears were positive in 68% of the effusions, air-dried Diff-Quik-stained smears in 66%, Cytospins in 83%, cell blocks in 85% and Millipore filters in 85%. Examination of one air-dried smear and one ethanol-fixed smear yielded a diagnostic sensitivity of 82%. Using a combination of two smears and one of three concentrating techniques (Cytospin, cell block or MIllipore filter) would have provided a diagnosis in over 90% of the effusions. The use of four or more preparations provided more sensitivity than did three preparations and decreased the likelihood that a malignant diagnosis would be based on the findings in only one preparation. The costs related to disposable materials and the College of American Pathologists (CAP) work load estimates for specimen preparation, staining and screening were compared. These data may be useful in developing optimal protocols for pleural effusion preparation in laboratories with specific work load requirements and limited resources.

Cost-Benefit Analysis↗
PubMed1990

A statistical analysis of the third UKEMS collaborative trial.

In the third UKEMS collaborative trial, nine laboratories optimized protocols for a variety of mammalian cell mutagenicity assays and used these protocols to test three reference mutagens. Data from the trial have been deposited with Mutagenesis under the scheme of the journal for a database of results of extensive mutagenicity testing programmes. A preliminary examination of the data using procedures advocated by the UKEMS guidelines on statistical evaluation of mutagenicity test data confirms that the assays are capable of giving clear, convincing data, in a form amenable to statistical analysis. The utility of the methods of analysis proposed by both the plate and fluctuation test statistical working groups was confirmed. In almost all cases variation between plates or trays from the same replicate treatment was little greater than Poisson or binomial, whereas variation between replicate treatments was substantially larger, confirming the recommendation of both groups for true independent replicates of each treatment. In six out of nine laboratories part of this variation between replicates was consistent for both viability and mutation, which might cause the guide-lines procedure for fluctuation test data to underestimate significance. In about half the cases examined, variation between experiments was significantly greater than variation between replicates within an experiment, which may create problems of interpretation. Supplementary tests, using the statistical package GLIM, suggested that although factors such as choice of expression time, level of S9, zero dose cloning efficiency or spontaneous mutation frequency might have an effect, they would not have been likely to interfere with detection of a mutagenic effect in the present data set.

Analysis of Variance↗
PubMedDate not supplied

OKT3 monoclonal antibody given for ten versus fourteen days as immunosuppressive prophylaxis in heart transplantation.

We have previously reported that murine antihuman monoclonal antibody OKT3 (Orthoclone OKT3) given for 14 days after heart transplantation is effective as immunosuppressive prophylaxis. The optimal protocol for OKT3 prophylaxis in heart transplantation is unknown, particularly the duration of OKT3 therapy. We conducted a consecutively allocated overlapping 6-month study with 68 heart transplant patients, comparing 14-day OKT3 (n = 34) to 10-day OKT3 treatment (n = 34). Both protocols included OKT3 given beginning 24 to 48 hours after operation, cyclosporine beginning on postoperative day 3, low-dosage steroids and azathioprine to prevent antibody production to OKT3, and a steroid pulse plus randomization to plus or minus vincristine after stopping OKT3. Pretransplant characteristics including age, sex, cause of congestive heart failure, and absence of positive pretransplant crossmatch were similar between the two groups. Although the infection rate was not significantly different between the two groups and mortality (one patient in each group) did not differ, 14-day prophylaxis decreased the number of treated rejection episodes per patient for the 6-month study (1.59 +/- 0.18 versus 2.24 +/- 0.19, p = 0.016). A 14-day course of OKT3 also decreased the risk of rejection during the 6-month follow-up period (p less than 0.05). In addition to having a decreased number of rejection episodes, patients in the 14-day protocol were also more likely to be withdrawn from maintenance steroids (79% versus 53%, p = 0.02). In conclusion, a measurable dose response efficacy can be demonstrated for 14-day versus 10-day OKT3 prophylaxis.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1989

A system for on-line detection and resolution of radiolabeled DNA molecules and its application to automated DNA sequence analysis.

We describe a system for the real-time detection of radioactively labeled DNA molecules in gel matrix, and we demonstrate the application of this system to DNA sequence analysis. DNA sequencing reactions prepared by the Sanger chain termination method are resolved by electrophoresis on 8% polyacrylamide gels. During electrophoresis the 32P-labeled DNA fragments are detected by solid state detectors positioned 22 cm from the top surface of the gel. This system is able to resolve a DNA sequence of 300 bases or greater. Optimized protocols that allow sequence information to be obtained from single stranded and double-stranded templates are described. A linear relationship exists between the input dpm and the integrated peak values over a 20-fold range indicating that accurate DNA quantitation is also possible using this system.

Automation↗
PubMed1985

Optimization of an immunotherapeutic protocol with poly(I,C)-LC.

The development of successful immunotherapeutic protocols requires new therapeutic strategies as well as the development of clinically predictive tumor models and protocols. A bell-shaped response curve is observed with many biological response modifiers (BRMs), not only for immunomodulation, but also for therapeutic activity. Although most BRMs, including poly(I,C)-LC, are toxic at high doses, the administration of nontoxic doses by an optimal protocol and route of injection results in significant therapeutic benefit and increased immunomodulation in tumor-bearing animals compared to the administration of a maximum tolerated dose (MTD). We suggest that Phase II clinical trials using an optimal immunomodulatory protocol may result in increased therapeutic activity compared to protocols based on the MTD.

Animals↗
PubMed1990

Optimization of survey protocols for MRI.

A method for evaluating the sensitivity of MRI protocols to changes in an arbitrary number of tissue parameters over a broad range of parameter values is presented. This analysis is useful for choosing an optimal basis set of images for either a "survey" protocol or tissue segmentation algorithms. A survey protocol is required when searching for a lesion of unknown type or location. Segmentation requires unique signal signatures for tissues that may cover a broad range of tissue parameter values. Data acquisition is modeled as a mapping of a domain of tissue parameter values into a signal manifold in a signal strength space defined by the MRI protocol. The efficacy of the protocol is evaluated by investigating the characteristics of the signal manifold. A figure of merit which maximizes the probability of discriminating each point in the domain of tissue parameters from all others is developed.

Clinical Protocols↗
PubMed1992

Detection protocols for biotinylated probes: optimization using multistep techniques.

Recent studies using biotinylated in situ hybridization (ISH) have utilized a wide range of detection protocols for the biotinylated hybrids, leading to conflicting reports in the literature regarding sensitivity. In this study we compared 11 different detection protocols for biotinylated ISH using a measles virus-specific RNA probe on formalin-fixed, paraffin-embedded central nervous system tissue infected with measles virus. Maximum sensitivity was achieved with five-step detection protocols incorporating the use of a monoclonal antibody to biotin. Single-step detection protocols were found to be insensitive, as shown by their failure to detect viral nucleic acid in infected white-matter cells. Only by increasing the number of steps in the detection protocols were these infected cells demonstrable. Unless pre-hybridization, hybridization, and detection protocols are optimized, the results obtained in pathogenicity studies using ISH could be misinterpreted, leading to false conclusions about nucleic acid distribution. This also applies to the ever-increasing use of ISH for diagnostic purposes.

Antibodies, Monoclonal↗
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↗
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↗