Increased 3H-thymidine Uptake Research Articles

Farnesylthiosalicylic acid induces caspase activation and apoptosis in glioblastoma cells

Primary glioblastomas (GBMs) commonly overexpress the oncogene epidermal growth factor receptor (EGFR), which leads to increased Ras activity. FTA, a novel Ras inhibitor, produced both time- and dose-dependent caspase-mediated apoptosis in GBM cell lines. EGFR-mediated increase in 3H-thymidine uptake was inhibited by FTA. FACS analysis was performed to determine the percent of apoptotic cells. The sub-Go population of GBM cells was increased from 4.5 to 13.8% (control) to over 45-53.6% in FTA-treated cells within 24 h. Furthermore, FTA also increased the activities of both caspase-3 and -9, and PARP cleavage. Treatment of GBMs with FTA before or after EGF addition to the cultures blocked phosphorylation of Akt and mitogen-activated protein kinases (MAPK). FTA also significantly reduced the amount of EGF-induced Ras-GTP as reflected by a decrease in the level of Ras bound to Raf-RBD-GST. This study demonstrates that inhibition of Ras methylation may provide a therapeutic target for the treatment of GBMs overexpressing EGFR.

4-Hydroxynonenal mediates cyclosporine promoted EBV-induced B cell transformation

Introduction. We had previously shown that cyclosporine A (CsA) directly promoted Epstein--Barr virus (EBV) infected human B cell (EBV-B cell) immortalization via oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end product of lipid peroxidation. We hypothesized that HNE mediates a direct oxidative stress promoting the effect of CsA on EBV-B cell transformation. Methods. Human B cells were purified from human spleen and incubated with EBV (B95-8) overnight for infection. To assay HNE production, EBV-B cells were treated with 0, 500, and 1000 ng/ml of CsA or with HNE (positive control) or DMSO (0.1%, vehicle control) for 10 min at 37°C. HNE--Protein adducts in the cell extract was assayed by slot-blot using polyclonal HNE antibody. For 3H-thymidine uptake and LMP1 assay, EBV-B cells were treated with HNE (1 μM) for 0, 0.5, 1, and 4 h at 37°C and cultured for 2 and 4 weeks. Cells were then assayed for expression of EBV oncogene LMP1 by using PE-conjugated anti-LMP1 antibody in flow cytometry and for 3H-thymidine uptake. Results. CsA at 500 and 1000 ng/ml significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units ± SE) of 251.3 ± 7.5 to 361.3 ± 9.7 and 342.7 ± 10.7, respectively ( P < 0.05, n = 3). EBV-B cells treated with physiological concentration of HNE (1 μM) for 0.5 h and 1 h and cultured for 2 weeks had significantly increased 3H-thymidine incorporation (cpm ± SE) over a control of 839.2 ± 54 to 1192 ± 41.6 and 1998 ± 24.8, respectively ( P < 0.02, n = 6). EBV-B cells treated with HNE (1 μM) for 0.5 and 1 h and cultured for 4 weeks also had significantly increased 3H-thymidine uptake over a control of 1035 ± 206 to 2022 ± 293 and 1941 ± 229, respectively ( P < 0.03, n = 6). HNE (1 μM) treated for 4 h did not have a significant effect on cell proliferation at 2 or 4 weeks. EBV-B cells treated with HNE (1 μM) for 1 h and subsequently cultured for 2 and 4 weeks had a significantly higher (>2.0 times ) LMP1 positive cell population over the control. Conclusion. This observation provides evidence for further understanding of CsA-induced oxidative stress and direct promotion of EBV related post-transplant lymphoproliferative disorder (PTLD).

Molecular mechanisms of endothelin-1-induced cell-cycle progression: involvement of extracellular signal-regulated kinase, protein kinase C, and phosphatidylinositol 3-kinase at distinct points.

