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Results for “T cell selection”

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At least 217 records · Page 12Linked to original sources
PubMed1986

Markedly altered membrane transport and intracellular binding of vincristine in multidrug-resistant Chinese hamster cells selected for resistance to vinca alkaloids.

Studies of a multidrug-resistant variant (DC-3F/VCRd-5L) of Chinese hamster lung cells selected for resistance to vinca alkaloids revealed marked alterations in transport and intracellular binding of [3H]vincristine compared to parental DC-3F cells. Influx of [3H]vincristine in DC-3F cells appears to be an equilibrating, but mediated, process. Although saturation kinetics for [3H]vincristine influx were not demonstrated, an extremely high temperature-dependence (Q10 27-37 degrees C = 5-6) and trans-inhibition of influx following preloading of cells with nonradioactive vincristine argue in favor of a carrier-mediated process. Efflux of [3H]vincristine from parental cells conformed to first-order kinetics (t1/2 37 degrees = 3.6 +/- 0.4) and exhibited a lower temperature-dependence (Q10 27-37 degrees C = 3-3.5) than influx. In variant vs. parental cells, influx of [3H]vincristine was reduced 24-fold and efflux was increased two-fold, accounting for the large (approximately 48-fold) reduction in steady-state level of exchangeable drug accumulating in variant cells. Otherwise, transport of [3H]vincristine in these cells showed characteristics similar to parental DC-3F cells. Also, the rate and amount of intracellular binding of [3H]vincristine in variant cells was almost 40-fold lower than in parental cells. These alterations in influx and efflux of [3H]vincristine and its intracellular binding appear to account, at least to a major extent, for the high level of resistance (2,750-fold) of this variant to vinca alkaloids. In contrast, cross-resistance of this variant to daunomycin (178-fold) could be explained only minimally by a transport alteration. Only a two-fold increase in efflux of [3H]daunomycin was demonstrated in variant vs. parental cells along with some decrease in intracellular binding. Influx of [3H]daunomycin was unaltered. In view of these results, we conclude that these two agents most likely do not share the same route for entry in these cells but might share the same efflux route.

Animals↗
PubMed2004

Improved beta-cell survival and reduced insulitis in a type 1 diabetic rat model after treatment with a beta-cell-selective K(ATP) channel opener.

Treatment with ATP-sensitive K(+) channel openers (KCOs) leads to inhibition of insulin secretion and metabolic "rest" in beta-cells. It is hypothesized that in type 1 diabetes this may reduce beta-cell death resulting from metabolic stress as well as reduce the immunogenicity of the beta-cells during autoimmune beta-cell destruction. We have investigated whether the beta-cell-selective KCO compound, NN414, can be used to improve beta-cell survival in DR-BB rats rendered diabetic by modulation of their immune system. The rats were treated three times daily on days 1-19 with NN414, diazoxide, or vehicle. On day 21, an intravenous glucose tolerance test was conducted to assess beta-cell function. Postmortem histological analysis of rats' pancreata assessed the degree of insulitis and beta-cell volume. Among NN414-treated rats, 46% (16 of 35) were found to have a beta-cell mass similar to that of nondiabetic controls and significant glucose-stimulated C-peptide values, whereas only 11% (4 of 36) of vehicle-treated rats possessed a normal beta-cell mass and function (P < 0.002, by chi(2) test). Furthermore, responsive NN414-treated rats were almost free of insulitis. Thus, this study demonstrated that treatment with KCO compounds can indeed lead to preservation of beta-cell function and reduction of insulitis in a rat diabetes model.

Animals↗
PubMed1995

High-speed photodamage cell selection using a frequency-doubled argon ion laser.

A flow cytometer was developed for the high-speed "sorting" of desired cells by selectively irradiating (zapping) the undesired cells from a population. After previous efforts to photoinactivate cells with photosensitizers had failed, it was decided to exploit the photosensitivity of the cell's DNA at 257 nm. It was shown that a 257 nm laser output power of 20-100 mW was sufficient to induce a 4.5 log cell kill after the cells were processed through a focused 257 nm laser beam. Experiments proved that the photodamage flow cytometer (ZAPPER) could selectively photoinactivate cells at rates over 22,000 events/s, and selection purities ranged from 81% to 100%. The yields of the desired cells depended on the selection mode. In the Enrichment mode, the zap laser was not aimed at the jet, and only undesired cells were exposed to a brief ultraviolet (UV) pulse after modulation of the UV laser beam. The yields of desired cells ranged from 95% to 105%. In the Purge mode, the zap laser beam was aimed onto the jet, and only desired cells were allowed to pass after deflection of the UV laser beam; the yields of desired cells ranged from 12% to 52%. The cause of the reduced yields in the PURGE mode was traced to the fact that the Electro-Optic Modulator was used to modulate the zap laser proved too slow for the intended application. The lifetime of the frequency-doubling crystal used for the generation of the 257 nm beam was found to be limited to several days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Myeloma cells selected for resistance to CD95-mediated apoptosis are not cross-resistant to cytotoxic drugs: evidence for independent mechanisms of caspase activation.

