DNA Synthesis In Lymphocytes Research Articles

ラット再生肝細胞の培養上清中に存在する増殖抑制因子

The auto-regulation mechanism of liver regeneration after partial hepatectomy was investigated in rats. Hepatocytes isolated from the individual remnant livers at various times after 70% removal of the liver were cultured, and determination of DNA synthesis of the cultured hepatocytes and collection of culture supernatants were performed. The maximum value of DNA synthesis was observed at 1 day after the operation and then gradually decreased, and the liver weights recovered rapidly during 2-4 days. The culture supernatants harvested from these primary cultured hepatocytes isolated from the rats at 2, 3 and 4 days after the operation suppressed dose-dependently DNA synthesis of hepatocytes which were isolated from normal rats and stimulated by epidermal growth factor (EGF) and insulin in vitro. The supernatants were non-cytotoxic against hepatocytes, and the activity of them was fairly stable for acid and heat treatments but not for proteolytic enzyme. No inhibition of the binding of EGF to the receptors on hepatocytes was observed. Furthermore, the factor showed an inhibitory activity of mitogen-induced DNA synthesis of human peripheral blood lymphocytes in culture. These results suggested that hepatocytes themselves might modulate the process of liver regeneration by secreting a growth inhibitory factor.

Recovery of Human Lymphocytes Damaged with γ-Radiation or Enzymatically Produced Oxygen Radicals: Different Effects of Poly(ADP-ribosyl)polymerase Inhibitors

Quiescent human lymphocytes were damaged in two different ways, both producing toxic oxygen radicals: xanthine oxidase plus hypoxanthine (XOD/HYP), or gamma-rays. Under conditions where DNA synthesis was reduced to 10-20% of control, inhibitors of poly(ADP-ribosyl)polymerase (ADPRP, an enzyme that becomes activated in the presence of DNA strand breaks) allowed lymphocytes to recover completely when the damage was caused by XOD/HYP, but they did not affect DNA synthesis of irradiated cells. However, a protective effect of ADPRP inhibitors was observed with irradiated lymphocytes receiving doses greater than or equal to 50 Gy. Unscheduled DNA synthesis was Unscheduled DNA synthesis was significantly increased when lymphocytes were damaged by high radiation doses in the presence of ADPRP inhibitors. We suggest that ionizing radiation does not stimulate poly(ADP-ribose) synthesis in lymphocytes at doses that impair lymphocyte DNA synthesis by 80-90%, while ADPRP may be involved in the repair of DNA lesions occurring at higher radiation doses.

Mutagenicity studies on paracetamol in human volunteers II. Unscheduled DNA synthesis and micronucleus test

The possible genotoxic effect of paracetamol (PC) was studied in a group of 11 healthy volunteers. PC was administered in the form of tablets 3 × 1000 mg in the course of 8 h. Blood samples and buccal mucosa cells were taken 0, 24, 72 and 168 h after the first administration of the drug. Each blood sample was used for the termination of the unscheduled DNA synthesis (UDS) in peripheral lymphocytes and ascorbemia in plasma. Buccal mucosa cells were analysed for micronuclei. After PC administration the level of UDS induced by MNNG was decreased to T/C = 4.11 ± 0.56 after 24 h vs. T/C = 5.02±0.47 ( p < 0.01) at 0 h. The frequency of micronucleated cells in the buccal mucosa was increased after 72 h to 0.38±0.07% vs. 0.19±0.06% ( p < 0.01) before PC administration. If PC was administered simultaneously with ascorbic acid (AA), also in a dose of 3 × 1000 mg, a decreased level of UDS was observed after 24, 72 and 168 h and the increased number of micronuclei was qualitatively the same as with PC alone: 0.38±0.09% after 72 h vs. 0.20±0.05% at 0 h AA did not decrease the genotoxic effect of PC, but prolonged the influence of PC on UDS.

Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by heparin.