Although it is well established that endothelin-1 (ET-1) has not only vasoconstrictive effects but also mitogenic effects, which seem to be implicated in vascular remodeling, little is known about the molecular mechanisms by which ET-1 induces cell-cycle progression. In this study, we examined the effects of ET-1 on the cell-cycle regulatory machinery, including cyclins, cyclin-dependent kinase (cdk), and cdk inhibitors in NIH3T3 cells. ET-1 increased cyclin D1 protein (5.1+/-1.9-fold increase, 8 hours after stimulation, P<0.05), cdk4 kinase activity (2.8+/-0. 5-fold increase, 12 hours after stimulation, P<0.01), and cdk2 kinase activity (2.1+/-0.4-fold increase, 16 hours after stimulation, P<0.05) in a time- and dose-dependent manner. ET-1-induced increase in cyclin D1 protein, and cdk4 kinase activity was not significantly inhibited by an inhibitor of the mitogen-activated protein kinase kinase 1/2, PD98059, nor by the protein kinase C inhibitor calphostin C, whereas ET-1-induced upregulation of cyclin D1 protein and cdk4 kinase activity was significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. In contrast, ET-1-induced activation of cdk2 kinase was significantly inhibited by PD98059, calphostin C, and LY294002. ET-1 increased 3H-thymidine uptake in a time-dependent fashion (0 hours, 4216+/-264 cpm per well; 8 hours, 5025+/-197 cpm per well; 16 hours, 9239+/-79 cpm per well, P<0.001 versus 0 hours). ET-1-induced increase in 3H-thymidine uptake was significantly inhibited by PD98059, calphostin C, and LY294002. These results suggest that ET-1-induced cell-cycle progression is, at least in part, mediated by the extracellular signal-regulated kinase, protein kinase C, and phosphatidylinositol 3-kinase and that those pathways may be involved in the progression of the cell cycle at distinct points.

The Fischer rat thyroid cell line FRTL-5 exhibits a nondiploid karyotype.

The FRTL-5 cell line is a stable thyroid cell line derived from the thyroid gland of the Fischer rat under defined culture conditions, which has been widely adopted as a model system for the study of thyroid cell function and for bioassay. While characterizing by flow cytometry FRTL-5 cells that were supplied to this laboratory by ATCC (American Type Cell Collection), we discovered that the cells (ATCC CRL8305) were not diploid, having approximately twofold the DNA content relative to a diploid control. The increase in DNA content also applied to cells originally supplied by the ATCC (described as passage 14) that when counted in metaphase had a modal chromosomal count of 84, indicating tetraploid status, double the expected 42 of a diploid rat cell. In a private communication, the ATCC confirmed these findings which nevertheless are contrary to previous literature reports where they were reported to be diploid. Tetraploid cells are less sensitive to thyrotropin (TSH) as measured by cyclic adenosine monophosphate (cAMP) production, compared with diploid cells (p = < 0.001). Despite similar 3H-thymidine uptake in 0.2% fetal calf serum, tetraploid cells show increased 3H-thymidine uptake in 5% fetal calf serum in the absence of TSH (p = 0.001). The origin of these chromosomal changes is unclear, but these findings must raise doubts regarding the suitability of the tetraploid FRTL-5 cell line as a model for studies of human or animal thyroid physiology.

Effect of glycated proteins on the matrix of glomerular epithelial cells.

In this study, cultured glomerular epithelial cells (GEC) were exposed to a diabetic milieu containing glycated proteins to determine whether such proteins cause metabolic alterations that may lead to defects seen in the extracellular matrix in diabetic nephropathy. Cultured glomerular epithelial cells were cloned and maintained in RPMI media containing 10% fetal bovine serum (FBS). The medium was changed to RPMI-1% glycated FBS (experimental) or RPMI-1% control FBS, and cells were incubated for 1 or 4 d. Mitogenicity was tested by 3H-thymidine uptake. The media were collected and analyzed for collagenase activity by a quantitative fluorescence assay and by zymography. The cell layers were processed for matrix antigen (collagen I, glomerular basement membrane antigens, laminin, and fibronectin) and for the proteins of the tight junction (cadherin, desmosomal protein) by quantitative immunoperoxidase and immunofluorescence. Cell lysates were tested for cadherin and desmosomal protein by immunoblotting. Cells were also grown on 0.2-microM filter membranes to test for permeability to 3H-inulin and 125I-albumin. Glycated FBS resulted in a 1.8-fold increase in 3H-thymidine uptake in subconfluent layers accompanied by an increase in cell number. The treatment caused accumulation of laminin (18% above control, P < 0.05) and basement membrane antigens (33% above control, P < 0.05). Collagen I and fibronectin were unchanged. Exposing cells to glycated FBS changed the distribution of cadherin from a linear to a diffuse pattern associated with a decrease in cadherin observed on immunoblots. The media of glycated FBS-treated cells contained 45% lower collagenase activity (72-, 92-, and 150-kD species). Permeability to inulin increased by 550% and to albumin by 320% in glycated FBS-treated monolayers compared with controls. It is concluded that glycated proteins increased the accumulation of matrix proteins in the GEC, associated with a concomitant depression in collagenase activity. There were qualitative and quantitative changes in the tight junction protein cadherin. These matrix changes resulted in a functional defect in the permselective properties of the GEC tight junctions and manifested as increased leakage of inulin and albumin. Thus, the GEC are metabolically sensitive to the presence of glycated proteins, and this could play a role in the pathogenesis of diabetic nephropathy.