We have previously shown that selection for resistance to the anthracenes, doxorubicin or mitoxantrone, results in coselection for resistance to CD95-mediated apoptosis (Landowski et al: Blood 89:1854, 1997). In the present study, we were interested in determining if the converse is also true; that is, does selection for CD95 resistance coselect for resistance to chemotherapeutic drugs. To address this question, we used two isogenic models of CD95-resistant versus CD95-sensitive cell lines: 8226/S myeloma cells selected for resistance to CD95-mediated apoptosis; and K562 cells expressing ectopic CD95. Repeated exposure of the CD95-sensitive human myeloma cell line, 8226/S, to agonistic anti-CD95 antibody resulted in a cell line devoid of CD95 receptor surface expression and completely resistant to CD95-mediated apoptosis. Multiple clonal populations derived from the CD95-resistant cell line showed no difference in sensitivity to doxorubicin, mitoxantrone, Ara-C, or etoposide, demonstrating that cross-resistance between Fas-mediated apoptosis and drug-induced apoptosis occurs only when cytotoxic drugs are used as the selecting agent. Using the inverse approach, we transfected the CD95-negative cell line, K562, with a CD95 expression vector. Clones expressing variable levels of cell-surface CD95 were isolated by limiting dilution, and analyzed for sensitivity to CD95-mediated apoptosis and response to chemotherapeutic drugs. We show that CD95 surface expression confers sensitivity to CD95-mediated apoptosis; however, it does not alter response to chemotherapeutic drugs. Similarly, doxorubicin-induced activation of caspases 3 and 8 was identical in the CD95-sensitive and CD95-resistant cell lines in both isogenic cell systems. In addition, prior treatment with the CD95 receptor-blocking antibody, ZB4, inhibited CD95-activated apoptosis in 8226/S cells, but had no effect on doxorubicin cytotoxicity. These results show that CD95 and chemotherapeutic drugs use common apoptotic effectors, but the point of convergence in these two pathways is downstream of CD95 receptor/ligand interaction.

Anthracenes↗
PubMed1997

Optimization of CD34+ cell selection using immunomagnetic beads: implications for use in cryopreserved peripheral blood stem cell collections.

Isolation of CD34+ cells from bone marrow, umbilical cord blood, and mobilized peripheral blood stem cell (PBSC) collections has many potential clinical benefits. The aim of this study was to evaluate the use of the ISOLEX 300 system to select hematopoietic precursors and determine the effectiveness at depleting contaminating tumor cells from cryopreserved/thawed PBSC. Median recovery of CD34+ cells and CFU-GM colonies was 71% and 51.5%, respectively, using a protocol optimized for our laboratory. A mean 2.9 log10 decrease in contaminating breast carcinoma cells was seen after the selection process. Selected CD34+ cells underwent a second round of cryopreservation/thawing while retaining 85.6% viability and 72.3% recovery of CFU-GM colonies.

Antibodies, Monoclonal↗
PubMed1990

Immunomagnetic purging of bone marrow: a model for negative cell selection.

Many in vitro techniques have been developed for removing cancer cells from the marrow of patients who are to undergo autologous bone marrow transplantation (ABMT). These purging techniques can be classified as immunological or pharmacological. The immunomagnetic technique has been widely used in neuroblastoma patients. It depends on an interaction between target neuroblastoma cells in the marrow and a complex of specific monoclonal antibodies and magnetized microspheres, the target cells being selectively removed by passage through a magnetic field. Laboratory studies with neuroblastoma and acute lymphoblastic leukemia cells have shown the high efficiency of this technique in selectively removing cancer cells while retaining adequate numbers of normal hematopoietic cells for subsequent reinfusion into the patient. Clinical studies in several hundred neuroblastoma patients, as well as small numbers of acute lymphoblastic leukemia, breast cancer, and myeloma patients, suggest that this is a clinically safe and effective technique. However, no clinical trial has been conducted comparing ABMT with and without in vitro marrow purging. Until such time, we will regard immunomagnetic purging as "standard of care" for neuroblastoma patients receiving ABMT.

Bone Marrow↗
PubMedDate not supplied

Transplantation of autologous peripheral blood progenitor cells: impact of CD34-cell selection on immunological reconstitution.

