Ginseng reduces the micronuclei yield in lymphocytes after irradiation

To assess the effect of Chinese ginseng in modifying the radiation-induced micronuclei (MN) yield in human G(o) peripheral blood lymphocytes (PBL), we conducted the cytokinesis-blocked (CB) MN assay in blood samples obtained from healthy volunteers (n=4). Before (137)Cs ex vivo irradiation, mononuclear cell cultures from each sample were incubated 24 h with different concentrations (0-2000 microg ml(-1)) of crude water extract of ginseng dry root. We found that (1) at 0 Gy and without the presence of ginseng, MN yield (mean+/-S.E.M.) was 11.7+/-2.7 per 1000 binucleated (BN) cells. Different concentrations of ginseng crude water extract did not affect the MN yields and the proliferative activity of PBL; (2) after 1 and 2 Gy exposure, radiation alone sharply increased the MN yields, respectively, to 119.6+/-17.4 and 340.5+/-20.9 per 1000 BN cells. However, treatment with ginseng for 24 h before radiation exposure, resulted in a significant linear decline of MN yields as ginseng concentration increases. Compared to radiation alone, the extent to which ginseng water extract reduced the MN yields induced by 1 Gy exposure was 46.0% at 1500 microg ml(-1) and 61.5% at 2000 microg ml(-1), and with 2 Gy exposure, it was 38.6% at 1500 microg ml(-1) and 46.5% at 2000 microg ml(-1); (3) MN data suggested a tendency for overdispersion relative to the Poisson model; and (4) over the different levels of ginseng concentrations, the trend in micronucleated BN index was as similar as that of the MN yields. These results indicated that (1) ginseng crude water extract exerts no apparent cytogentic effect on human PBL at concentrations up to 2000 microg ml(-1) as evaluated by the CBMN assay; and (2) the protection of ginseng water extract against (137)Cs-induced MN in human PBL is concentration-dependence. Therefore, our findings indicated that ginseng may have therapeutic value as a possible radioprotector for normal tissue during radiotherapy of cancer patients.     

Composition of splenic T lymphocytes in allophenic mice.

Guinea pig peritoneal macrophages were activated in vitro by culturing with MAF (macrophage activating factor)-containing fractions from stimulated lymphocytes. These macrophage preparations demonstrate a 60% increase in the production of prostaglandins of the E series (PGE) when compared with macrophages cultured with fractions from unstimulated lymphocytes. PGE accumulation in macrophage cultures is maximal after 24 hr with MAF; tumor cytotoxicity is also maximal at this time. The final PGE concentration in cultures of activated macrophages averaged 3 × 10^?8M.     

The quantitative analysis of micronucleus induction in human lymphocytes cultured in vitro (preconditions for making modified micronucleus assay)

The new modification of the method of micronucleus (MN) detection without cytochalasin-B is used in this paper. The code name of the method is called "method of micronucleus detection in mononucleated cells". The basis of this method is that it makes possible to analyze MN and chromosome aberrations (CA) at the same slides. To confirm the true supposition of the authors about correlation between MN quantity and chromosome/chromatid type aberrations so called "coefficient of transformation" was calculated and it was 7.9 +/- 0.41 for the chromosome type aberrations and over 67.2 +/- 30.2 for chromatid type aberrations. Mutagenic action of gamma-irradiation and 8-methoxypsoralen (8-MOP), activated with long-wave UV-light was estimated for the first cell cycle mitosis. When compared in straight experiments results of gamma-induced MN were received by two methods: the method of cytokinesis-block (commonly used) and by the suggested method, the "coefficient of transformation" of CA into MN was 3.6, when cytochalasin-B was used and 6.7 without using it. The total data give a possibility to make a new cytological micronucleus test for mutagens revealing. As we think the modified test is more simple, more reliable less laborious and less expensive.     

