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At least 631 records · Page 35Linked to original sources
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↗
PubMed1994

Optimal method for RNA extraction from mouse glomeruli.

Extraction of RNA has been described for rat and rabbit glomeruli but not for mouse glomeruli. Due to their small size, mouse glomeruli cannot be isolated by relatively simple sieving techniques. Based on recently reported methods for the isolation of mouse glomeruli, we developed an RNA isolation technique by performing comparative methodological studies. Two standard RNA extraction methods were compared. In addition in separate experiments the influence was studied of protease inhibitors and freezing and thawing of whole kidney prior to glomeruli isolation, on the yield and degradation of RNA. Therefore kidneys were perfused with 10 ml 0.01 M PBS containing 1.25% Fe3O4 through the aorta. Kidneys were decapsulated and passed through a 75-microns metal screen. After pelletting and washing, tubes were placed against a magnet and pelleted glomeruli were washed three times. In a second experiment protease inhibitors were added to the PBS. As a third method, kidneys were frozen before the isolation of glomeruli. From isolated glomeruli RNA was extracted using either caesium chloride or lithium chloride method. The yields of RNA (OD 260) were highest using the lithium chloride method. Hybridization of Northern blots of extracted RNA with cDNA probes showed the best results when RNA was extracted using the lithium chloride method, while the caesium chloride method led to considerable degradation of RNA. Freezing of kidney tissue prior to RNA extraction led to the virtual absence of any signal. We then applied this method successfully in an in-vivo model of experimental lupus nephritis. This is the first description of an optimal protocol for the extraction of RNA from mouse glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Recovery of DNA, RNA and protein from gels with microconcentrators.

The use of a new product, Microcon/Micropure (a centrifugal ultrafiltration device combined with a microporus insert), for the purification of DNA, RNA, peptides and proteins from gels is described. Using this system, DNA can be recovered from agarose gel in concentrated, contamination-free form in only 15 min. Results of studies on the effects of fragment size and various pretreatment of the gel slice on DNA recovery are presented. The Microcan/Micropure combination can also be used for the recovery of macromolecules from polyacrylamide gels. Optimized protocols for the recovery of RNA, oligonucleotides and proteins from polyacrylamide gels using a crush and elute method, along with a study of critical parameters, are presented.

Centrifugation↗
PubMed1995

Reproducible amplification of RAPD markers from vertebrate DNA.

Randomly amplified polymorphic DNA (RAPD) is a modification of PCR that uses short, randomly generated primers to amplify genomic DNA. Generally, many bands of mixed intensity (i.e., strong, faint, fuzzy or sharp) are generated with each primer. Mixed-intensity bands are inherent with the RAPD technique because (i) the target DNAs are undefined, (ii) one or more copies of the target DNA may exist per genome and (iii) the percentage of hybridization of primer to target may vary. The problem of mixed-intensity band exacerbates the well-known sensitivity of PCRs to reagent and template concentrations, pH and other reaction parameters. These complications have discouraged many investigators from using RAPD. Our goal was to optimize the RAPD amplification conditions for vertebrate DNA. We present the optimized protocol along with an experimental strategy for obtaining reproducible, interpretable RAPD banding patterns in vertebrates.

Animals↗
PubMed1994

Heparin administration and monitoring for neuroangiography.

PURPOSE: To establish the optimal protocol of heparin administration during interventional neuroradiology. METHODS: We assessed 100 cases of neuroangiography, including endovascular surgery, and measured activated coagulation time before and 5 minutes after heparin administration, and before and 5 minutes after protamine neutralization. In some cases actual heparin concentration was assayed using a chromogenic substrate technique. RESULTS: The actual plasma heparin concentration significantly correlated with the dose of heparin administered intravenously (r = .98; P < .0001) and changes in activated coagulation time (r = .85; P < .0001). The change in activated coagulation time significantly correlated with the dose of heparin injected intravenously (r = .54, P < .0001). The ratio of change in activated coagulation time significantly correlated with time elapsed after heparin administration (r = -.70, P < .0001). CONCLUSIONS: The activated coagulation time is useful in monitoring administration and neutralization of heparin during neuroangiography, and a bolus injection of 60 U/kg heparin should be adequate to carry out neuroangiography for 75 minutes safely, even for endovascular surgery.

