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Results for “protocol optimization”

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PubMed2002

Optimization of gene transfer into primitive human hematopoietic cells of granulocyte-colony stimulating factor-mobilized peripheral blood using low-dose cytokines and comparison of a gibbon ape leukemia virus versus an RD114-pseudotyped retroviral vector.

Primitive human hematopoietic cells in granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) are more difficult to transduce compared to cells from umbilical cord blood. Based on the hypothesis that MPB cells may require different stimulation for efficient retroviral infection, we compared several culture conditions known to induce cycling of primitive hematopoietic cells. MPB-derived CD34(+) cells were stimulated in the presence or absence of the murine fetal liver cell line AFT024 in trans-wells with G-CSF, stem cell factor (SCF), and thrombopoietin (TPO) (G/S/T; 100 ng/ml) or Flt3-L, SCF, interleukin (IL)-7, and TPO (F/S/7/T; 10-20 ng/ml), and transduced using a GaLV-pseudotyped retroviral vector expressing the enhanced green fluorescence protein (eGFP). Compared to cultures without stroma, the presence of AFT024 increased the number of transduced colony-forming cells (CFC) by 3.5-fold (with G/S/T), long-term culture-initiating cells (LTC-IC) by 4.6-fold (with F/S/7/T), and nonobese diabetic/severe immunodeficiency disease (NOD/SCID)-repopulating cells (SRC) by 6.8-fold (with F/S/7/T). Similar numbers of long-term culture-initiating cells (LTC-IC) and SRC could be transduced using AFT024-conditioned medium (AFT-CM) or a defined medium that had been supplemented with factors identified in AFT-CM. Finally, using our best condition based on transduction with the gibbon ape leukemia virus (GaLV)-pseudotyped vector, we demonstrate a 33-fold higher level of gene transfer (p < 0.001) in SRC using an RD114-pseudotyped vector. In summary, using an optimized protocol with low doses of cytokines, and transduction with an RD114 compared to a GaLV-pseudotyped retroviral vector, the overall number of transduced cells in NOD/SCID mice could be improved 144-fold, with a gene-transfer efficiency in SRC of 16.3% (13.3-19.9; n = 6).

Animals↗

PubMed2003

Production and application of polyclonal antibody to human thyroid transcription factor 2 reveals thyroid transcription factor 2 protein expression in adult thyroid and hair follicles and prepubertal testis.

Germline mutations in thyroid transcription factor 2 (TTF2) cause thyroid agenesis, spiky hair, and cleft palate, indicating thyroidal and extrathyroidal expression. We sought to investigate this by producing and applying an antibody to human TTF2. The coding region of human TTF2 was cloned into a bacterial expression vector, production of the soluble TTF2 protein optimized, and pure TTF2 obtained by nickel chromatography. Rabbits were immunized and the resulting TTF2 polyclonal titrated on formalin-fixed, paraffin-embedded sections of thyroid. The optimized protocol was applied to a range of tissues. Nine milligrams of TTF2 protein was obtained per liter of culture and a high-titer antibody produced. This displayed specific staining of thyroid follicular cell nuclei/cytoplasm and not of the interstitium, connective tissue, smooth muscle, or endothelium. No staining was obtained with the preimmune serum in the same conditions, or with the majority of other tissues tested with the TTF2 polyclonal. The exceptions were testis and skin, in which nuclear TTF2 immunoreactivity was present in the seminiferous tubules and cells in the follicular outer root sheath, respectively. In conclusion, we have produced a polyclonal antibody for human TTF2 and demonstrated immunoreactivity for this transcription factor in adult human thyroid and hair follicles and prepubertal testis.

Adolescent↗

PubMed2003

Surgical skill facilitation in videoscopic open surgery.