Peripheral blood progenitor cells (PBPC) represent an ideal source of stem cells for autologous transplantation because of technical advantages and more favourable engraftment kinetics. The reconstituion of a functional immune system occurs earlier in patients transplanted with cytokine-mobilized autologous PBPC compared with bone marrow; because of the greater T-cell content in PBPC products, donor-derived antigen-specific T-cells transferred with the graft might contribute to short-term immunity in transplant recipients. Despite a prompt reconstitution of B- and T-cell numbers, both B- and T-cell function are profoundly impaired for a prolonged period of time after PBPC infusion. The positive selection of CD34+ cells might provide effective tumor cell purging without compromising hematopoietic recovery in patients with acute leukemia, multiple myeloma, breast cancer and non-Hodgkin's lymphoma, whose autografts have been reported to contain malignant cells which might promote disease relapse. However, the incidence of viral infections in the early posttransplant period might be increased after CD34-selected compared with unmanipulated PBPC transplants, as a result of the lack of accessory and immune cells in the graft. The purpose of this review is to provide an update on immunological reconstitution after transplantation of autologous PBPC; in particular, emphasis will be placed on the mechanisms of immune dysfunction after the infusion of unmanipulated and CD34-selected autografts.

Antigens, CD34↗
PubMed1992

Collateral sensitivity to nitrosoureas in multidrug-resistant cells selected with verapamil.

We have examined the effects of the nitrosoureas, streptozotocin (STZ) and 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), on a human multiple myeloma cell line, RPMI 8226, and its drug-resistant variants. Cell lines selected for doxorubicin (DOX) resistance alone displayed a STZ and BCNU cytotoxicity profile similar to that of the parent cell line. In contrast, two of the drug-resistant variants selected with DOX plus verapamil, an agent which inhibits P-glycoprotein-mediated multidrug resistance, displayed a collateral sensitivity to STZ and BCNU. Verapamil was included in the selection protocol because it has been shown to inhibit the P-glycoprotein-mediated multidrug resistance phenotype and is now in clinical trials as a chemosensitizing agent. The collateral sensitivity to these nitrosoureas seen in the DOX plus verapamil-selected cell lines is due to the functional loss of a DNA repair molecule, O6-Methylguanine DNA methyltransferase (MGMT). The functional loss of MGMT is secondary to the loss of MGMT gene expression. The loss of MGMT gene expression is not due to loss or gross rearrangement of the MGMT-coding region. If this selection pressure applied in vitro reflects the in vivo situation, then new chemotherapeutic strategies may be devised to exploit this phenomenon. These cell lines will serve as useful models for delineating mechanisms which govern MGMT expression.

Base Sequence↗
PubMed1994

[T cell selection and tolerance induction in the thymus].

T cells play a major role in the immune system. While developing in the thymus, useful T cells are selected for expansion and harmful ones are deleted or inactivated. During this process, it is the T cells themselves which are doing the learning as opposed to the thymus doing the teaching. Both clonal deletion and clonal anergy are mechanisms of tolerance, the former being thymic, and thus central, while the latter is peripheral. Following a signal from the T cell receptor, anergy or deletion is triggered, if there are no co-stimulatory factors. Here, the outcome is determined by binding intensity. However, if the signal from the T cell receptor is accompanied by the required additional co-stimulatory signals, then the T cell is rescued from apoptosis and proliferates.

Animals↗
PubMed1984

Immunospecific vesicle targeting facilitates fusion with selected cell populations.

Antibody-directed targeting of vesicles to cells dramatically enhances polyethylene glycol-mediated fusion and microinjection. Sealed erythrocyte ghosts or liposomes, containing fluorescent bovine serum albumin, were targeted to murine spleen and thymus cells, and to lymphocyte and monocyte cell lines. In all cases, targeted cell populations showed substantial levels of microinjection, whereas populations treated with the fusogen in the absence of targeting were not significantly microinjected. Attachment of vesicles to selected cells was achieved by first labelling the cells with biotin-modified antibody and then treating them with avidin-coupled sealed ghosts or liposomes. Another approach to the promotion of selective fusion aims to alter the cell recognition properties of Sendai virus so that its fusogenic activity may be redirected to specific cellular targets. The agglutination and fusion of red cells by UV-inactivated Sendai virus were completely blocked by low concentrations of a Fab preparation of a monoclonal antibody against the viral haemagglutinin (HN) sites. Agglutination and fusion activity were restored in the presence of Fab-anti-HN by providing an alternative recognition system, namely, when the virus had been coupled with biotin and the red cells with avidin. Methods for facilitating microinjection by specifically directing vesicles to target cells may be particularly useful in overcoming barriers to the transfer of genes into lymphocytes by standard transfection techniques.

