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At least 253 records · Page 14Linked to original sources
PubMed1980

In vitro generation of cytotoxic lymphocytes against radiation- and radiation leukemia virus-induced tumors. III. Suppression of anti-tumor immunity in vitro by lymphocytes of mice undergoing radiation leukemia virus-induced leukemogenesis.

Adult C57BL/6 mice exposed to fractionated irradiation or inoculated with the radiation leukemia virus (RadLV), develop high incidence (80-100%) of lymphatic leukemias within 3-6 mo. RadLV-induced lymphomas can elicit cytotoxic responses in vitro in lymphocytes of preimmunized syngeneic mice, a reaction that is dependent on the expression of membrane-associated viral antigenicity. As soon as 5 d after RadLV inoculation, and during the entire leukemogenic process, suppressor T cells are detectable in the spleen that are capable of specifically abrogating generation of syngeneic anti-tumor cytotoxic cells in vitro. Mice exposed to fractionated x irradiation do not develop suppressor cells and their splenocytes may be stimulated in vitro to generate cytotoxicity toward RadLV-induced leukemias. These findings suggest that although RadLV has been isolated from radiation-induced leukemias, x-ray- and RadLV-induced leukemogenesis do not seem to involve a common viral etiology, and that induction of suppressor cells during RadLV leukemogenesis may be essential for tumor progression.

Animals↗
PubMed2001

In vitro models, in vivo models, and pharmacokinetics: what can we learn from in vitro models?

In vitro pharmacokinetic models of infection can make an important contribution to the study of the pharmacodynamic properties of an antibacterial agent. In conjunction with animal and human pharmacodynamic evaluations, they provide data to allow for the optimization of drug dosing regimens. In vitro models can be used simply to describe the effect of a drug on a bacterial population as well as to provide data for more-analytical studies, including hypothesis testing. Analytical study designs provide information on the pharmacodynamic parameter best related to the chosen outcome, as well as its magnitude. Factors such as the characteristics of the model (method of drug removal, inoculum density, and growth phase), doses simulated, species and susceptibility range of bacteria, and methods and analytical tools used to measure antibacterial effect will have an effect on the conclusions drawn. In vitro models have an important future role in ensuring antibiotic efficacy and in reducing the risks of resistance.

Animals↗
PubMed2004

In vitro selection of restriction endonucleases by in vitro compartmentalization.

Restriction endonucleases are widely used in laboratory applications from recombinant DNA technology to diagnostics, but engineering of restriction enzymes by structure-guided design and in vivo directed evolution is at an early stage. Here, we report the use of an in vitro compartmentalization system for completely in vitro selection of restriction enzymes. Compartmentalization of a single gene in a rabbit reticulocyte in vitro transcription/translation system serves to isolate individually synthesized enzymes from each other. In each compartment, an active enzyme cleaves only its own encoding gene, whereas genes encoding inactive enzymes remain intact. Affinity selection of the cleaved DNA encoding active restriction endonucleases was accomplished by the use of streptavidin-immobilized beads and dUTP-biotin, which was efficiently incorporated into the cohesive end of the cleaved DNA using a DNA polymerase. We confirmed that genes encoding active restriction endonuclease FokI could be selected from a randomized library. This method overcomes the limitations of current in vivo technologies and should prove useful for rapid screening and evolution of novel restriction enzymes from diverse mutant libraries, as well as for studies of catalytic and evolutionary mechanisms of restriction enzymes.

Amino Acid Sequence↗
PubMed2006

Particokinetics in vitro: dosimetry considerations for in vitro nanoparticle toxicity assessments.