The operating room (OR) was traditionally characterized as a closed environment, in which the view of the operative field was available to the surgeon and assistant only. In laparoscopy, integration of technology into the surgical theatre has transformed surgical procedures into minimally invasive events, with viewing of the surgical field using endoscopic cameras. Similar technical advances to the open surgical environment will allow visualization and coordination of finer surgical maneuvers on standard video monitors. The objective of this study was to develop optimal protocols for performing basic open surgical maneuvers without direct viewing of the operating field, instead watching a monitor that displays the image of the surgical field captured by an endoscopic camera. The AESOP robotic arm and Alpha Virtual Port (Computer Motion, Goleta, California) were used to hold the endoscopic camera in different positions relative to the surgeon and the operative table. The surgeons conducting the study evaluated six such different setups. Based on the average time to complete the task in each of these setups and the ease of adaptation to the new working conditions, we concluded that at least one of these setups could be translated into the OR. The advantages of integrating video image enhancement over classical open surgery (OS) are that the surgical field can be magnified to perform finer maneuvers, and to share views of the surgical field with additional clinicians and trainees.

Clinical Competence↗

PubMed2000

The beta-galactosidase gene as a marker for hematopoietic reconstitution: individual cell analysis in a murine bone marrow transplantation model.

We have used a simple, single-gene retrovirus carrying the Escherichia coli beta-galactosidase reporter gene (lacZ), termed LlacZ. This virus was found to infect immortalized myeloid and lymphoid precursor/leukemic cell lines efficiently as well as primary murine bone marrow clonogenic progenitors, without apparent modulation of growth or phenotype. Following infection of bone marrow cells, a significant proportion of progenitors--36% of lineage-negative cells with low levels of c-kit expression (lin-/c-kit(lo)) known to be enriched with pluripotent hemopoietic stem cells, and 19% of Sca1-positive cells known to be enriched with transplantable cells with lymphomyeloid-reconstituting ability--were shown to express lacZ. Use of an LlacZ-infected population of post 5-fluorouracil bone marrow cells to reconstitute lethally irradiated mice demonstrated the presence of lacZ-expressing cells in the spleen at day 12 post-transplantation with provirus detected in individual spleen colonies (CFU-S). In the long term (3-6 months following transplantation), lacZ expression was detected in hematopoietic tissues of all recipient mice. The use of two-color in situ and flow cytometry analysis combined with lineage-specific antibodies showed lacZ expression in both myeloid and lymphoid cells in spleen and bone marrow. In addition, lacZ-expressing cells were detected in secondary recipient mice injected with bone marrow cells derived from primary LlacZ recipients. Overall, these data show the efficacy of a single gene vector for stem cell transduction, the utility of beta-galactosidase as a single cell marker for stem cell transduction and reconstitution ability, and the need for protocol optimization to see high-level multilineage gene expression.

Animals↗

PubMed2001

Current use of HER2 tests.

Reliable detection of HER2 overexpression is important for the success of trastuzumab (Herceptin) therapy. Several methods are available for measuring HER2 expression at the DNA, RNA or protein level. The method most frequently employed is immunohistochemical (IHC) detection of the HER2 receptor in paraffin sections. Advantages include the precise localization of the HER2 protein, the availability of paraffin material and the ease of the procedure. However, IHC can be influenced by the sensitivity/specificity of the antibody, tissue treatment and, in particular, subjective assessment. These disadvantages do not exist in the detection of gene amplification by fluorescence in situ hybridization (FISH) or polymerase chain reaction. However, FISH requires expensive equipment that is not widely available in pathology laboratories. Another approach quantitates shed HER2 antigen in the serum by an enzyme-linked immunosorbent assay. The key advantage of this method is the ease of sampling blood, however, serum HER2 concentrations do not accurately reflect the tumor status. Furthermore, this method does not register single-cell expression, which is important for therapeutic decision making. For routine diagnostics, the combination of IHC and FISH is useful. In addition to improving the accuracy and comparability of HER2 assays, these optimized protocols may further enhance the efficacy of trastuzumab therapy by selecting those patients most likely to respond.

Biomarkers, Tumor↗

PubMed1986

Use of an orthogonal design method to study two-stage chemical carcinogenesis in BALB/3T3 cells.