The immunomodulating effects of heparin and natural and synthetic heparinoids (which are now undergoing clinical trials for the treatment of AIDS) on cellular immunity (DNA synthesis and cytotoxic responses of mouse lymphocytes to allogeneic cells and histocompatible tumors) were studied. The results showed that (1) high and low m.w. heparin enhanced mouse antitumor and antiallogeneic cell responses in vitro; (2) other sulfated heparinoids did not have this enhancing activity and some of them (including dextran sulfate) totally suppressed generation of cytotoxic cells; (3) these immunomodulating activities of heparin and heparinoids did not correlate with their anticoagulant effects, degree of sulfation, and mitogenic activity; (4) heparin did not increase the production of IL-2 and did not enhance the action of IL-2 on the cells in MLC, heparin also had no effect on the growth-promoting activity of IL-2 on cloned cytotoxic T cells; (5) heparin had a synergistic enhancing effect with IL-1 on the generation of cytotoxic cells in MLC; and (6) heparin abolished endothelial cell growth factor-induced suppression of cytotoxic response. The latter two effects by themselves, however, could not fully explain the entire immunoenhancing activity of heparin. These results indicate that heparin and heparinoids have multiple effects on the immune system and that some of them can enhance, whereas others can suppress cell-mediated responses.

Full replacement of 2-mercaptoethanol by cysteine plus selenium compounds in augmenting DNA synthesis of mitogen-stimulated mouse spleen lymphocytes.

Mouse spleen lymphocytes require 2-mercaptoethanol for maximal mitogenic activation in vitro. Previous studies indicate that the lymphocytes are defective in the cystine transport activity and that they require 2-mercaptoethanol to utilize cystine. 2-Mercaptoethanol catalytically carries cysteine moiety into the cells in a mixed disulfide form. Because cysteine is easily oxidized to cysteine in the culture medium, it has been not easy to precisely examine the effect of near-physiological concentrations of cysteine on the activation of lymphocytes. By controlling the cysteine content in the medium, we have reviewed the effect of cysteine to see if cysteine replaces 2-mercaptoethanol in enhancing the DNA synthesis of lipopolysaccharide-stimulated lymphocytes. It was found that cysteine was less effective than 2-mercaptoethanol, and that cysteine fully replaced 2-mercaptoethanol when a selenium compound was supplemented. The effects of cysteine and selenium compounds were apparently independent and additive. Among the selenium compounds examined, sodium selenite and L-selenocystine were much more effective in stimulating DNA synthesis than sodium selenate and L-selenomethionine.

Evaluation of antihuman T lymphocyte saporin immunotoxins potentially useful in human transplantation.

We have synthesized 3 immunotoxins (ITs) by covalently coupling the saporin-6 hemitoxin (SAP) to OKT11, SOT3, and SOT1a murine monoclonal antibodies that recognize human T lymphocyte CD2, CD3, and CD5 surface antigens, respectively. The resulting ITs, referred to as OKT11-SAP, SOT3-SAP, and SOT1a-SAP, are equally effective in inhibiting eukaryotic protein synthesis in a cell-free system, and all 3 ITs bind to human T lymphocytes in an almost comparable manner. However, these reagents differ markedly in their ability to kill target T lymphocytes as assessed by measuring the inhibition of DNA synthesis and growth of clonable T lymphocytes in response to mitogenic and allogeneic stimuli. Whereas the anti-CD2 IT, OKT11-SAP, shows moderate cytotoxicity against T lymphocytes, the anti-CD3 IT, SOT3-SAP, and the anti-CD5 IT, SOT1a-SAP, are highly effective in eliminating the same target cells. The concentrations inhibiting 50% (IC50) of T lymphocyte DNA synthesis are 60 nM, 4.5 nM, and 1.4 nM for OKT11-SAP, SOT3-SAP, and SOT1a-SAP, respectively. Among 3 tested lysosomotropic amines, i.e., ammonium chloride, chloroquine, and amantadine, the latter only moderately potentiates the cytotoxicity of SOT1a-SAP (IC50 0.36 nM). We show that the conditions under which T lymphocyte killing is accomplished require less than 10 min exposure of T lymphocytes to the ITs, in the absence of adjuvant molecules artificially added to the incubation medium and at physiologic culture pH. These experimental characteristics of unprecedented closeness to a physiologic in-vivo model are likely to reflect the biophysical properties of the SAP moiety of the ITs. We conclude that clinical studies are warranted to define the advantage of using SAP ITs over previously described immunoconjugates.