The cytokinesis-block micronucleus assay as a biological dosimeter in spleen and peripheral blood lymphocytes of the mouse following acute whole-body irradiation

To evaluate the application of the cytokinesis-block (CB) micronucleus (MN) assay as a biological dosimeter following in vivo exposure to ionising radiation we determined the micronucleus frequency in spleen and peripheral blood lymphocytes of the mouse, serially, for 14 days following acute whole-body irradiation. The baseline MN frequency of spleen lymphocytes (7.86 +/- 0.68, mean +/- 1 SD) was significantly (p less than 0.001) elevated when compared to that for peripheral blood lymphocytes (4.10 +/- 0.53). Immediately after irradiation there was a substantial dose-related increase in MN, but the MN frequencies in spleen lymphocytes (120.2 +/- 9.4 for 1 Gy; 409.5 +/- 38.4 for 2 Gy) were significantly (p less than 0.009) elevated compared to those in peripheral blood lymphocytes (78.0 +/- 7.0 for 1 Gy; 200.2 +/- 10.9 for 2 Gy). During the 14 days after irradiation, the MN frequency in spleen lymphocytes declined gradually to approximately half of the value observed immediately after irradiation. By contrast the MN frequency in peripheral blood lymphocytes increased during the week after irradiation, but ultimately MN frequencies in blood and spleen became approximately the same by day 14. Study of isolated murine lymphocytes irradiated in vitro showed that the number of MN generated by a given dose of radiation was approximately 2-3 times greater than the number generated by in vivo irradiation. These results suggest that measurement of MN in vivo after irradiation can be used as an in vivo dosimeter. However, precise dosimetry is probably affected by factors such as kinetic changes in different lymphocyte populations and possibly by in vivo factors which influence sensitivity of cells to radiation.     

Mitogen induction of ion channels in murine T lymphocytes

Using gigohm-seal recording, we studied ion channel expression in resting and activated T lymphocytes from mice. Both the number of channels per cell and the predominant type of K+ channel depend upon the state of activation of the cell. Unstimulated T cells express small numbers of K+ channels, typically a dozen per cell, and are heterogeneous, usually expressing either type n or type l K+ channels (see DeCoursey, T. E., K. G. Chandy, S. Gupta, and M. D. Cahalan. 1987. Journal of General Physiology. 89:379-404). 1 d after stimulation by the murine T cell mitogen concanavalin A, large numbers of type n K+ channels appear in enlarged, activated cells. Type n channels appear in activated cells with a time course consistent with that reported for mitogen-induced enhancement of protein synthesis. Voltage-gated tetrodotoxin-sensitive Na+ channels present in about one-third of unstimulated cells from the MRL-n strain are increased approximately 10-fold after activation.     

All-trans retinoic acid stimulates IL-2-mediated proliferation of human T lymphocytes: early induction of cyclin D3

Vitamin A is established as an important immune regulator, but the mechanisms whereby vitamin A regulates T cell biology are poorly defined. In this study, we show that an active metabolite of vitamin A, all-trans retinoic acid (RA), potently stimulates T cell proliferation by modulating IL-2-mediated signaling downstream of IL-2R and independent of the induction of IL-2. Thus, at concentrations as low as 0.1 nM, RA enhanced the division of normal human T lymphocytes that were simultaneously stimulated with anti-CD3 mAbs and saturating concentrations of IL-2. At the optimal concentration of RA (50 nM), a 3-fold increase in T cell proliferation was observed. The induced proliferation was preceded by increased phosphorylation of the retinoblastoma protein and enhanced G1- to S-phase progression. Interestingly, the promitogenic effect of RA was found to be particularly directed toward increased expression of cyclin D3 at both the mRNA and protein level. Furthermore, the stimulatory effect of RA on cyclin D3 expression as well as on cell proliferation was completely abolished in the presence of the JAK inhibitor AG-490 or blocking IL-2R alpha mAbs, and RA also enhanced cyclin D3 expression and T cell proliferation in the presence of IL-2 alone. Finally, we showed that the proliferative effect of RA was mimicked by agonists of the retinoic acid receptor (RAR) and completely inhibited by a RAR-selective antagonist. In conclusion, our results indicate that RA, via RAR, stimulates IL-2-induced signaling in a JAK-dependent manner to enhance cyclin D3 expression and thereby promote T cell proliferation.     