An orthogonal design method was used to study two-stage chemical carcinogenesis in BALB/3T3 cells. Four factors were studied: different concentrations of the initiator N-methyl-N-nitro-N'-nitrosoguanidine (MNNG); different concentrations of the promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA); different concentrations of fetal calf serum (FCS) and different times at which TPA was added after cell initiation. From the results of three experiments designed by Table L9(3(4)), L8(2(7)) and L16(4(5)), 0.3 microgram/ml MNNG was the highest possible initiating concentration and 0.25 microgram/ml TPA was the minimum effective concentration for promoting activity. There is synergy between MNNG and TPA, the optimum combination in sequential treatment being 0.3 microgram/ml MNNG and 0.25 microgram/ml TPA. The 10% FCS standard concentration was the optimal one; however, below 5% few foci appeared. The time at which TPA was added had little effect on cloning efficiency and transformation frequency. So the use of this orthogonal design in cell culture has many advantages: several factors can be tested simultaneously; it is easy to find the optimal protocol conditions and the dose-response relationship is stable, which enables the reproducibility to be improved. In addition, the different tables proposed may help to reveal unexpected problems.

Animals↗

PubMed1994

DNA sequencing using ultra small amounts of reagents and template.

One of the key points in the genome project is finding ways to reduce the running cost in DNA sequencing. One way is to use a highly-sensitive fluorescent DNA sequencer, where only trace amounts of template DNA and reagents are needed. An experimental protocol optimized for the trace amounts of DNA analysis was established by using the hybridization reaction rate coefficient of primers on template DNA, which was estimated to be 7.5 x 10(5) M-1sec-1 at 37 degrees C. One femtomole of template DNA with 0.001 unit of modified T7 DNA polymerase (Sequenase Ver. 2.0) and also 0.45 fmol of M13 template DNA with 0.01 unit of Taq DNA polymerase were enough to sequence DNA of up to 400 bases.

Bacteriophage M13↗

PubMed1998

Oocyte cryopreservation.

Cryopreservation of human oocytes has been employed with little success in clinical practice, even though it may solve the legal and ethical problems linked to embryo freezing. Various attempts to cryopreserve human oocytes have mostly been unsuccessful, leading to low oocyte survival rates after thawing, and the search for an optimal protocol for oocyte cryopreservation remains elusive. A preliminary study was undertaken to evaluate some of the factors influencing the survival rate of human oocytes and the efficiency of intracytoplasmic sperm injection (ICSI) as an insemination procedure. A total of 38 women with tubal infertility were enrolled in the study. The cryopreservation procedure consisted of a slow freeze-rapid thawing technique using 1,2 propanediol and sucrose as cryoprotectants. The overall oocyte survival rate was approximately 60%. A better survival rate was obtained when the oocytes were cryopreserved in the presence of partially removed cumulus oophorus rather than in the presence of totally enzymatically removed cumulus oophorus. The cryoprotectant concentration and the equilibration time also appear to influence the oocyte survival rate. ICSI may be an efficient method of achieving a satisfactory outcome in terms of fertilization in cryopreserved human oocytes. Embryonic morphological quality does not seem to be compromised by cryopreservation. In conclusion, these data show that cryopreservation may ensure that the integrity of the human oocyte is adequate for normal fertilization and embryo development.

Adult↗

PubMed2004

Evaluation of methods for extracting Xylella fastidiosa DNA from the glassy-winged sharpshooter.