Cerebral Angiography↗
PubMed1993

[Preventive treatment of superficial tumors of the bladder using intravesical BCG. Review].

Local immunotherapy with BCG was first described by Zbar in an experimental model. The clinical use of BCG in human bladder carcinoma was proposed by Morales in 1976. Since then randomised studies have confirmed the value of BCG against recurrence and progression in superficial transitional cell carcinoma of the bladder (Lamm, Herr). However the optimal protocol has not been defined and the mode of action of BCG remains obscure. In this paper, the authors review the clinical results of BCG and its complications then focus on recent immunological data concerning its mode of action. Finally a practical protocol is proposed.

Administration, Intravesical↗
PubMed1995

[Three-dimensional helical CT angiography of the abdomen].

To evaluate the quality of three-dimensional (3D) images of the abdominal vasculature acquired using enhanced helical CT, 3D reconstructions were performed for 43 examinations (38 patients). Twenty-one of 43 examinations were also reconstructed by Maximum Intensity Projection (MIP). The CT scanner employed was the Toshiba Xforce. Helical CT data were acquired using up to 20 continuous 1.5-sec rotations with an X-ray beam width of 5 mm and a couchtop movement speed of 5 to 10 mm/1.5 sec. Axial images were reconstructed at a section interval of 2 mm. Optimal protocol on enhanced helical CT was as follows: Iopamidol 300 mg I/ml was administered intravenously using a biphasic technique (3-4 ml/sec for the initial 100 ml, followed by 0.7-1.5 ml for the remaining 50 ml), and delay times of the early and late phases were 25-35 and 90 sec, respectively. Aortic branches were clearly demonstrated on early phase, while portal branches were well defined on late phase. In the visualization of abdominal vessels, 3D images were nearly equal to MIP images. However, for anteroposterior images, MIP images were superior to 3D images in quality, because 3D images had some longitudinal direction artifacts. Three-dimensional images were considered to be useful for correctly evaluating overlapping abdominal vasculatures. From the above results, 3D and MIP images of the abdominal vasculature obtained using enhanced helical CT were considered to compensate for each other.

Abdomen↗
PubMed1995

[Therapeutic strategy in breast cancer].

The authors present the actual concepts of the therapeutic strategy for the breast cancer. The choice of the optimal protocol treatment is based on a complete and correct pretherapeutic evaluation. This implies the staging using TNM/ UICC/ 1987 system (explained in the text) and the definition of the prognostic factors: axillary lymph node involvement, other pathological patterns, the situation of the hormonal receptors and the cell proliferation index. For the stages I-II the strategy of the treatment include: modified radical mastectomy, postoperative irradiation in well defined cases and the adjuvant systemic treatment using chemotherapy and hormonal therapy. The laparoscopic ovariectomy is a safe and simple technique. For the local advanced cancer (IIIA and IIIB) the treatment begins with a systemic aggressive approach, the surgery being applied following the tumoral regression. In the stage IV the complex palliative treatment is indicated.

Breast Neoplasms↗
PubMed1996

The present status of hyperthermia in Japan.