DNA synthesis, cell proliferation index in normal and abnormal gallbladder epithelium.

The observation of mitotic figures in the epithelium of the normal gallbladder is exceptional because cell renewal is occurring at a very slow rate. It is only after using 3H-thymidine and autoradiography to observe the cells in DNA synthesis that evidence of a significant epithelial cell replication has been provided. Because numerous mitotic figures and increased 3H-thymidine uptake have been observed after intraluminal introduction of foreign bodies or after ligation of the common bile duct in animals, mechanical distension has been supposed to represent an important trigger factor of cell proliferation in this hollow organ. An increased epithelial cell renewal was also observed in human gallbladders of patients with a complete obstruction of the common bile duct causing the distension. However, the absence of correlation between the degree of gallbladder distension and the proliferative response was suggesting that factors other than distension could be involved. In studies on experimental lithiasis cell proliferation appeared to be enhanced in the gallbladder epithelium of mice fed on a cholesterol-cholic acid-rich lithogenic diet. The fact that the increase in proliferative activity was preceding the formation of gallstones was another indication that factors other than mechanical stimulation by stretching or by the stones may stimulate cell renewal in this organ. Factors in the bile of animals receiving a lithogenic diet could be involved which might cause cellular death and, hence, a regenerative reaction. Direct mitogenic effect of an unknown factor in the bile of these animals is an alternative possibility. On the other hand the stimulating effect of postprandial hormones on gallbladder cell renewal suggested by the observation of a DNA synthesis peak after feeding has been established. Synthetic cholecystokinin analogues have been shown to increase the proliferative activity and to induce epithelial hyperplasia in this organ. In one recent study using fundusectomy to increase the serum gastrin levels, a significant proliferative stimulation in the gallbladder was also observed. In human gallbladder mitotic activity in gallbladders with gallstones in much higher than in the controls. No correlation between stone number, weight or volume and the proliferative activity was put in evidence, whereas cell renewal appeared to be more influenced by the composition of the stones than by their physical presence. Epithelial DNA synthesis activity was, namely, much higher in gallbladders with cholesterol stones than in those with pigment stones. Whether increased cell turnover and, hence, cellular shedding into the lumen could represent a nucleating factor for cholesterol stones is an attractive working hypothesis. Considering the very high frequency of gallstones in man and also the frequent association of gallbladder cancer and lithiasis, further studies on mitotic activity in this organ are required. In conclusion, data from animal experiments and in vitro studies on human gallbladders indicate that gallbladder epithelial cell proliferation may be influenced by several mechanical, chemical and hormonal factors. The list of these factors is still incomplete while their possible role in gallbladder disease is a fascinating exploration field for future research.