Simultaneous detection of DNA strand breaks and unscheduled DNA synthesis in mutagen-treated human lymphocytes in the absence of hydroxyurea

Human lymphocytes in the quiescent state were exposed to UVC radiation. After irradiation the cells were allowed to repair for various times in the presence of [ 3H]thymidine or [ 3H]deoxycytidine in the culture medium. Hydroxyurea was not used to suppress semiconservative DNA replication in the small number of growing cells. After incubation DNA strand breaks were detected by the DNA-unwinding method and the amount of 3H incorporation in DNA was measured by liquid scintillation counting. The results show that the yield of DNA strand breaks and the amount of unscheduled DNA synthesis (UDS) can be measured from the same lymphocyte sample. A low background 3H incorporation in untreated cells could be achieved even in the absence of hydroxyurea. This requires, however, that 3H incorporation is measured only in the double-stranded DNA and that [ 3H]dCyd is used instead of [ 3H]dThd as the labelled deoxynucleoside.

Early effects of right or left cerebral cortex ablation on mitogen-induced spleen lymphocyte DNA synthesis

The cerebral neocortex has been shown to modulate the immune system in an asymmetrical way. In mice, ablation of the left cortex decreases whereas a symmetrical right lesion enhances B and T cell-mediated responses measured at 6–8 weeks after lesioning. In order to study the possibility of neuronal reorganisation during the post-operative period, immunological parameters were determined as early as 2 weeks after right or left cortical ablation. Right lesions depressed lymphocyte DNA synthesis induced by concanavalin A (ConA) and phytohemagglutinin whereas left lesions only depressed ConA-induced blastogenesis. Right or left lesions had no effect on lipopolysaccharide-induced proliferation of B lymphocytes as well as on serum antibody levels. Comparisons between early and late effects of right or left cortical ablation on the immune system showed that each hemicortex differentially modulates lymphocyte sub-populations but also that the neuronal reorganisation following surgery can be different according to the side of cortex lesion.

An extracellular role for calmodulin-like activity in cell proliferation.

1. Addition of extracellular pure pig brain calmodulin was found to modulate DNA synthesis and cell proliferation in K562 human leukaemic lymphocytes. At lower cell densities calmodulin significantly stimulated [3H]thymidine uptake; at higher densities it decreased it. 2. A protein biochemically indistinguishable from calmodulin was detected in the cell-conditioned media of rapidly dividing K562 cells. The concentration of calmodulin-like activity found in the conditioned media of these and a range of other normal and neoplastic cells (250-1636 ng/ml) was of the same order as would stimulate DNA synthesis in subconfluent cells. 3. Amounts of extracellular calmodulin-like activity and immunoreactivity varied during cell growth from low to high density, a peak of extracellular calmodulin preceding DNA synthesis in synchronized K562 cells. Extracellular calmodulin concentrations did not correlate with the presence of lactate dehydrogenase in the medium. 4. Inhibition of extracellular calmodulin activity by calmodulin antagonist immobilized on agarose beads, or by antibody to calmodulin, significantly decreased DNA synthesis. 5. These data strongly suggest that calmodulin or a very closely related protein can influence mitosis through an extracellular mechanism.