The research on hyperthermia in Japan was started by the Hyperthermia Study Group in 1978. Six years later, in 1984, the Japanese Society of Hyperthermic Oncology (JSHO) was established. More and more research has been conducted since then. At present, 215 units of heating equipment are installed for use. Among these, 24% are microwave heating equipment, and 66% are radiofrequency (RF) capacitive heating equipment. A nation-wide survey has revealed that about 60% of hyperthermia therapy involves the treatment of deep-seated tumours by RF capacitive heating and RF intracavitary heating. The treatment of superficial tumours by microwave heating represents another 12.5%. Most of the clinical application in the United States and in Europe is microwave heating of superficial tumours. The different modalities of treatment were thermoradiotherapy in 57.5%, thermochemotherapy in 22.6% and thermochemoradiotherapy in 14.5% of the cases surveyed. Compared to other countries, Japan has the highest number of hyperthermia equipment installed, and the most doctors involved in hyperthermia therapy. The main reasons for the advanced state of hyperthermia research in Japan include the development of excellent heating equipment, high membership in JSHO, grant-in-aid by the Japanese government, and coverage by insurance for this form of therapy. Based on 33 papers selected from two books which the author had edited, the optimal protocol, effectiveness and indication for the use of hyperthermia has been established.

Combined Modality Therapy↗
PubMed1996

Anti-CD4 monoclonal antibody therapy.

Because they can be selected to target only cells which are crucial for rejection, monoclonal antibodies (mAbs) offer enormous potential for specific manipulation of the immune response. Interest in the clinical potential of anti-CD4 mAbs has been heightened by the demonstration, in experimental models, that such therapy can produce long-term donor-specific non-responsiveness. Early clinical trials using two murine anti-CD4 mAbs (BL4 and mT-151) were discouraging, with over 50% of recipients suffering early rejection episodes. Another murine preparation, OKT4A, was initially found to prolong allograft survival in non-human primates. Limited clinical trials revealed that this mAb was well tolerated, that most recipients produced an antimurine response, and that only 26% of patients suffered rejection episodes during the first 3 post-operative months. Another murine preparation, Max. 16H5, has been reported to reverse late onset acute rejection episodes as effectively as, but more safely than, conventional immunosuppression. More recent interest has focused upon humanized recombinants of these earlier murine anti-CD4 preparations. cMT-412, has been studied in recipients of heart or heart-lung allografts. These patients were observed to have less frequent and markedly delayed rejection episodes, fewer infectious complications, and better overall survival than that observed in an ATG-treated control group. Further studies are thus being undertaken. A CDR grafted IgG4 preparation of OKT4A has also been studied. This molecule (OKTcdr4a) contains only 8% of the parent murine sequence while retaining the binding affinity of OKT4A for the human CD4 antigen. In a pilot trial, biopsy-proven reversible rejection episodes were observed in 2/11 (18%) of renal allograft recipients. There were no allograft failures and no antibody response to the mAb. These and other trials emphasize the intense interest in immunosuppressive regimens incorporating anti-CD4 mAbs as well as the difficulties encountered in defining optimal protocols. Nevertheless, the impressive results observed in rodent and non-human primate models suggest that these agents are likely to play an important role in future immunosuppressive protocols, particularly those designed to induce tolerance.

Animals↗
PubMed1996

Evaluation of chromogenic substrates for measurement of protease production by biocontrol strains of Trichoderma.

Four chromogenic substrates were compared, and methods were developed for measuring protease activity from fungi. Digestion of azoalbumin, a water-soluble substrate, resulted in dye release most closely proportional to enzyme activity. Substrates insoluble in water were advantageous for time-course studies, and azocoll was more sensitive to digestion and easier to handle than hide powder azure. The optimal pH was 7 for measurement of extracellular protease activity from the Trichoderma strains. Addition of calcium or serine protease inhibitors did not affect crude protease activity. The optimized protocol was used to demonstrate that specific activity of proteases produced by the strains of Trichoderma tested did not correlate with their known biocontrol ability.

Albumins↗
PubMed1998

Tumor pretargeting for radioimmunodetection and radioimmunotherapy.