In situ assessment of cell viability within biodegradable polylactic acid polymer matrices

Efforts to expand treatment options for articular cartilage repair have increasingly focussed on the implantation of cell polymer constructs. Primary cells cultured from perichondrium, a chondrogenic tissue, were found to survive in vitro within a biodegradable porous polylactic acid matrix. The novel application of an in situ fluorescent double-stain protocol to cell polymer constructs was supported by increased 3H-thymidine uptake and the ability of cell seeded polylactic acid to form first passage explant cultures. This in situ viability staining technique allowed for rapid determination of cell viability and, in conjunction with confocal microscopy, assessment of cellular distribution within a biodegradable scaffold. Advantages of using this method over histological and electron microscopic analysis include in situ observation, absence of distortion in scaffold architecture due to polymer dissolution and disruption during processing, and obtaining a viability assessment within 30 min. Potential applications of this protocol as a screening tool for laboratory engineered tissues and in the evaluation of cellular injury in natural tissues are discussed.

Stimulation of proliferative activity of human natural killers (CD16+CD56+ cells) by recombinant interleukin-3in vitro

The proliferative activity of human natural killers (CD16+CD56+ cells) in the presence of 100 and 1000 IU/ml human recombinant interleukin-3 is investigatedin vitro. It is shown that recombinant interleukin-3 reliably enhances natural killer proliferation, causing a 9–15.2-fold increase of3H-thymidine uptake by CD16+CD56+ cells both in complete culture medium and in conditioned medium. The effect of the factor is 3.9–6.4 and 3.6–8.9-fold more potent than that of recombinant interleukin-2 and granulocyte-macrophage colony-stimulating factor, respectively, in the same doses.

Enhanced cellular response in mice treated with a Brucella antigen-liposome mixture.

The capacity of liposomes constituted by dycetyl-phosphate (0.009 mM), cholesterol (0.017 mM), lecithin (0.003 mM), and myristic (0.1 mM), stearic (0.1 mM), or oleic acid (0.1 mM) to modify the lymphocyte response to Brucella melitensis antigens in mice was studied. Mice treated with antigens mixed with liposomes containing myristic, stearic or oleic acid had higher antibody titres than mice given antigen suspended in a saline solution. Liposomes alone, without Brucella antigens, resulted in increased 3H-thymidine incorporation by lymphocytes both in vivo and in vitro. The addition of polyclonal activators (LPS and ConA) caused a further increase of 3H-thymidine uptake. Moreover, spleen lymphocytes from mice inoculated with Brucella antigens mixed with the liposomes had a significantly lower population of B lymphocytes (10%), and a notable increase in the Tc lymphocytes (20%). Autoradiography of sections of popliteal ganglia of treated mice showed that the radioactivity was concentrated mainly in the membrane structures of the cell.

Enhanced cellular response in mice treated with a Brucella antigen-liposome mixture

The capacity of liposomes constituted by dycetyl-phosphate (0.009 mM), cholesterol (0.017 nM), lecithin (0.003 mM), and myristic (0.1 mM), stearic (0.1 mM), or oleic acid (0.1 mM) to modify the lymphocyte response to Brucella melitensis antigens in mice was studied. Mice treated with antigens mixed with liposomes containing myristic, stearic or oleic acid had higher antibody titres than mice given antigen suspended in a saline solution. Liposomes alone, without Brucella antigens, resulted in increased 3H-thymidine incorporation by lymphocytes both in vivo and in vitro. The addition of polyclonal activators (LPS and ConA) caused a further increase of 3H-thymidine uptake. Moreover, spleen lymphocytes from mice inoculated with Brucella antigens mixed with the liposomes had a significantly lower population of B lymphocytes (10%), and a notable increase in the T c lymphocytes (20%). Autoradiography of sections of popliteal ganglia of treated mice showed that the radioactivity was concentrated mainly in the membrane structures of the cell.

Alpha‐interferon (α‐IFN) protects B‐chronic lymphocytic leukaemia cells from apoptotic cell death in vitro