IL 2 receptor induction on human T lymphocytes: role for IL 2 and monocytes

In this report we studied the requirements for the activation and proliferation of highly purified human T lymphocytes. Purified T cells incubated for 3 days with PHA neither proliferate nor express IL 2 receptors as detected by FACS analysis with the use of anti-Tac antibodies. However, purified T cells incubated with Con A or anti-T3 moAb do not proliferate, albeit 30 to 35% T cells express Tac epitopes. The addition of IL 2, either natural purified or recombinant, resulted in both the appearance of Tac antigen and the proliferation of PHA-activated T cells. Much to our surprise, IL 2 did not induce proliferation of Tac-positive T cells activated by Con A or soluble anti-T3 unless monocytes were added to the cultures. These data suggested that two classes of IL 2 receptors might exist on T cells, one of which was not functionally involved in T cell proliferation. In keeping with this interpretation, we have been able to demonstrate, using a radiolabeled IL 2 binding assay, that anti-T3 moAb induced almost exclusively IL 2 receptors of low affinity (Kd = 30 to 70 X 10(-9) M) and that additional signals, provided by monocytes, are required for the acquisition of high affinity receptors. IL 2 itself can induce high affinity receptors on PHA-stimulated T cells but not on cells activated by Con A or anti-T3. In this latter case the physical presence of monocytes is required and cannot be substituted by IL 1, thus indicating a previously unreported role for monocytes. It is postulated that the contact of monocytes with T cells induces a switch from an inactive low affinity conformation of the IL 2 receptor to a functional high affinity one.     

Ascorbic acid reduces accumulation of [3H]spiperone in mouse striatum in vivo

[3H]Spiperone was administered (20 microCi/kg, 0.0003 mg/kg, sc) to mice. In agreement with other published reports, 2 hr later the accumulation of tritium was three to four times greater in the corpus striatum than in the cerebellum. Ascorbic acid (100, 1000, 2000 mg/kg, ip, 30 min) reduced the 2-hr accumulation in the corpus striatum 16, 42, and 63%, respectively, with only the highest dose producing any significant (18%) reduction in the cerebellum. The effect was still evident in striatum 18 hr after a single dose of 1000 mg/kg. Striatal minces taken from mice treated 1 or 2 hr earlier with ascorbic acid (2000 mg/kg, ip) showed no reduction in [3H]spiperone binding. However, preincubation of striatal minces for 2 hr with ascorbic acid (10(-3) M) produced an 82% reduction in specific binding while not having any effect on nonspecific binding. While it cannot be certain that the reduction of striatal [3H]spiperone concentrations after ascorbic acid in vivo was not a result of some nonspecific alteration in the pharmacokinetics of [3H]spiperone, the in vitro observation strongly suggests that it resulted from an alteration of binding characteristics at the receptor level.     

Chromatid-type aberrations following irradiation in G0 lymphocytes with heavy ions

We describe a low level of chromatid-type aberrations which included the relatively rare isochromatid/chromatid triradial in peripheral blood lymphocytes that were irradiated, ostensibly in G0, with accelerated heavy (12)C ions. These were produced only at the energies of 69 MeV/n (34.6 keV/microm), almost absent at the energy of either 58.6 MeV/n (46.07 keV/ microm) or 19.3 MeV/n (97 keV/microm), nor were they found after low-LET X-rays. Mechanisms potentially responsible for their formation are discussed.     