An immunoperoxidase-autoradiography double labeling method for analysis of lymphocyte activation markers and DNA synthesis

An immunoperoxidase-autoradiography double labeling method for analysis of lymphocyte activation markers and DNA synthesis is described. For this study expression of MHC locus II coded Ia antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein was analyzed using monoclonal antibodies in avidin-biotin-peroxidase complex staining combined with visualization of [ 3H]thymidine incorporation by autoradiography. Compared to spontaneous [ 3H]thymidine incorporation assay information is obtained at single cell level. In contrast to blast indexes calculated from MGG stained preparates, information on the expression of various functional cell surface structures as well as DNA synthesis is also obtained. Double-assay for lymphocyte phenotype and DNA synthesis by flow cytometry might be preferred to light microscopy, but the widespread use of immunoperoxidase staining and autoradiography may make this new kind of approach more easily available. Other advantages worth considering are the possibility of transporting, staining and counterstaining as microscope slides and the permanent nature of the documentation and morphological information obtained. In our experience, this method seems to be useful for studying resting peripheral blood lymphocytes as well as mitogen and antigen induced changes in the lymphocyte activation state.

Evidence that transferrin supports cell proliferation by supplying iron for DNA synthesis

Transferrin is essential for cell proliferation and it was suggested that it may trigger a proliferative response following its interaction with receptors, serving as a growth factor. However, since the only clearly defined function of transferrin is iron transport, it may merely serve as an iron donor. To further clarify this issue, we took advantage of an iron chelate, ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH), which we developed and previously demonstrated to efficiently supply iron to cells without using physiological transferrin receptor pathway. As expected, we observed that blocking monoclonal antibodies against transferrin receptors inhibited proliferation of both Raji and murine erythroleukemia cells. This inhibited cell growth was rescued upon the addition of Fe-SIH which was also shown to deliver iron to Raji cells in the presence of blocking anti-transferrin receptor antibodies. Moreover, blocking anti-transferrin receptor antibodies inhibited [ 3H]thymidine incorporation into DNA and this inhibition could be overcome by added Fe-SIH. In addition, Fe-SIH slightly stimulated, while SIH (an iron chelator) significantly inhibited, DNA synthesis in phytohemagglutinin-stimulated peripheral blood lymphocytes. Taken together, these results indicate that the only function of transferrin in supporting cell proliferation is to supply cells with iron.

Transforming growth factor-beta is a potent immunosuppressive agent that inhibits IL-1-dependent lymphocyte proliferation.

Transforming growth factor-beta (TGF-beta), a product of neoplastic and hemopoietic cells, is a bifunctional regulator of the immune response. At femtomolar concentrations, TGF-beta stimulates monocyte migration, and picomolar quantities induce synthesis of monocyte growth factors, including IL-1, that may promote tissue repair by regulating fibrosis and angiogenesis. Paradoxically, TGF-beta at picomolar concentrations also blocks the ability of IL-1 to stimulate lymphocyte proliferation. At 0.01 to 1.0 ng/ml, TGF-beta 1 and its homologue, TGF-beta 2, suppress the IL-1-dependent murine thymocyte proliferation assay. TGF-beta also inhibits human peripheral blood T lymphocyte mitogenesis. Inhibition of cell division appears to occur after activation of the lymphocytes inasmuch as neither gene expression nor translation of IL-2R is suppressed. Furthermore, TGF-beta does not block synthesis of IL-2. Therefore, TGF-beta 1 and TGF-beta 2 likely act at a site distal to IL-1 to block lymphocyte DNA synthesis. These findings suggest that TGF-beta secreted in an inflammatory site may be beneficial in diminishing lymphocyte function while promoting fibrosis and tissue repair. However, TGF-beta generated by neoplastic tissues may provide a mechanism for unrestricted tumor cell growth through its selective immunosuppressive effects.

Human B-cell differentiation by Fc fragment of IgG: I. Fc fragment from human IgG induces plasma cell generation but cannot induce lymphocyte proliferation

Circulating mononuclear cells (MNC) from normal donors were examined for lymphocyte proliferation and plasma cell differentiation following stimulation by Fc and Fab fragments or by intact IgG. Lymphocyte differentiation and DNA synthesis were examined as a function of culture duration and concentration of Fc, Fab fragments, and IgG. Plasma cells containing intracytoplasmic Ig were demonstrated by immunofluorescence with a polyvalent antiserum to human immunoglobulin and with specific antisera (anti-μ, -γ, -α, -δ, -κ, and -λ chains). DNA synthesis of mononuclear cells cultures was analyzed by measuring [ 3H]thymidine incorporation. The results indicated that only the Fc fragments are able to induce the differentiation of B cells. The polyclonal plasma cell response to Fc fragments was dose dependent, peaked on the sixth day of culture, and was isotypically diverse (IgM > IgA > IgG). This activity requires the presence of T helper cells and monocytes. In contrast, the Fc fragments were unable to induce a proliferative response.