B-chronic lymphocytic leukaemia cells (CLL) are prone to apoptotic cell death when cultured in vitro. Apoptosis and loss of the bcl-2 protein is prevented in CLL cells cultured in the presence of interleukin-4. In this study we analysed the effects of alpha-IFN on the DNA fragmentation, bcl-2 protein levels and cell survival in purified B-cells from 16 CLL patients. Alpha-IFN (10(3) U/ml) reduced the degree of spontaneous DNA fragmentation of CLL cells after a 30 h culture period (from a mean of 22.2% in control cultures to 10.5%, P < 0.01). This inhibition was accompanied by preservation of bcl-2 protein and an increased survival of CLL cells compared to control cultures. In parallel, alpha-IFN inhibited hydrocortisone induced DNA fragmentation in CLL cells. The effects of alpha-IFN on DNA synthesis of CLL cells were variable (in two patients a decrease and in seven an increase in 3H-thymidine uptake) and did not correlate with the effect on DNA fragmentation. In conclusion, our data suggest that alpha-IFN, like IL-4 and gamma-IFN, inhibits apoptosis of CLL cells. These in vitro data indicate that the clinical responses of some CLL patients to alpha-IFN cannot be explained by a direct cytotoxic effect of alpha-IFN on circulating CLL cells. Alternatively, alpha-IFN may inhibit the proliferation of the small fraction of clonogenic CLL progenitors, or interfere with cellular interactions necessary for the survival and growth of CLL cells.

The Influence of Acute Overdistension on Rat Bladder Function and DNA Synthesis

Prolonged micturition problems are often encountered after long-term bladder overdistension caused by urinary retention. In animal studies, damage to the bladder wall innervation has been found following overdistension. Experimentally, acute overdistension has also been implicated in the pathogenesis of the response to partial outlet obstruction. In the present study we investigated the influence of overdistension on micturition volume and frequency, on in vitro bladder function using the whole bladder model and on 3H-thymidine uptake, localization and DNA synthesis.Overdistension was induced for 3 hours by forced diuresis and balloon obstruction. Another group of rats was catheterized for 3 hours but received no diuretic, nor was the balloon inflated. An additional group of controls was neither anesthetized nor catheterized. Overdistension caused a gradual increase in bladder mass which was maximal at 7 days. During the first 24 hours following overdistension, the frequency of micturition decreased, but normalized thereafter. A progressive decrease in the response to field stimulation was noted between 16 hours and 7 days following overdistension and remained at this level until 21 days. There were, however, no significant differences in the responses to carbachol, ATP and KCl. There was a 30% reduction in the ability of field stimulation to empty the bladder 16 hours after overdistension, but no impairment of the emptying ability of carbachol. Overdistension was followed by a significant increase in 3H-thymidine uptake, which was maximal at 2 days. 3H-thymidine labelling increased rapidly after overdistension and was maximal within 16 hours in the urothelium. In smooth muscle, connective tissue and lamina propria, maximal labelling occurred at 2 days. Catheterization alone caused a mild distension which was associated with a small, but statistically significant, increase in 3H-thymidine incorporation into DNA within 16 hours. The labelling was located primarily in the urothelium.Overdistension causes a proliferative reaction within the bladder wall. Its initial effects occur within the urothelium, and the later involvement of the subendothelial smooth muscle and connective tissue is directly proportional to the degree of bladder distension. Three weeks following overdistension, the bladder’s functional state was not completely recovered, although the urinary bladder was found to have a good capacity to adapt and compensate for the stress-induced changes caused by overdistension. It is, therefore, clear that overdistension may have long-lasting effects on the bladder.

Fibroblasts induce heparin synthesis in chondroitin sulfate E containing human bone marrow-derived mast cells

Human bone marrow-derived mast cells (hBMMCs), differentiated in vitro in suspension culture and under the influence of human peripheral blood mononuclear cells conditioned medium (hCM), were tested for their response to recombinant human interleukin-3 (rhIL-3) and for their behavior in different microenvironments. The hBMMCs were incubated in the presence of rhIL-3 and the changes in their proliferation rate were determined. Recombinant hIL-3 induced a more than sixfold increase in 3H-thymidine uptake into the hBMMC DNA in a dose-dependent manner. Human CM used as a control for proliferation response induced a more than eightfold maximal proliferation rate increase. Rabbit anti-rhIL-3 completely inhibited hBMMC 3H-thymidine uptake induced by rhIL-3 and decreased the hCM-induced proliferation by approximately 50%. These hBMMCs were cocultured with four different mytomicin C-treated cell monolayers and assayed for phenotypic changes. After only 2 days in coculture with either embryonic mouse skin-derived fibroblasts (MESFs) or human skin-derived fibroblasts (HSFs), a marked increase in granule number and density was noted on staining with toluidine blue. Mast cells that initially stained alcian blue+/safranin- at day 0 of coculture became alcian blue+/safranin+ during the coculture period. Human BMMC proteoglycan synthesis shifted from approximately 85% chondroitin sulfate E to approximately 60% heparin within 14 to 19 days of coculture with the MESF monolayer and to approximately 50% heparin within 19 days of coculture with the HSF monolayer. None of the above- mentioned changes were noted in cocultures of hBMMCs with 3T3 cell line fibroblast monolayers or in cocultures with bovine vascular endothelium (BVE) cell monolayers. These results demonstrate microenvironmental effects exerted by the MESF and HSF monolayers on IL-3-dependent hBMMCs similar to those reported in the conversion of murine mast cell phenotype.