The rapid growth in the use of in vitro methods for nanoparticle toxicity assessment has proceeded with limited consideration of the unique kinetics of these materials in solution. Particles in general and nanoparticles specifically, diffuse, settle, and agglomerate in cell culture media as a function of systemic and particle properties: media density and viscosity and particle size, shape, charge and density, for example. Cellular dose then is also a function of these factors as they determine the rate of transport of nanoparticles to cells in culture. Here we develop and apply the principles of dosimetry in vitro and outline an approach for simulation of nanoparticle particokinetics in cell culture systems. We illustrate that where equal mass concentrations (mug/ml) imply equal doses for dissimilar materials, the corresponding particle number or surface area concentration doses differ by orders of magnitude. More importantly, when rates of diffusional and gravitational particle delivery are accounted for, trends and magnitude of the cellular dose as a function of particle size and density differ significantly from those implied by "concentration" doses. For example, 15-nm silver nanoparticles appear approximately 4000 times more potent than micron-sized cadmium oxide particles on a cm(2)/ml media basis, but are only approximately 50 times more potent when differences in delivery to adherent cells are considered. We conclude that simple surrogates of dose can cause significant misinterpretation of response and uptake data for nanoparticles in vitro. Incorporating particokinetics and principles of dosimetry would significantly improve the basis for nanoparticle toxicity assessment, increasing the predictive power and scalability of such assays.

Cells, Cultured↗
PubMed1988

Drug-induced in vitro tolerance to allogeneic antigens. II. Further analysis of in vitro-tolerized spleen cells in a fully allogeneic murine combination.

C3H/HeSlc (C3H, H-2k) spleen cells were made tolerant in vitro to C57BL/6CrSlc (B6, H-2b) at the cell-mediated cytotoxicity (CMC) level by in vitro stimulation for 48 hr with mitomycin C (MMC)-treated B6 spleen cells, and treatment with 5 micrograms/ml of 5-fluorouracil for a further 9 hr. These cells were given intraperitoneally to neonate (C3HxB6) F1 mice to examine whether these tolerized spleen cells would cause lethal graft-versus-host disease (GVHD). Despite the lack of CMC, the tolerized C3H spleen cells caused lethal GVHD in most of the neonate F1 mice. Evaluating from various immune parameters, it was evident that T cell populations responsible for IL-2 production, cytostasis, and delayed footpad reaction (DFR) were retained intact after in vitro tolerance induction, probably because of their less-proliferative characteristics in response to fully allogeneic antigen stimulation, and were considered to be responsible for lethal GVHD. Contribution of natural killer (NK) cells to lethal GVHD was not ruled out.

Animals↗
PubMed1989

Effect of a single dose of beta,beta'-iminodipropionitrile in vivo on the properties of neurofilaments in vitro: comparison with the effect of iminodipropionitrile added directly to neurofilaments in vitro.

The biochemical properties of neurofilaments isolated from control and iminodipropionitrile-treated rats were compared with regard to autophosphorylation capacity, hydrolysis of ATP, and the formation of a viscous gel between filaments. Both preparations exhibited a similar polypeptide composition, and no covalent cross-linking between neurofilament subunits was induced by iminodipropionitrile in vivo. An ATPase activity, systematically present in all preparations, was unaffected by the administration of iminodipropionitrile to the rats. Conversely, the autophosphorylation of neurofilament subunits in vitro was significantly higher in preparations from iminodipropionitrile-treated rats than from control animals, with a marked increase of the phosphorylation of a high molecular weight neurofilament-associated protein. Iminodipropionitrile provoked a higher gelation capacity of neurofilaments as measured in vitro, with a lower critical concentration for the preparation from treated animals. A similar increased interaction was obtained with millimolar concentrations of iminodipropionitrile added to bovine neurofilaments in vitro, involving likely neurofilament-associated molecules, because the effect of the drug was lost after their extraction by 0.8 M KCl. These results support the hypothesis that iminodipropionitrile interferes with the neurofilament networks through a preferential interaction with the neurofilament-associated proteins, resulting in a change in their properties and consequently in an increased capacity of interaction between the polymers.

Adenosine Triphosphatases↗
PubMed1993

In vitro assay for the Bacillus subtilis signal peptidase SipS: systems for efficient in vitro transcription-translation and processing of precursors of secreted proteins.

The signal peptidase (SPase) SipS of Bacillus subtilis is responsible for the processing of precursors of secreted proteins. It differs from the SPases of Gram-negative bacteria in structure and specificity. To assay the activity of SipS in vitro, two efficient transcription-translation systems for the synthesis of radio-labelled precursors were developed. The systems were completely derived from B. subtilis. Post-translational in vitro processing of pre-staphylokinase by SipS was demonstrated. SipS activity was stimulated in vitro by several non-ionic detergents, whereas it was not affected by a large variety of proteinase inhibitors. SipS shares the latter property with other SPases.