Animals↗
PubMed1994

L1210 cells selected for resistance to methoxymorpholinyl doxorubicin appear specifically resistant to this class of morpholinyl derivatives.

We investigated the mechanism of resistance in murine L1210 leukaemia cells selected after treatment with FCE 23762 methoxymorpholinyl doxorubicin: (MMRDX), a methoxymorpholinyl derivative of doxorubicin active in vitro and in vivo on multidrug-resistant (mdr) cells, currently undergoing phase I clinical trials. The resistant subline obtained after repeated in vitro treatments, L1210/MMRDX, is resistant in vitro and in vivo to all tested methoxymorpholinyl derivatives and to cyanomorpholinyl doxorubicin, but shows resistance to morpholinyl derivatives only in vivo or following their activation with rat S9-liver fractions in vitro. L1210/MMRDX cells are sensitive to classic mdr- and altered topoisomerase (AT)-mdr-associated drugs. These cells do not appear to overexpress the mdr1 gene, nor do they exhibit impaired intracellular drug accumulation and efflux or altered levels of glutathione and glutathione S-transferase. The extent of DNA single-strand break formation and, after microsomal activation, of DNA interstrand cross-links after treatment with MMRDX was similar in the parent and the resistant subline. The mechanism of resistance in L1210/MMRDX cells remains to be identified but may prove a novel one, highly specific for this class of mdr-active anthracyclines.

Animals↗
PubMed1979

Resistance to bromodeoxyuridine mutagenesis and toxicity in mammalian cells selected for resistance to hydroxyurea.

Mutant cell lines resistant to hydroxyurea (HU), an inhibitor of the enzyme ribonucleotide reductase, were selected from a line of Syrian hamster melanoma cells. Mutant lines were selected for resistance to 0.3 mM HU, and from these lines, second-step mutants were selected for resistance to 1.9 mM HU. The HUr lines were tested ffor their responses to 5-bromodeoxyuridine (brdU), in terms of toxicity, mutagenesis, and incorporation of BrdU into DNA. All of the HUr lines showed increased resistance to the toxic effects of BrdU. in addition, the HUr lines all were resistant to BrdU mutagenesis. Overall, there was good correlation among the levels of resistance to HU toxicity, BrdU toxicity, and BrdU mutagenesis in the HUr lines. These tests were carried out under conditions such that the parental and HUr cells incorporated equal amounts of BrdU into nuclear DNA. Therefore, the resistance of the HUr cells to the effects of BrdU cannot be attributed to decreased incorporation of BrdU into DNA. These results suggest that the HUr cells have an lateration in ribonucleotide reductase activity that simultaneously confers resistance to HU and BrdU. The properties of the HUr cells suggest that the perturbation of deoxcytidine metabolism by BrdU. The properties of the HUr cells suggest that the perturbation of deoxcytidine metabolism by BrdU triphosphate inhibition of ribonucleotide reductase activity plays a key role in the toxic and mutagenic effects of BrdU in mammalian cells.

Animals↗
PubMed1987

Local cortical lesions abolish lateral inhibition at direction selective cells in cat visual cortex.

Many cells in the cat visual cortex display a strong selectivity for the direction of motion of an optimally oriented stimulus. Postsynaptic inhibition has been suggested to generate this direction selectivity in simple cells, but the intracortical pathways involved have not been identified. While continuously recording from simple cells in layers 4 and 6, we have inactivated the superficial cortical layers in small regions 0.4-2.5 mm from the cortical column under study by using heat lesions, localized cooling or gamma-aminobutyric acid (GABA) microiontophoresis. When inactivation affected cortical regions retinotopically representing motion in the non-preferred direction towards the receptive field, the responses to movement in this direction increased, and the recorded cells lost direction selectivity due to loss of inhibition. Our results indicate that direction selectivity of simple cells involves asymmetric inhibition of predictable cortical topography.

Animals↗
PubMed2001

Cell-selective intracellular delivery of a foreign enzyme to endothelium in vivo using vascular immunotargeting.