Fibroblasts Induce Heparin Synthesis in Chondroitin Sulfate E Containing Human Bone Marrow-Derived Mast Cells

Human bone marrow-derived mast cells (hBMMCs), differentiated in vitro in suspension culture and under the influence of human peripheral blood mononuclear cells conditioned medium (hCM), were tested for their response to recombinant human interleukin-3 (rhIL-3) and for their behavior in different microenvironments. The hBMMCs were incubated in the presence of rhIL-3 and the changes in their proliferation rate were determined. Recombinant hIL-3 induced a more than sixfold increase in 3H-thymidine uptake into the hBMMC DNA in a dose-dependent manner. Human CM used as a control for proliferation response induced a more than eightfold maximal proliferation rate increase. Rabbit anti-rhIL-3 completely inhibited hBMMC 3H-thymidine uptake induced by rhIL-3 and decreased the hCM-induced proliferation by approximately 50%. These hBMMCs were cocultured with four different mytomicin C-treated cell monolayers and assayed for phenotypic changes. After only 2 days in coculture with either embryonic mouse skin-derived fibroblasts (MESFs) or human skin-derived fibroblasts (HSFs), a marked increase in granule number and density was noted on staining with toluidine blue. Mast cells that initially stained alcian blue+/safranin- at day 0 of coculture became alcian blue+/safranin+ during the coculture period. Human BMMC proteoglycan synthesis shifted from approximately 85% chondroitin sulfate E to approximately 60% heparin within 14 to 19 days of coculture with the MESF monolayer and to approximately 50% heparin within 19 days of coculture with the HSF monolayer. None of the above-mentioned changes were noted in cocultures of hBMMCs with 3T3 cell line fibroblast monolayers or in cocultures with bovine vascular endothelium (BVE) cell monolayers. These results demonstrate microenvironmental effects exerted by the MESF and HSF monolayers on IL-3-dependent hBMMCs similar to those reported in the conversion of murine mast cell phenotype.

Immunopathological signs in mice treated with mercury compounds — I. Identification by the popliteal lymph node assay of responder and nonreponder strains

In the present study we screened mice from 22 different inbred strains for potential differences in their immunological reaction to HgCl 2. As a rapid screening test, we used the popliteal lymph node assay (PLNA). Mice were injected s.c. into one hind footpad with 3–60 μg of Hg 2+, given as HgCl 2 in phosphate-buffered saline (PBS); contralateral hindfeet remained uninjected. Control mice received PBS alone. On day 6 the weights of the draining and contralateral PLN were determined and the PLN index calculated. While we found linear dose-response curves in some strains, these curves had a different shape in others. Out of the total of 21 euthymic strains tested only strain DBA/2 (H-2 d) proved to be a nonresponder to HgCl 2; it remained a nonresponder over the whole dose range (3–180 μg Hg 2+) and period of time (days 2–12) studied. The other H-2 d strains tested, i.e. NZB, BALB/c and B10.D2/n, showed absent or low PLN responses only in the lower dose range (3–30 μg Hg 2+). F 1 hybrids of strain DBA/2 and the responder strain C57BL/6 gave an intermediate response. While C3H nu/nu mice failed to respond to HgCl 2, C3H + / nu mice did. The weight increase of the draining PLN after HgCl 2 injection was preceded in time by an increased 3H thymidine uptake by the PLN. Histologically, enlarged PLNs revealed increased cellularity in both the T-cell and the B-cell areas. When CH 3HgCl, instead of HgCl 2, was injected all three strains tested, including DBA/2, responded by PLN enlargement. We conclude that (1) HgCl 2 is an immunostimulatory agent in mice in that that it induces T-cell-dependent enlargement of the draining PLN upon local injection, (2) there are striking genetic differences between inbred strains of mice in the PLN response to HgCl 2, but these differences are not paralleled by similar differences in the response to CH 3HgCl, (3) responsiveness to HgCl 2 appears to be inherited in a codominant fashion, and (4) there is suggestive evidence that the observed genetic differences are determined by both H-2 and non-H-2 genes.