Bacillus subtilis↗
PubMed1997

The in vitro adhesion of Candida albicans to buccal epithelial cells (BEC) from diabetic and non-diabetic individuals after in vivo and in vitro application of nystatin.

Buccal epithelial cells (BEC) from 12 patients with diabetes mellitus and 12 age- and sex-matched non-diabetic subjects were tested in vitro for adhesion of Candida albicans following exposure to nystatin both in vitro and in vivo. Adhesion was significantly reduced (P < 0.002) to cells from both the diabetic and non-diabetic subjects after in vitro exposure to nystatin, but the reduction in adhesion was variable (5.0-50.7% in control subjects and 0.5-48.4% in diabetic subjects) and equivalent between the two groups. In vivo exposure to nystatin produced no overall significant reduction in candidal adhesion to cells from either diabetic or control subjects.

Adult↗
PubMed2002

An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution.

The "in vitro virus" is a molecular construct to perform evolutionary protein engineering. The "virion (=viral particle)" (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this "viral genome" was demonstrated.

Journal Article↗
PubMed2004

In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes.

To develop a reliable follicle culture system, mouse preantral follicles 150-200 microm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro.

Animals↗
PubMed1980

In vivo or in vitro modulating effects of vincristine on the generation of allogeneic cytotoxic lymphocytes in vitro.

Cytotoxic lymphocytes carrying the H-2(i5) haplotype were generated in vitro against alloantigens specified by the IC-S-G-D regions of H-2. Responder lymphocytes were collected from donors B10.A(5R) non-treated or treated with 3 or 0.6 mg/Kg, ip of Vincristinee (VCR) and incubated with irradiated B10 spleen cells. High doses of VCR produced depression of the cytotoxic response (CR), whereas 0.6 mg/Kg of the drug produced substantial increase of the response. Further experiments were conducted adding VCR in vitro to the tissue culture during the generation of cytotoxic lymphocytes. Depression of CR occurred following treatment with high concentrations of VCR. On the other hand, marked increase of CR was found using responder lymphocytes treated in vitro with low concentrations (i.e. 0.016 - 0.0032 microgram/ml) of the drug. The mechanism of VCR-induced enhancement of CR has not been elucidated. However, the present data show the possibility of increasing host immune responses by treatment of donor mice or of responder lymphocytes with VCR at selected doses or concentrations.

Animals↗
PubMed1999

Possible mechanism for in vitro complement activation in blood and plasma samples: futhan/EDTA controls in vitro complement activation.

BACKGROUND: Ongoing in vitro complement (C) activation in citrate or EDTA plasma has prevented an accurate analysis of C-activation products generated in vivo. The aim of this study was to characterize handling and storage conditions required to prevent in vitro C activation in blood and plasma samples collected with Futhan/EDTA. METHODS: Biotrak(TM) RIAs were used to quantitatively measure C3a and C4a in blood and/or plasma samples from healthy individuals (controls) and from liver transplant patients. Blood samples were routinely drawn into either EDTA (1 g/L) tubes or into tubes containing both EDTA (1 g/L) and Futhan (0.1 g/L) and immediately centrifuged at 2000g for 15 min at 4 degrees C. RESULTS: In controls, C4a, but not C3a, in fresh samples (time 0) was higher in EDTA plasma than in Futhan/EDTA plasma (n = 20; P = 0.002). Futhan/EDTA prevented C3a and C4a generation in blood and plasma samples held at room temperature (22-23 degrees C) for 1 h and in plasma held for 24 h at 4 degrees C or -70 degrees C. The mean C3a concentration (1.76 mg/L; n = 19) at time 0 in EDTA plasma samples from liver transplant patients was significantly higher than for controls (0.34 mg/L; n = 11). In these patients, the mean C3a in EDTA samples increased to 13.8 mg/L after 60 min at room temperature, but there was no change in the C3a concentration of an EDTA plasma from a control. In the patients, C3a concentrations were lower in Futhan/EDTA plasma than in EDTA at time 0 and after 60 min at room temperature (1.40 and 2.02 mg/L, respectively). The mean patient C4a was 4.02 mg/L in EDTA plasma at time 0 vs 0.24 mg/L for controls; it increased to 16.9 mg/L after 60 min at room temperature compared with 0.76 mg/L for controls. The mean patient C4a was 0.83 mg/L in Futhan/EDTA plasma at time 0 vs 0.1 mg/L for controls. Neither patient nor control C4a concentrations increased vs time in Futhan/EDTA. CONCLUSION: The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.