Lymphocyte Activation in Oral Lichen Planus In Situ

The current study evaluated the activation of T-cell mediated cellular immune response in the oral lesions in lichen planus (OLP). Analysis was made of the ultramorphology, lymphocyte activation marker expression, DNA synthesis, and gamma-interferon production of the inflammatory cells in OLP lesions. According to these four different aspects of lymphocyte activation, only a minor fraction, 5% at the most, of all T-cells in situ were activated. This minor fraction, however, not the resting T-cells without these signs of activation, may decide the outcome of the local immune-inflammatory process in OLP. Of the inflammatory cells in situ, however, 81 +/- 5% were Ia positive. This finding may be the result of an Ia inducing capacity of the gamma-interferon produced locally by the activated T-cells.

Differences in immunological susceptibility to cadmium toxicity between two rat strains as demonstrated with cell biological methods. Effect of cadmium on DNA synthesis of thymus lymphocytes

When 2 inbred rat strains, the Brown-Norway rat and the Lewis rat were exposed to the same amount of CdCl 2 for 15 days, a completely different immunological reaction pattern could be demonstrated. Despite the same amount of intrathymic cadmium in both strains, the Brown-Norway rat showed a significant decrease in thymocytes in the S-phase and a significant increase of thymocytes in the G2 phase and mitosis, in contrast with findings in the Lewis rats. A new method for estimating subtle forms of thymus atrophy showed a slight decrease in the number of the smallest thymocytes in the Brown-Norway rat after exposure to cadmium, in contrast with that in the Lewis rat. Evidence is presented that the approximately 1.7 times larger number of thymocytes/mg thymus in the Lewis rat, compared to the Brown-Norway rat, as well as the approximately 2.5 times lower proliferation rate of the thymocytes, and an approximately 1.5 times higher metallothionein content of the thymus medulla epithelial cells in the Lewis rat, might be responsible for the observed difference in toxicity. The zinc content of the thymus was not significantly decreased by exposure to CdCl 2, and did not differ significantly between both strains.

Interleukin-1-like activity produced by hybrids constructed with Epstein-Barr-virus-transformed human B lymphocytes and mouse myeloma cells.

Peripheral blood B lymphocytes from a donor with positive tuberculin skin test reaction were transformed into lymphoblastoid cell lines by Epstein-Barr virus and then fused by polyethylene glycol with mouse myeloma cells. Human-mouse hybrid cells producing human IgM monoclonal antibody to purified protein derivative of tuberculin were isolated, and the concentrated supernatant of one of these cell hybrids was tested for the capacity of interfering with DNA synthesis of human and mouse lymphocytes. The hybrid cell supernatant was found to contain soluble factors that increased DNA synthesis in unstimulated human and mouse lymphocytes and that, conversely, decreased DNA synthesis in concanavalin-A-stimulated cells. Gel filtration experiments showed that these antagonistic activities were due to at least two different factors, one of which resembled human interleukin-1 in biochemical and biological properties.