生体活性ガラスのリンパ球免疫機能に対する影響

Since the effects of bioactive glass on host defence mechanisms in the oral region are still unknown, we investigated the in vitro effects of bioactive glass on lymphocyte functions, particularly lipopolysaccharide (LPS)-induced responses.Spleen cells were cultured with mitogen (LPS, lipid A-associated protein or phytohemagglutinin-P) on the surface of bioactive glass and other experimental materials (Al2O3 ceramic, Hydroxyapatite, Silica glass) underlaid in a 96-well multiplate. The effects of materials on lymphocyte functions were evaluated by three parameters, i. e., mitogen induced enhancement of 3H-thymidine uptake, and LPS-induced increment of IgM producing cells and augmentation of polyclonal IgM production. Results were as follows;1. Three mitogens increased 3H-thymidine uptake by spleen cells with dose dependency. Bioactive glass reduced the mitogen-induced uptake of 3H-thymidine by spleen cells. Bioactive glass was found to be most effective in response to LPS among the three mitogens.2. Number of polyclonal IgM producing cells was increased depending upon the dose of LPS. Bioactive glass inhibited the LPS-induced increase of the number of polyclonal IgM producing cells.3. However, bioactive glass was found to enhance LPS-induced production of polyclonal IgM-antibody.These results suggest that bioactive glass, when closely cultured with lymphocytes, suppresses mitogen-induced proliferation of lymphocytes, but enhances LPS-induced production of polyclonal IgM-antibody.

Dose and time related effects of T-2 toxin on mitogenic response of murine splenic cells in vitro

The effects of T-2 toxin in vitro on murine splenic cells were investigated. Cells from syngeneic male NFS/N mice were cultured with three concentrations of either lipopolysaccharide (LPS), pokeweed mitogen (PWM), phytohaemagglutinin (PHA), or concanavalin A (Con A), for 48 h. T-2 toxin (10 −12−10 −8M) was added at the beginning or 24 h after initiation of the cultures. Exposure to T-2 toxin (10 −11−10 −10M) after 24 h increased 3H-thymidine uptake by splenic cells. Stimulation by PWM increased dramatically while the response to LPS was increased to a lesser extent in this system. Conversely, exposure to T-2 toxin decreased T-cell responses to both PHA and Con A. Exposing cells to T-2 toxin at the initiation of culture increased Con A stimulation; LPS and PWM responses decreased following 48 h exposure. When splenic cells were pre-treated for 60 or 90 min at 0°C with 10 −9M T-2 toxin the responses to both PWM and LPS increased. Increased 3H-thymidine uptake was also observed following 90 min exposure to T-2 toxin alone at either 0 or 37°C. The increased PWM response and the mitogenic phenomena observed when cells were exposed only to T-2 toxin were consistent with previously observed effects of in vivo T-2 toxin exposure.

The effects of C-reactive protein on human cell-mediated cytotoxicity.