The recent spread of the plant pathogenic bacterium Xylclla fastidiosa Wells et al. by an invasive vector species, Homalodisca coagulata Say, in southern California has resulted in new epidemics of Pierce's disease of grapevine. Our goal is to develop an efficient method to detect low titers of X. fastidiosa in H. coagulata that is amenable to large sample sizes for epidemiological studies. Detection of the plant pathogenic bacterium X. fastidiosa in its insect vector is complicated by low titers of bacteria, difficulty in releasing it from the insect mouthparts and foregut, and the presence of substances in the insect that inhibit polymerase chain reaction (PCr). To select the optimal protocol for DNA extraction to be used with PCR, we compared three standard methods and 11 commercially available kits for relative efficiency of X. fastidiosa DNA extraction in the presence of insect tissue. All of the protocols tested were proficient at extracting DNA from pure bacterial culture (1 x 10(5) cells), and all but one protocol successfully extracted sufficient bacterial DNA in the presence of insect tissue. Three DNA extraction techniques, immunomagnetic separation, the DNeasy Tissue kit (Qiagen, Hercules, CA), and Genomic DNA Purification kit (Fermentus, Hanover, MD), were compared more closely using a dilution series of X. fastidiosa (5000-0 cells) with and without insect tissue present. The DNeasy Tissue kit was the best kit tested, allowing detection of 5 x 10(3) X. fastidiosa cells with an insect head background.

Animals↗

PubMed2001

Toxicity and health-related quality of life during and after high dose rate brachytherapy followed by external beam radiotherapy for prostate cancer.

BACKGROUND: The optimal protocol for combining high dose rate brachytherapy and external beam irradiation as treatment for localized prostate cancer is unknown. Toxicity rates and clinical and biochemical outcomes should be evaluated to validate the current treatment protocol. METHODS: Fifty-eight patients were treated for prostate cancer with high dose rate brachytherapy followed by 30 Gy of external beam radiation therapy. Toxicity during treatment and for 12-18 months thereafter, and treatment-related morbidity, were evaluated. Physician-assessed treatment-related toxicity was graded at the time of occurrence using the Radiation Therapy Oncology Group morbidity criteria. Four separate self-administered questionnaires were used to collect longitudinally demographic data and general and prostate disease-related measures of quality of life. RESULTS: Various degrees of rectal bleeding due to radiation proctitis were experienced by 13 patients (22%) at a median time of 11 months. Two of these patients needed hospitalization to undergo laser coagulation of the rectal mucosa. Study patients had statistically significant decreases in five SF-36 domains during the first month of treatment. All measures recovered by 12 months. Sexual function was not affected by irradiation. Lower urinary tract symptoms assessed by IPSS/QOL scores worsened significantly during the first month of treatment but later recovered to baseline levels. Physician-assessed RTOG scores failed to detect these changes. CONCLUSIONS: Morbidity associated with combined radiation therapy was greatest during the first month of treatment and affected quality of life significantly. Most measures recovered to baseline levels by 12 months following radiation therapy. Although the current protocol appears acceptable, measures should be taken to decrease treatment-related morbidity further.

Adult↗

PubMed2005

Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca).

In white spruce, an improvement of somatic embryo number and quality can be achieved through experimental manipulations of the endogenous levels of reduced (GSH) and oxidized (GSSG) glutathione. An optimal protocol for embryo production included an initial application of GSH in the maturation medium, followed by replacement with GSSG during the remaining maturation period. Under these conditions, the overall embryo population more than doubled, and the percentage of fully developed embryos increased from 22% to almost 70%. These embryos showed improved post-embryonic growth and conversion frequency. Structural studies revealed remarkable differences between embryo types, especially in storage product deposition pattern and organization of the shoot apical meristem (SAM). Compared with their control counterparts, glutathione-treated embryos accumulated a larger amount of starch during the early stages of development, and more protein and lipid bodies during the second half of development. Differences were also noted in the organization of SAMs. Shoot meristems of control embryos were poorly organized and were characterized by the presence of intercellular spaces, which caused separation of the subapical cells. Glutathione-treated embryos had well-organized meristems composed of tightly packed cells which lack large vacuoles. The improved organization of the shoot apical meristems in treated embryos was ascribed to a lower production of ethylene. Differences in meristem structure between control and treated embryos were also related to the localization pattern of HBK1, a shoot apical meristem 'molecular marker' gene with preferential expression to the meristematic cells of the shoot pole. Expression of this gene, which was localized to the apical cells in control embryos, was extended to the subapical cells of treated embryos. Overall, it appears that meristem integrity and embryo quality are under the direct control of the glutathione redox state.