UNLABELLED: The limited success of the sole use of monoclonal antibodies for cancer detection and treatment has led to the development of multistep methods using antibodies in conjunction with low molecular weight agents. For tumor pretargeting, it is important to optimize dose and schedule of relevant agents and to understand barriers to targeted delivery. Here, we address these issues for the anti-carcinoembryonic antigen bifunctional antibody-hapten and the streptavidinylated antibody-biotin systems using a recently developed physiologically based pharmacokinetic model. METHODS: For baseline conditions of a standard 70-kg man with a 20-g tumor embedded in the liver, the model was used in conjunction with the Medical Internal Radiation Dosimetry schema to: estimate absorbed doses in tumor and normal tissues; determine the dose dependence of effector agent accumulation in tumor; simulate tumor-to-background effector agent uptake ratio; and calculate the therapeutic ratio for different antibody forms and radionuclides. Alternative drug administration schemes and variable tumor physiological conditions were considered. RESULTS: Model simulations showed that 131I-labeled biotin with the streptavidinylated F(ab')2 provided the highest therapeutic ratio under the optimized conditions. The simulations also showed that biotin with the bifunctional streptavidinylated immunoglobulin G provided the highest tumor-to-liver uptake ratio during the early period. Sensitivity analysis showed that antibody extravasation was the major factor limiting the accretion of the effector agent in tumor, whereas antigen expression in normal tissues and tumor antigen shedding had little effect on the absorbed doses. CONCLUSION: Tumor pretargeting should provide a definite advantage over direct antibody targeting with up to a 200% increase in tumor-to-background ratio in radioimmunodetection and up to a 76% increase in tumor-to-bone marrow therapeutic ratio in radioimmunotherapy. Rapid antibody clearance from the bloodstream before effector agent injection is expected to improve the therapeutic ratio marginally (3%-10%). However, continuous plasmapheresis dramatically increased the tumor-to-background ratio by a factor of 10 in RAID and the tumor-to-bone marrow therapeutic ratio by more than 110% for short-lived radionuclides in RAIT. Apart from drastic measures such as extended plasmapheresis, pretargeting selectivity was neither sensitive enough for radioimmunodetection nor effective enough for radioimmunotherapy in patients with typical solid tumors even using the optimized protocols.

Animals↗
PubMed1998

Rapid and reliable assessment of volume percentage of epithelium in borderline and invasive ovarian tumors.

OBJECTIVE: To analyze factors determining intraobserver and interobserver reproducibility of stereology in borderline and invasive ovarian tumors. STUDY DESIGN: Fast and simple assessment of VPE was possible by using a highly automated interactive video overlay system suitable for application in a routine pathology laboratory. The point distance of a Weibel grid and the number of fields of vision per area of interest required to obtain good reproducibility were investigated. In addition, intraobserver and interobserver reproducibility was assessed, and the results of the improved technique were compared to those of the classical method. RESULTS: The experiments showed that intraobserver and interobserver variations in volume percentage of epithelium (VPE) assessments in a given case were caused mainly by high field-to-field variation and not so much by differences in the precision of assessment in a single field of vision. Therefore, many fields but only few points per field need to be measured to obtain, in a short time, a precise estimate of VPE in the measurement area of a tumor. From these results, an optimized protocol for VPE assessment was constructed. Using this protocol, nine observers independently assessed VPE in seven cases. Counting only one point in each of 100 systematically randomly sampled fields of vision (corresponding to a point distance of +/- 560 microns) yielded high intraobserver reproducibility (coefficient of variation [CV], 4%; R, .99; range, 0.98-1.00) and interobserver reproducibility (CV, 6%; R, .98; range, 0.97-1.00) in a short time. Assessment of one case took approximately three minutes, and the observers experienced the work as pleasant. CONCLUSION: VPE assessed with systematic random sampling, using a grid with one point per field of vision and counting 100 hits, yields an inexpensive, fast and highly reproducible measure of an important prognostic variable in ovarian tumors. This assessment can be performed easily in a routine pathology laboratory.

Epithelial Cells↗
PubMed1998

Low-dose ultraviolet exposure early in development can lead to widespread melanoma in the opossum model.