Adult↗
PubMed1999

In vitro and in vivo evaluation of a new polysulfone membrane for hemodialysis. Reference methodology and clinical results. (Part 1: in vitro study).

Different high flux membranes have been recently developed. The present study is aimed at describing the technical features and the clinical performances of a new high flux polysulfone membrane (T-sulfone, Toray, Japan). The study has been carried out on two different dialyzers (surface area = 1.3 and 1.8 m2). The filters have been tested in vitro under definite experimental conditions. The hydraulic flow resistance, the pressure drop in the blood compartment and the hydraulic permeability have been determined in a wide range of in vitro experimental conditions. The in vitro sieving coefficients for various solutes have also been determined utilizing human blood. Hydraulic permeability was found in the range of 28.4 ml/h/mm Hg/m2 and sieving coefficients were between 0.96 and 1.0 for all low molecular weight solutes. The sieving coefficient for inulin was 0.95. The pressure drop in the filter at 300 ml/min of blood flow was 95 mm Hg for the 1.3 m2 and 57 mm Hg for the 1.8 m2. The filters are then designed to operate in the presence of high blood flows without excessive resistance in the blood compartment. The blood compartment analyzed by means of a special radiological sequence obtained with a helical scanner after dye injection confirmed the homogeneous distribution of the blood flow in several cross sections of the bundle. Adequate distribution of dialysate was confirmed with a similar method applied to the dialysate compartment. The new imaging techniques utilized were greatly helpful to determine adequacy of filter design and flows distribution.

Biocompatible Materials↗
PubMedDate not supplied

In vitro histoculture of human tumors with fluorescent dye end-points measured by confocal microscopy: high correlation of in vitro and in vivo chemosensitivity.

Tumors from 40 patients and 7 established human xenograft tumor lines were grown in three-dimensional histoculture. A Viable-Cell-Index (VCI) based on fluorescent dyes and Growth Fraction Index (GFI) based on [3H]thymidine incorporation were measured by confocal microscopy and histological autoradiography, respectively, after treatment with cytotoxic agents. Chemosensitivity in vitro with the two methods was correlated with chemosensitivity of the same set of human xenografted tumor lines grown in nude mice. The percent accuracy of in vitro to in vivo correlation with VCI (73%) was higher than GFI (63%). The number of false positives with VCI was 12.1% (4/33), and with GFI was 31.3% (10/32). The results thus indicated that in vitro histoculture with fluorescent vital-dye end-points to measure cell viability is of potential use to determine tumor chemosensitivity.

Animals↗
PubMed1991

Lack of in vitro and in vitro effects of fenbendazole on phase I and phase II biotransformation enzymes in rats, mice and chickens.

Intraperitoneal administration of 10 mg fenbendazole/kg bw daily for 5 d caused no significant alterations in the activities of hepatic microsomal drug-metabolizing enzymes viz aminopyrine N-demethylase, aniline hydroxylase and cytosolic glutathione S-transferase in rats, mice and chickens. Similarly no significant difference in the amount of microsomal cytochrome P-450 and NADPH-cytochrome c reductase was found between control and treated animals. In vitro incubation of fenbendazole with rat, mouse and chicken microsomes suggests that the drug neither binds to microsomal protein cytochrome P-450 nor inhibits the activities of aminopyrine N-demethylase and aniline hydroxylase. Similarly in vitro addition of fenbendazole to cytosolic glutathione S-transferase from the above species did not alter the activity of this enzyme. The results indicate that fenbendazole does not alter the activity of hepatic microsomal monooxygenase system significantly in rats, mice and chickens at a dosage level of 10 mg/kg body weight. In vitro studies also indicate that fenbendazole does not interact with the hepatic microsomal monooxygenase system, indicating it is not a substrate for cytochrome P-450-dependent monooxygenase system.