H-2 antigen requirements in the in vitro induction of SV40-specific cytotoxic T lymphocytes

T cytotoxic cells generated to syngeneic SV40 virus transformants lyse only SV40 target cells that are syngeneic at the H-2 locus. In contrast, SV40-specific tumor transplantation immunity shows no requirements for syngeneic H-2. Inoculation of allogeneic or even xenogeneic transformants will confer immunity to a challenge of syngeneic SV40 tumor cells. The experiments described here represent an attempt to reconcile these apparently conflicting observations. In our hands, generation of SV40-specific T cytotoxic cells in vitro requires both in vivo priming and secondary in vitro sensitization. We have found that priming for a secondary syngeneic-restricted response requires only that the cell employed be SV40 transformed. That is, priming may be accomplished with syngeneic, allogeneic, or xenogeneic SV40 transformants. Thus, the apparent lack of H-2 restriction in vivo immunity does not eliminate a role for the H-2-restricted cytotoxic T cell in tumor transplantation immunity.     

Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice

Iron is a potent oxidant that can lead to the formation of genotoxic lipid peroxides. Ascorbic acid, which enhances dietary iron absorption, has been suggested to enhance the oxidant effects of iron and to directly lead to the formation of lipid peroxides. The combined effects of dietary iron and ascorbic acid on genotoxicity were investigated by measuring the frequency of micronuclei in the bone marrow cells of C3H/He mice. In addition, liver iron concentration was measured in all treated groups. Three weeks old mice were fed diets for 3 weeks containing iron at 100 or 300 mg/kg diet in the form of FeSO(4) that were supplemented either with or without ascorbic acid (15 g/kg diet). The results of the bone marrow micronucleus test revealed that the high iron diet resulted in an increased frequency of micronucleated polychromatic erythrocytes (MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet did not show any effect on incidence of MnPCEs and protected against the increased frequency of MnPCEs induced by the high iron diet. However, liver iron concentration was significantly increased only in the high iron treated and ascorbic acid supplemented group as compared to all other groups. These results demonstrate that ascorbic acid protects against the clastogenic effects of iron.     

IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.     

Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice

Iron is a potent oxidant that can lead to the formation of genotoxic lipid peroxides. Ascorbic acid, which enhances dietary iron absorption, has been suggested to enhance the oxidant effects of iron and to directly lead to the formation of lipid peroxides. The combined effects of dietary iron and ascorbic acid on genotoxicity were investigated by measuring the frequency of micronuclei in the bone marrow cells of C3H/He mice. In addition, liver iron concentration was measured in all treated groups. Three weeks old mice were fed diets for 3 weeks containing iron at 100 or 300 mg/kg diet in the form of FeSO₄ that were supplemented either with or without ascorbic acid (15 g/kg diet). The results of the bone marrow micronucleus test revealed that the high iron diet resulted in an increased frequency of micronucleated polychromatic erythrocytes (MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet did not show any effect on incidence of MnPCEs and protected against the increased frequency of MnPCEs induced by the high iron diet. However, liver iron concentration was significantly increased only in the high iron treated and ascorbic acid supplemented group as compared to all other groups. These results demonstrate that ascorbic acid protects against the clastogenic effects of iron.     

Differential influence of 2'-deoxyguanosine on the induction and expression of suppressor T lymphocytes in vivo

Subcutaneous (sc) immunization of mice with allogeneic spleen cells can induce delayed-type hypersensitivity (DTH) to histocompatibility antigens. Intravenous immunization with irradiated allogeneic spleen cells, on the other hand, induces suppressor T (Ts) lymphocytes. These Ts cells are capable of suppressing the host-versus-graft (HvG) DTH reactivity which normally arises after sc immunization. Moreover they can suppress the development of antihost DTH effector T cells during graft-versus-host (GvH) reactions. These models for HvG and GvH DTH reactivity were used to study the influence of 2'-deoxyguanosine (dGuo) on the induction, further development, and expression of Ts cells in vivo. It was found that administration of dGuo inhibits the proliferation-dependent induction and further development of Ts cells, but not the suppression mediated by already activated Ts cells.     

Immune competence of splenic lymphocytes following graft-vs-host disease in mouse allogeneic radiation chimeras.