C-reactive protein (CRP) is a trace serum protein that increases markedly in concentration during inflammatory reactions. Although CRP, in the presence of a multivalent ligand, binds in vitro to a small percentage of peripheral blood lymphocytes from normal donors and is present on natural killer (NK) cells, exogenous addition of CRP has few effects on human lymphocytes. CRP causes minimal enhancement of proliferation in a mixed lymphocyte culture and a slight increase in 3H-thymidine uptake by unstimulated cells. The most significant effect of CRP is a substantial increase in cell-mediated cytotoxicity (CMC). In this publication, we show that CRP dramatically enhances the alloantigen-activated cytotoxic response only when it is present at the initiation of culture and that pretreatment of responder cells with CRP will not produce enhancement. Although the CMC enhancement generated the presence of CRP is not antigen specific, it is mediated by a T cell, and neither NK-like cells nor monocytes are involved in mediating CRP enhanced killing.

3H‐thymidine uptake in B cell lymphomas — Relationship to treatment response and survival

Cell suspensions were obtained from biopsy tissue from 149 patients with B cell lymphomas and analysed with regard to DNA-synthesis as assessed by 3H-thymidine uptake, response to therapy and survival. The 3H-thymidine uptake was significantly increased in lymphomas of high versus low grade malignancy (p = 0.0001), in patients with stage I and II versus stage III and IV (p = 0.014), and in patients with general symptoms (p = 0.0025) as opposed to asymptomatic cases. The complete response rate was significantly higher in patients with increased thymidine uptake than in those with low uptake, 26/51 (51%) cases versus 24/83 (29%) cases, respectively (p = 0.014). 55 patients with increased 3H-thymidine uptake survived for significantly shorter times than (94 patients) with low uptake (p = 0.0056). Furthermore, a markedly larger group of high-risk patients was identified by the 3H-thymidine assay than by histopathology alone, 55 cases versus 23 cases, respectively. Among the patients (126 cases) with low grade tumours, those with increased 3H-thymidine uptake (40 cases) had poorer outcome than those with low uptake (86 cases) (p = 0.045). The data suggest that DNA-synthesis in this study, as assessed by 3H-thymidine uptake, is an independent indicator of survival in NHL. Furthermore, it may be a useful parameter in laying down guidelines for therapy in B cell neoplasms, especially in low grade tumours.

Sodium orthovanadate stimulation of DNA synthesis in Nakano mouse lens epithelial cells in serum-free medium.

Quiescent cultured Nakano mouse lens cells incubated for 40 hours with sodium orthovanadate incorporated 3H-thymidine at an accelerated rate; the greatest response occurred at 20 microM vanadate, whereas by 2 microM an incorporation rate equivalent to unstimulated cells was noted. Microscopic examination of the cells revealed that those exposed to concentrations of vanadate greater than 100 microM had lysed by the end of the 40-hour incubation. Reduction in vanadate exposure time to 1 hour caused the cells to incorporate the greatest amount of 3H-thymidine at a vanadate concentration of 200 microM to 500 microM. Half-maximum incorporation of 3H-thymidine (after a 40-hour incubation) was induced by a 2-hour incubation with 20 microM vanadate. Studies with insulin showed that while 20 ng/ml insulin alone did not increase 3H-thymidine incorporation, 20 ng/ml insulin in combination with 20 microM vanadate resulted in a significant increase in 3H-thymidine uptake over cells exposed to only vanadate. Insulin alone will increase cell number and insulin with vanadate are synergistic in the stimulation of DNA synthesis, but the two together show no further increase in cell number over that produced by insulin alone. Thus, vanadate can increase progression from G1/G0 to S-phase, but cannot stimulate cells to divide. Studies designed to detect DNA damage and repair rather than S-phase DNA synthesis demonstrated that vanadate was not causing increased 3H-thymidine uptake by damaging DNA. Cell counts revealed that vanadate, while able to induce DNA synthesis, does not induce mitosis. Autoradiography and equilibrium sedimentation experiments demonstrated that gene amplification was not occurring. A known vanadate exchange inhibitor blocked the ability of vanadate to increase 3H-thymidine incorporation which is consistent with the idea that cellular internalization of vanadate is required for this effect to be seen. 86Rb+ uptake experiments demonstrate that the vanadate concentration inducing 50% inhibition of (Na+, K+)ATPase is nearly two orders of magnitude more concentrated that vanadate concentrations shown capable of inducing 3H-thymidine uptake. This strongly suggests that (Na+, K+)ATPase inhibition is not the central mechanism by which DNA synthesis is stimulated by vanadate.