Vascular immunotargeting, the administration of drugs conjugated with antibodies to endothelial surface antigens, has the potential for drug delivery to the endothelium. Our previous cell culture studies showed that biotinylated antibodies to PECAM-1 (a highly expressed endothelial surface antigen) coupled with streptavidin (SA, a cross-linking protein that facilitates anti-PECAM internalization and targeting) may provide a carrier for the intracellular delivery of therapeutic enzymes. This paper describes the PECAM-directed vascular immunotargeting of a reporter enzyme (beta-galactosidase, beta-Gal) in intact animals. Intravenous injection of [125I]SA-beta-Gal conjugated with either anti-PECAM or IgG led to a high 125I uptake in liver and spleen, yet beta-Gal activity in these organs rapidly declined to the background levels, suggesting rapid degradation of the conjugates. In contrast, anti-PECAM/[125I]SA-beta-Gal, but not IgG/[125I]SA-beta-Gal, accumulated in the lungs (36.0+/-1.3 vs. 3.9+/-0.6% injected dose/g) and induced a marked elevation of beta-Gal activity in the lung tissue persisting for up to 8 h after injection (10-fold elevation 4 h postinjection). Using histochemical detection, the beta-Gal activity in the lungs was detected in the endothelial cells of capillaries and large vessels. The anti-PECAM carrier also provided 125I uptake and beta-Gal activity in the renal glomeruli. Predominant intracellular localization of anti-PECAM/SA-beta-Gal was documented in the PECAM-expressing cells in culture by confocal microscopy and in the pulmonary endothelium by electron microscopy. Therefore, vascular immunotargeting is a feasible strategy for cell-selective, intracellular delivery of an active foreign enzyme to endothelial cells in vivo, and thus may be potentially useful for the treatment of acute pulmonary or vascular diseases.

Animals↗
PubMed2000

Human papillomavirus type 16-immortalized endocervical cells selected for resistance to cisplatin are malignantly transformed and have a multidrug resistance phenotype.

Cis-diamminedichloroplatinum (II) (cisplatin, CDDP) is a highly effective chemotherapeutic agent against cervical cancer, but drug resistance is a major obstacle in its clinical application. The mechanism of drug resistance in human cervical cancer is not well understood. Here, we established an in vitro endocervical, cisplatin-resistant cell system that mimics the development of cisplatin resistance in the human cervix. Human papillomavirus (HPV) type 16-immortalized human endocervical cells (HEN-16-2) were treated with cisplatin, and the cisplatin-selected cells (HEN-16-2/CDDP) were resistant to cisplatin, paclitaxel, actinomycin D, doxorubicin, etoposide, and 5-fluorouracil, thus demonstrating a multidrug resistance (MDR) phenotype. Furthermore, compared with a similar passage of drug-sensitive HEN-16-2 cells, HEN-16-2/CDDP cells exhibited the general growth characteristics of cancer cell lines: faster growth in medium containing serum and high calcium levels, higher saturation density, anchorage-independent growth, and formation of tumors in nude mice. These results provided the first in vitro evidence that cisplatin selection can transform HPV-immortalized endocervical cells and cause a phenotype of MDR.

Animals↗
PubMedDate not supplied

Influence of antigen processing on thymic T-cell selection.

The design of a specific blocking peptide for the immunosuppressive therapy of an autoimmune disease requires the identification of peptides of an autoantigen that are physiologically processed in vivo and bind to MHC-encoded membrane glycoproteins. However, knowledge of how an antigen is physiologically processed by antigen-presenting cells (APC) in vivo, particularly in the thymus, is lacking. It is also unknown whether the processing of an antigen by different APC in the thymus can influence thymic T-cell selection. This is an important consideration for attempts to delete or inactivate autoreactive T cells that elicit autoimmune disease. To address these issues, we investigated the processing of biosynthetically labelled recombinant human insulin (rHI), a model autoantigen, injected into mice and characterized the insulin peptides associated with MHC class II molecules on thymic epithelial cells and dendritic cells. These APC were found to differ in the way they process insulin. The detection of MHC-class-II-bound insulin peptides on the surface of the epithelial cells but not the dendritic cells correlated with their capacity to either present or not present insulin to T cells, respectively. Thus, antigen processing may control the appearance of different peptide-MHC class II complexes on thymic APC that mediate positive and negative selection, and thereby influence the development of the T-cell repertoire. Our findings could have important bearing on the future design of synthetic blocking peptides that reduce or eliminate the onset of autoimmune disease.

Animals↗
PubMed1977

Selective cell loss in the lateral geniculate nucleus of adult cats following binocular lid suture.

Two weeks of binocular lid suture in adult cats caused selective cell loss in lamina A1 of the lateral geniculate nucleus. The loss was manifested as a shift in the latency distribution of LGN cell responses to optic chiasm shock. Proportionately fewer LGN cells with slowly conducting retinal afferents (X-cell range) were in evidence following binocular suture. This change was observed only in lamina A1 while laminae A and C retained normal latency distributions. These observations confirm and extend recent reports of the susceptibility of adult cat visual system to environmental modification.

Animals↗