Stimulation of DNA synthesis in murine lymphocytes by the drug swainsonine: Immunomodulatory properties

Swainsonine, an inhibitor of Golgi α-mannosidase II, has recently been shown to have potent antimetastatic activity in experimental metastasis assays (1–4). In the case of systemic administration, the possible mechanism of action is unknown; the results reported here indicate that it can be explained at least in part by swainsonine stimulation of lymphocyte proliferation. In the present experiments, the standard in vitro mitogenic stimulation assay was used to test the effect of swainsonine on spleen cells. Treatment of spleen cell cultures with the optimum concentrations of the drug enhanced proliferation by 80–146%, as measured by ( 3H) thymidine incorporation, relative to untreated cultures. Similarly, when spleen cell cultures were prepared from mice maintained on swainsonine-supplemented drinking water, proliferation was stimulated at least 3-fold relative to cultures derived from animals maintained on regular water. The enhanced mitogenesis is apparently not directly related to increased expression of Concanavalin A (Con A) binding sites, since swainsonine induced mitogenesis is not inhibited by α-methylmannoside in contrast to Con A induced mitogenesis which is completely inhibited. These results suggest that the antimetastatic effect of systemically administered swainsonine is at least in part related to its ability to enhance proliferation of those specific cell populations involved in immune surveillance. This represents the first demonstration of a mechanism of action of swainsonine on the immune system.

Effects of aspirin-like drugs on mitogen-stimulated DNA synthesis of lymphocytes from pregnant rats and offspring.

Previous studies have shown that salicylates and protein-calorie malnutrition compromise immunological responses in humans and experimental animals. The present study compared the effects of prenatal normal and low protein diets, with and without aspirin-like drug treatments, on lymphocyte blastogenesis measured by tritiated thymidine uptake for DNA synthesis in splenic lymphocytes from pregnant rats and their offspring following stimulation with the mitogens concanavalin A, phytohemagglutinin, and pokeweed mitogen. Aspirin treatment was associated with increased lymphocyte thymidine uptake for blastogenesis in pregnant rats fed the normal protein control diet and their offspring. The phytohemagglutinin-stimulated increase detected in offspring lymphocytes could not be statistically guaranteed. A low protein diet alone and a normal protein diet combined with salicylamide treatment was associated with decreased blastogenesis in pregnant rats but not in their offspring. Salicylamide or aspirin combined with a low-protein diet decreased blastogenesis in both dams and their offspring. Aspirin combined with a normal protein diet did not adversely affect blastogenesis in either pregnant rats or their offspring. This study suggests that low dietary protein and aspirin-like drugs may independently decrease lymphocyte blastogenesis of pregnant rats and in combination they may also reduce lymphocyte blastogenesis in offspring. The significance of increased lymphocyte blastogenesis in both mothers and offspring following aspirin treatment of pregnant rats fed a normal protein diet is unclear.

7] Purification of staphylococcal enterotoxins

Publisher Summary This chapter describes the purification of Staphylococcal Enterotoxins. Six distinct enterotoxins (A, B, C l , C 2 , D, and E) are produced by Staphylococcus aureus and account for the majority of food poisoning in the United States. The purification of the enterotoxins is based on classical ion-exchange and gel filtration techniques. However, newer methodologies, such as fast-protein liquid chromatography (FPLC), are applied to the purification of these toxins. Although these toxins are termed enterotoxins, it is a misnomer, because none produces the classic ileal loop response. The enterotoxins are also shown to be powerful mitogens and are able to induce DNA synthesis and interferon synthesis in lymphocytes. Enterotoxin A is among the most effective T-cell mitogens that stimulate interferon production in mouse spleen cell cultures. However, it seems clear that these mitogenic activities are not associated with either major antigenic regions of the molecules or with their site of action for emesis and diarrhea.

Bovine platelet-derived factor showing mitogenic activity on bovine leukemia virus-infected lymphocytes.

The platelet-derived factor (PDF) which has the suppressive effect against plasma blocking factor (PBF), was further characterized and partially purified. PDF was pH and heat stable, but it was rendered unstable by treatment with proteolytic enzymes. The biological activity of PDF was associated with four entities of molecular weights; 150, 000 (Fr.I), 130, 000 (Fr.II), 85, 000 (Fr.III) and 50, 000 (Fr.IV) determined by gel filtration in 1M acetic acid. The fraction IV showed the highest suppressive effect against PBF as well as the highest stimulative activity of DNA synthesis in bovine lymphocytes and murine or bovine fibroblasts.