Suckling young of opossums (Monodelphis domestica) were exposed to ultraviolet radiation (UVR, predominantly UVB: 290-320 nm) in part to determine an optimal protocol for induction and progression of melanoma in this species. In all, 620 litters were introduced to one of seven protocols. The lowest dose (175 J/m2) administered three times a week for almost three weeks led to the highest incidence of melanotic lesions with melanoma potential (8.1%) among young (5-month-old) adults. Among 101 much older animals (> 17 months at necropsy), 43% showed metastatic melanoma to the lymph nodes and almost one-third of these had progressed to widespread dissemination. Three of the latter animals, from a total of 13 obtained so far, were selected for detailed histological examination of disseminated disease. At necropsy, all three showed widespread metastases beyond the lymph nodes to the spleen, lungs, and other distant sites. Histological changes typical of malignant melanoma included junctional activity, mitotic figures, and nerve and vessel invasion. This novel finding leads us to conclude that UVR can act as a complete carcinogen for progression to widely disseminated disease and that exposure of sucklings can lead, in old age, to widespread metastatic melanoma in this model. The results are thus not inconsistent with the view that, in humans, early exposure to sunlight might act as an initiating factor in a later progression to malignant melanoma.

Age Factors↗
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↗
PubMed2001

Tips and step-by-step protocol for the optimization of important factors affecting cellular enzyme-linked immunosorbent assay (CELISA).

CELISA, or cellular enzyme-linked immunosorbent assay, is a powerful and easy to use technique to study cell surface antigens under different stimulations. Nevertheless, some factors must be discussed and optimized prior to reaching a reproducible CELISA. These include the choice of cell density, fixative agent, blocking agent, culture medium, optimal antibody dilutions, and incubation time. In this paper, we first present a short review of some references devoted to CELISA by means of a comparison of these parameters, followed by their description. Then, we describe and study these different parameters using practical examples comparing TNF-induced ICAM-1 expression as an end point, on HBL melanoma and HUVEC. These cell lines were also chosen because they differ in their ability to grow as discontinuous and continuous layers, respectively. Furthermore, we designed a comprehensive flow chart, as well as a complete step-by-step protocol for CELISA optimization.

Antigens, Surface↗
PubMed2003

[Dosage regimen optimization in cancer chemotherapy using a mathematical model].

In cancer chemotherapy, it is important to design treatment strategies that ensure a desired rate of tumor cell kill without unacceptable toxicity. To optimize treatment, we used a mathematical model describing the pharmacokinetics of anticancer drugs, antitumor efficacy, and drug toxicity. This model was associated with constraints on the allowed plasma concentrations, drug exposure, and leukopenia. Given a schedule of drug administrations, the mathematical model optimized the drug doses that could minimize the tumor burden while limiting toxicity on the white blood cells. Simulation suggests that the optimal drug administration is an initial high dose chemotherapy up to saturation of constraints associated with normal cell toxicity followed by a maintenance continuous infusion at a moderate rate. Data related to etoposide investigations were next used in a feasibility study. Simulations made with the usual clinical protocols and optimized protocols revealed that model-based optimal drug doses lead to greater cytoreduction. Also, examples showed how to use this new approach for the dose ranging problem and they evaluated the sensitivity of the optimized protocols with respect to the clinical constraints.

Antineoplastic Agents↗
PubMed1984

A sequential protocol for the optimization of diagnostic procedures in hepatology.

The aim of this paper is to present a sequential protocol to be used in clinical practice for the detection of liver diseases, based on clinical history, physical examination, and laboratory investigation. The evaluation of the protocol efficacy was carried out on a sample of 288 subjects (92 normal, and 196 affected by various liver alterations) by comparing the obtained results with reference classifications independently performed on the basis of all available subject records (including many more laboratory tests than the ones used in the protocol). A cost-benefit analysis was also carried out, based on the resident population of Regione Piemonte, comparing the protocol with two other simpler ones. Results show that the proposed protocol allows a considerable reduction of costs, showing a high estimated efficacy for clinical use.

Adolescent↗