The abnormal immune response of long-term mouse allogeneic chimeras is reflected by qualitative deficiencies in either T or B lymphocytes. The present study was undertaken to determine if a relationship existed between the severity of graft-vs-host disease (GVHD) that these animals had experienced and a functional defect in either the T or B cell population. The in vitro PFC response of chimera spleen cells to sheep red blood cells (SRBC) was evaluated in the presence of normal T or B lymphocytes 4 to 8 months after marrow transplantation and well beyond the GVHD period. In an analysis of several different allogeneic radiation chimeras, our results showed no relationship between the severity of GVHD experienced and the immunologic capacity of either T or B cells. Thus, different chimera combinations showing similar degrees of GVHD were functionally deficient in one or the other of these two cells types or both with no apparent predilection for abnormality in either population. In examining the quantitative in vitro PFC response to sheep RBC by spleen cells from individual chimeras, we found that the number of PFC formed was related to the severity of GVHD experienced by that animal. A general relationship between severity of GVHD and PFC capacity may also exist between chimeras of different genetic combinations. However, this relationship is not precise since gross exceptions occur. Our results, although documenting further the qualitative abnormalities in T and/or B lymphocytes of radiation chimeras, do not reveal the factor or mechanisms by which these cells are made unresponsive. It is suggested that the tolerance-inducing mechanism of these animals, whether it be humoral blocking factors or suppressor cells, is in some way interfering with the collaboration of T and B cells for antibody production.     

Ascorbic acid inhibits apoptosis induced by X irradiation in HL60 myeloid leukemia cells.

Exposure of cells to ionizing radiation can cause apoptosis. Since antioxidants have been shown to protect against radiation-induced apoptosis, in this study we have evaluated the putative protective effect of ascorbate against radiation-induced apoptosis as well as the production of peroxides in the cells. HL60 cells transport the oxidized form of ascorbic acid, dehydroascorbic acid (DHA), and accumulate reduced ascorbate. Exposure of the cells to 5-40 Gy X radiation resulted in induction of apoptosis. Preincubation of the cells with DHA reduced the level of apoptosis after exposure to 5-20 Gy. Exposure of the cells to 5 or 20 Gy X radiation did not affect the intracellular concentration of peroxides, while phorbol myristate acetate (PMA), which is known to induce production of H(2)O(2) in cells (and served as a control), resulted in an increase in peroxides and a decrease in intracellular ascorbate. Irradiation of the cells with 1-3 Gy resulted in up-regulation of expression of BCL2 without affecting the level of apoptosis. At higher doses of radiation, enhanced BCL2 expression did not prevent radiation-induced apoptosis. Loading of the cells with ascorbate prior to their exposure to 1-3 Gy X radiation did not affect the enhanced BCL2 expression observed in the irradiated cells. At higher doses of radiation, ascorbate decreased apoptosis and restored the level of BCL2 in the cells. Exposure of the cells to 3-20 Gy X radiation enhanced the cell surface expression of TNFRSF6 (formerly known as Fas/APO-1) antigen and enhanced anti-TNFRSF6 antibody-induced apoptosis of the cells. Ascorbate loading did not affect expression of TNFRSF6 and did not overcome the anti-TNFRSF6 antibody-induced apoptosis. In conclusion, our data demonstrate that exposure of HL60 cells to radiation enhanced BCL2 and TNFRSF6 expression. Ascorbate did not affect BCL2 or TNFRSF6 expression. We therefore conclude that it protects HL60 cells against radiation-induced apoptosis, although the mechanisms of protection must still be elucidated.     