Germination↗

PubMed2000

Detection and characterization of micronuclei in a murine liver epithelial cell line, by application of the in vitro cytokinesis block MN assay and PRINS.

The cytokinesis block micronucleus assay was applied to murine cell line C6, derived from fetal liver, after an optimal protocol had been designed. Micronucleus frequencies were assayed after exposure to three concentrations of colcemid or diepoxybutane. Two-colour primed in situ DNA synthesis (PRINS) was applied to simultaneously label telomeric and centromeric (minor satellite DNA) sequences. Both chemicals induced a highly significant increase in MN and the effect was dose dependent. Diepoxybutane did not appear to significantly increase the frequency of centromere-positive micronuclei. Colcemid, as expected, induced high frequencies of centromere-positive micronuclei at all concentrations tested; in addition a significant increase in centromere-negative micronuclei was observed at 10(-5) M. Many centromere-positive micronuclei carried three or four telomeres, thus indicating that a duplicated (non-disjoined) chromosome with two chromatids was contained in the micronucleus. This observation leads to the conclusion that micronuclei deriving from missegregation could be due to errors occurring before the onset of anaphase. The results obtained on C6 cells are in good agreement with those obtained on other cell systems, indicating that this cell line can be considered for in vitro aneuploidy evaluation.

Anaphase↗

PubMed2004

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH.

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.

Cell Line, Tumor↗

PubMed1995

Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol.

Use of a cryoprotective agent is indispensable to prevent injury to human spermatozoa during the cryopreservation process. However, addition of cryoprotective agents to spermatozoa before cooling and their removal after warming may create severe osmotic stress for the cells, resulting in injury. The objective of this study was to test the hypothesis that the degree (or magnitude) of human sperm volume excursion can be used as an independent indicator to evaluate and predict possible osmotic injury to spermatozoa during the addition and removal of cryoprotective agents. Glycerol was used as a model cryoprotective agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypo-osmotic solutions (iso-osmotic medium diluted with water) and to shrinkage in hyperosmotic solutions (iso-osmotic medium with sucrose) were determined. Sperm plasma membrane integrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosomotic exposure. The result indicate firstly that motility was much more sensitive to anisosmotic conditions than membrane integrity, and secondly that motility was substantially more sensitive to hypotonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or shrinking) was determined. The second step, using these sperm volume excursion limits and previously measured glycerol and water permeability coefficients of human spermatozoa, was to predict, by computer simulation, the cell osmotic injury caused by different procedures for the addition and removal of glycerol. The predicted sperm injury was confirmed by experiment. Based on this study, an analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.

Buffers↗

PubMed2002

Recommendations for sentinel lymph node processing in breast cancer.

The status of the sentinel lymph node (SLN) has been shown to accurately reflect the presence or absence of metastases in the axilla in patients with breast cancer. This study was designed to determine the optimal protocol for SLN processing. A total of 173 SLNs from 96 breast cancer patients who had successful SLN localization and underwent completion axillary node dissection were identified. All SLNs were negative for metastases by initial routine histologic evaluation. The nodes were submitted in a total of 300 blocks. Each block was serially sectioned to produce 10 levels. Pan-cytokeratin stain was performed on levels 3 and 8. All other levels were stained with hematoxylin and eosin. Metastases were identified in 22 SLNs from 19 patients by examining all 10 levels. The first two hematoxylin and eosin- or the first cytokeratin-stained levels were positive for metastases in 21 (95.5%) of the 22 positive SLNs. Two additional hematoxylin and eosin-stained and one cytokeratin-stained levels of each SLN correctly identified the status of the node in 94 (97.9%) of 96 patients. Therefore, we recommend that after an initial hematoxylin and eosin-stained section, two additional hematoxylin and eosin-stained sections and one cytokeratin-stained section should be evaluated.