Aminopyrine N-Demethylase↗
PubMed1991

Effect of hyperthermia on both primary proliferation and self-renewal of human leukemic progenitor cells in vitro: its application to in vitro purging.

Self-renewal, as defined by the capacity to yield new colonies following replating, is an important function of leukemic progenitors (L-CFU) to originate self-maintaining clones. In this work, we studied the effect of hyperthermia (41-44 degrees C) on the growth of human L-CFU derived primary colonies and of secondary colonies formed by replating to evaluate the purging effect of human L-CFU by heat. The survival curves clearly demonstrated much greater hyperthermic sensitivity of L-CFU compared to normal granulocyte-macrophage progenitors (CFU-GM) at all temperatures (41-44 degrees C) studied. At 42 degrees C or higher, L-CFU decreased (by more than 2 log reduction) dramatically and therefore were unable to form colonies in vitro. At 42 degrees C and 43 degrees C, 65 and 30%, respectively, of CFU-GM obtained from remission and normal marrows were left after 1 h exposure. At 44 degrees C, however, CFU-GM derived colonies disappeared after a 2 h exposure. In the four available patients with acute myelogenous leukemia, secondary colonies formed by replating were decreased in proportion to the decreasing primary colonies during heating (42 and 43 degrees C). However, their self-renewal capacity was retained in vitro until the primary colonies disappeared. These observations suggest that heat exposure at 43 degrees C for 1 h could be the most effective conditions for in vitro purging of human L-CFU because of the wide difference between surviving fractions of CFU-GM and L-CFU.

Bone Marrow Transplantation↗
PubMed1980

Vitamin A receptor in cancers and in vitro-transformed epithelial cells of the rat urinary bladder: its implication in the preventive effect of vitamin A on in vitro keratinization.

Cellular retinol-binding protein (CRBP) in cancers and in vitro-transformed epithelial cells of the ACI/N rat urinary bladder was assayed with radiolabeled retinol by sucrose density gradient centrifugation. CRBP was detected in abundance in tumor tissues of all 5 transplantation bladder cancer lines. However, this protein was below the limits of detection in bladder cancer cells of culture lines that had originated from the same primary cancers as the corresponding transplantation lines. CRBP was detected in small quantity in in vitro-transformed epithelial cells of a culture line but it was absent in another line of similar origin. The addition of retinol to the culture medium prevented the in vitro keratinization of CRBP-positive cells but it had no effect on the keratinization of CRBP-negative cells.

Animals↗
PubMed1995

Azidothymidine and interferon-alpha in vitro effects on hematopoiesis: protective in vitro activity of IL-1 and GM-CSF.

Preclinical and clinical studies with an azidothymidine (AZT)/interferon-alpha (IFN-alpha) combination resulted in a marked and synergistic antiretroviral activity. The administration of the two drugs in HIV-seropositive patients affected with Kaposi's sarcoma, however, induced neutropenia, thrombocytopenia, and, in some cases, anemia. A possible means to improve the therapeutic index of AZT and/or IFN-alpha in AIDS patients could be the addition of hematopoietic growth factors. In vitro activity of cytokines on the hematotoxicity of the AZT-IFN-alpha association has not yet been studied. We have performed an in vitro study to evaluate the toxicity of AZT, IFN-alpha, or both on peripheral blood hematopoietic progenitors (CFU-GM and BFU-E) and to assess the activity of interleukin 1 (IL-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), or both in modifying AZT-IFN-alpha hematotoxicity. Results indicate that AZT, IFN-alpha, and combinations of the two have a dose-dependent inhibitory effect on the in vitro growth of peripheral blood hematopoietic progenitors. Combinations of AZT and IFN-alpha inhibited CFU-GM and BFU-E proliferation in an additive manner. Neither IL-1 nor GM-CSF alone was able to induce a significant reduction of AZT-induced damage. Only the addition to the cultures of both cytokines partially curbed the antiproliferative activity of AZT at low dosages.

Antimetabolites↗