Effects of ghrelin on the proliferation and secretion of splenic T lymphocytes in mice

Ghrelin, a novel gut--brain peptide predominantly produced by the stomach, displays strong growth hormone (GH)-releasing activity mediated by the hypothalamus-pituitary GH secretagogue receptor (GHS-R). Recently, the ghrelin receptor has also been detected in peripheral systems including immune tissues, suggesting that ghrelin may play an important role in the regulation of immune function. In this paper, we assessed the presence and function of the ghrelin receptor in murine splenic T cells. The enriched T cells express the mRNA of ghrelin and ghrelin receptor mRNA, and there is a significantly positive correlation between them. Moreover, we showed that ghrelin dose-dependently inhibits proliferation of splenic T cells when they are costimulated by anti-CD3. In addition, ghrelin suppressed Th(1) (IL-2 and IFN-gamma) and Th(2) (IL-4 and IL-10) cytokines mRNA expression. These results demonstrate the presence of the ghrelin receptor in murine spleen T lymphocytes and a functional role of ghrelin as a modulator of lymphocyte function. This function of ghrelin may have some relevance to the pathophysiology of immunologic alterations related to metabolism.     

Increases in microtubule assembly and in tubulin content in mitogenically stimulated mouse splenic T lymphocytes

We have examined the changes in the microtubule and tubulin contents in populations of mouse splenic T lymphocytes stimulated by the mitogen concanavalin A. Indirect immunofluorescence staining with antiserum to tubulin indicated that a more extensive microtubule network was assembled from the centrosome in those cells which had increased in size in response to the mitogen. Direct counts of microtubules from electron micrographs of the centrosome regions of cells showed approximately a 2-fold increase in microtubule number in 48 h stimulated populations and up to a 5-fold increase in the large, fully stimulated, blast cells. Determinations of tubulin and actin contents were made by the measurement of peptides specific to those proteins. As a percentage of total cell protein both of these cytoskeletal proteins increased during the first 24 h of stimulation. Tubulin increased 50% by 24 h and remained high in populations stimulated for 48 h. The tubulin content per cell increased 2.5-fold, from 0.20 to 0.51 μg/10⁶ cells, in the 48 h stimulated population. An increase in tubulin content was also seen following the stimulation of nude mouse B lymphocyte populations and of total splenic lymphocyte populations. Our results show that during lymphocyte stimulation there is a large increase in the numbers of microtubules assembled which is correlated with, and appears dependent on, a similar large increase in the cellular tubulin content.     

The role of major histocompatibility complex expression on head and neck cancer cells in the induction of autologous cytotoxic T lymphocytes

Using head and neck tumors, we studied the role of HLA class I and DR antigens on tumor cells in cytotoxic T lymphocyte (CTL) induction. Expression of major histocompatibility complex (MHC) antigens was investigated by two-color flow cytometry analysis and for this study we used the tumor cells, over 50% of which expressed both HLA class I and DR antigens on their surface. In seven cases, tumor cells were divided into three groups according to the specificity of monoclonal antibodies (mAb) to MHC to study the role of MHC antigens on tumor cells in CTL induction: one was not blocked (MHC double-positive tumor), a second was blocked by anti-class I mAb (class-Ingative DR-positive tumor) and third was blocked by anti-DR mAb (class-I-positive DR-negative tumor). Subsequently, these tumors were used to stimulate an autologous mixed lymphocyte/tumor cell culture for 5 days (MLTC) followed by further cultivation with interleukin-2 for 12 days. The induced autologous tumor killer cells were most cytotoxic when non-treated tumors, which consist mainly of cells that are both HLA-class I and DR-positive, were used as stimulator cells. When the tumor cells blocked by anti-DR mAb were used as stimulators, autologous tumor killer activity was lower than that induced by tumor cells blocked by anti-class-I mAb. Moreover, cytolysis by autologous tumor killer cells induced by stimulation of non-treated tumor cells was blocked during the effector phase, 26.6%–42.3% and 32.7%–53.8% by anti-class-I and anti-DR mAb respectively, suggesting that majority of the autologous tumor killer cells are MHC-restricted CD8⁺ or CD4⁺ CTL. These results suggest that both MHC class I and class II antigens on head and neck tumor cells play a critical role in inducing CTL.