Adult↗

PubMed2000

Large-scale identification of differentially expressed genes during neurogenesis.

We report here a modified mRNA differential display method and its application for the analysis of differential gene expression in NGF-treated PC12 cells and in embryonic rat spinal cord. The optimized protocol is based on low fidelity priming of multiple cDNAs followed by high fidelity amplification. In PC12 cells induction by nerve growth factor (NGF) altered the expression of 4% of the 466 transcripts evaluated. During neurogenesis of the spinal cord we found that 30% of the 288 examined products changed. The differential expression of the characterized genes was confirmed by independent quantitative PCR. We conclude this method is suitable for the identification of increases and decreases of mRNA levels and allows the discovery of differentially expressed unknown transcripts.

Animals↗

PubMedDate not supplied

Peptide vaccination for patients with melanoma and other types of cancer based on pre-existing peptide-specific ctotoxic T-lymphocyte precursors in the periphery.

Identification of antigenic peptides expressed on cancer cells enables us to treat cancer patients with peptide-based immunotherapy. Although optimal protocols for peptide-based vaccines have not yet been elucidated, boosting the immune system could be a better approach than priming the immune system to elicit prompt and potent peptide-specific T-cell responses in cancer patients. With this possibility in mind, the authors undertook a clinical trial in which cancer patients were vaccinated with peptides (maximum 4) after confirmation of pre-existing peptide-specific cytotoxic T-lymphocyte (CTL) precursors in the periphery. Fourteen patients (seven with melanoma and seven with other types of cancer) positive for either HLA-A24 or HLA-A2 were enrolled in this study. Fourteen and 16 peptides were used to screen for HLA-A24+ and HLA-A2+ patients, respectively. The vaccination was well tolerated, and the only adverse effects were local pain and fever. Kinetic analysis revealed that peptide-reactive CTLs increased after peptide vaccination in 7 of 14 patients. Immunoglobulin G (IgG) reactive to the administered peptides was detected in 2 patients before vaccination, although it became detectable in 8 of the other 12 patients after the peptide vaccination. Stable disease for more than 6 months was observed in five patients (one with melanoma and four with other types of cancer); all of these patients showed increased levels of peptide-specific IgG. These results indicate that peptide vaccination of patients showing evidence of pre-existing peptide-specific CTL precursors can be applied in further clinical trials aimed at the treatment of melanoma and other types of cancer.

Adult↗

PubMed2000

Timing of the hepatic arterial phase during contrast-enhanced computed tomography of the liver: assessment of normal values in 25 volunteers.

OBJECTIVES: To define normal values of the beginning and duration of the hepatic arterial phase (HAP) during contrast-enhanced computed tomography (CT). METHODS: Twenty-five volunteers (16 men, 9 women; mean age, 60.0 years) without history or suspicion of liver disease were examined with dynamic single-section CT. Scanning was performed at a single level that included the liver, aorta, and portal vein. A series of 25 scans was obtained over a period of 88 seconds (1 baseline scan followed by 16 scans every 2 seconds and 8 scans every 7 seconds) beginning with the injection of a bolus of contrast agent (40 mL, 10 mL/s) and a 40-mL NaCl bolus chaser. Contrast enhancement in the liver, aorta, and portal vein was measured with regions of interest, and time-density curves were obtained. These data were processed with a pharmacodynamic fitting program and the duration of the HAP was calculated. The onsets of the HAP and the portal venous phase were assessed as lag times, referring to the beginning of enhancement in the abdominal aorta. RESULTS: The mean lag time of the HAP was 5.4 seconds after the aorta and the mean duration was 8.6 seconds. The mean lag time of the portal venous phase was 13.9 seconds after the aorta. CONCLUSIONS: These data can be used to optimize protocols for routine CT. Because of the short duration of the HAP, imaging of the entire liver during this phase is possible only with multidetector CT scanners.

Adult↗