Umbilical Cord Leucocytes transformed by Lymphoid Cell Filtrates from Healthy People

SEVERAL investigators have shown that a cell-free filtrate, or lethally irradiated whole cells, from Burkitt's lymphoma or cells from acute leukaemic blood can transform human leucocytes in vitro^1–6. Transformed leucocytes can multiply rapidly and, perhaps indefinitely, in vitro. Recently, my associates and I reported that a cell-free filtrate prepared from the Kaplan cell—an established line derived from the blood of an infectious mononucleosis patient—can also transform leucocytes⁷. Furthermore, we described a simple procedure for the investigation of leucocyte transformation⁸. Using this technique, it has been possible to show that a cell-free filtrate prepared from lymphoid cell lines of healthy individuals can transform umbilical cord blood leucocytes.     

Formation of Paul-Bunnell antibodies by cultures of lymphocytes from infectious mononucleosis.

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10⁷ cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.     

Nonspecific cytotoxic effects of antigen-transformed lymphocytes. Kinetics, cell-requirements and the role of recruitment

PPD-stimulated human peripheral blood lymphocytes have been shown to exert a nonspecific cytotoxic effect on allogeneic and xenogeneic target cells labeled with ⁵¹Cr. Chromium release induced by washed transformed lymphocytes was linear with time. At lymphocyte to target cell ratios of 120:1, significant killing could be demonstrated as early as $\text{1}\text{2}$ hr. At 8 hr, killing occurred with ratios as low as 3:1. The cytotoxic effect was related to the ability of the lymphocytes to transform to PPD, but reached an earlier peak than either DNA or RNA synthesis. Supernatants from lymphocyte cultures were not cytotoxic: instead, viable transformed cells were required. Partial removal of macrophages from the original cultures increased the cytotoxic effect.Lymphocytes could also be nonspecifically recruited to exert a cytotoxic effect by a mitogenic-like factor produced in transforming cultures. Preliminary evidence suggested that this factor acted independently of PPD and of histocompatibility antigens. These results are discussed with reference to possible amplification mechanisms for late cytotoxic effects, and to delayed hypersensitivity reactions in vivo.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

Chicken Harder's gland: evidence for a relatively pure bursa-dependent lymphoid cell population

Theophylline, caffeine, and dibutyryl cAMP, agents that elevate intracellular levels of cyclic AMP, were found to inhibit cytotoxin elaboration by PHA-stimulated human lymphocytes. Cytotoxins in diluted supernatants from 3-day lymphocyte cultures were assayed by ⁵¹Cr release from L cells. Addition of the agents to the lymphocyte cultures inhibited cytotoxin elaboration 70–90% at 10^?3M and 20–50% at 10^?5M. In addition, it was found that the inhibition is reversible and occurs at a step other than the initial mitogen triggering.The inhibition of cytotoxin elaboration by these agents correlates strikingly with their inhibition of lymphocyte-mediated target cell lysis. The results are consistent with the hypothesis that cytotoxin is the mediator of target cell killing by lymphocytes.     

Photodynamic activity of anthraquinones isolated from Heterophyllaea pustulata Hook f. (Rubiaceae) on MCF-7c3 breast cancer cells

Searching for agents that could be effective in the treatment of cancer, special highlight has focused on the study of numerous plant-derived compounds. We previously demonstrated that anthraquinones (AQs) isolated from a vegetal species: Heterophyllaea pustulata Hook f. (Rubiaceae), such as rubiadin, rubiadin-1-methyl ether, soranjidiol, soranjidiol-1-methyl ether exhibit photosensitizing properties without antecedents as photodynamic agents in malignant cells. In the present study, we investigated the potential role of these AQs as a phototoxic agent against human breast carcinoma using MCF-7c3 cells. All AQs exhibited significant photocytotoxicity on cancer cells at the concentration of 100 μM with 1 J/cm² light dose, resulting soranjidiol-1-methyl ether in complete cell destruction. The observed cellular killing by photoactivated AQs exhibited close relation with singlet oxygen production, except for soranjidiol-1-methyl ether, where cell viability decrease is in relation to uptake by tumor cells.     

Impaired cellular immunecompetence in infectious mononucleosis as assessed by a local graft-versus-host reaction in rats and restoration of the immunecompetence by trypsin.

Lymphocytes from 11 patients with acute infectious mononucleosis were tested for functional capacity by means of a local graft-versus-host reaction (GVHR) and for T lymphocyte markers by the spontaneous rosette-formation test (E rosette). All of the patients showed an increased percentage (49–75%) and high absolute numbers (2078–8736/mm³) of E rosette-forming cells but no functional activity, i.e., a negative GVHR. Repeated examinations performed in four patients at intervals up to 6 months after the initial test showed a significant drop in the number of E rosette-forming cells although the GVHR remained negative. Trypsinization of lymphocytes performed in nine of the 11 patients resulted in recovery of immunecompetence of the T lymphocytes in five; in contrast, trypsinization of normal control lymphocyte abolished their ability to mount a normal GVHR. In view of the number of cases in which infectious mononucleosis has been followed by lymphoproliferative diseases, the importance of long-term follow-up in patients who have undergone an episode of infectious mononucleosis is stressed.     

Dissociation of in vitro lymphocyte transformation from production of a mononuclear leucocyte chemotactic factor in agammaglobulinemic chickens

Cultures of splenic and peripheral lymphocytes from normal chickens immunized intravenously with Brucella abortus organisms were stimulated by this antigen to incorporate significantly greater amounts of ³H-thymidine and ¹⁴C-leucine than lymphocytes from unimmunized animals. Lymphocytes from immunized agammaglobulinemic chickens were unresponsive to Brucella. This defect could not be corrected by the addition of either normal nonimmune or irradiated normal immune spleen cells to cultures of ag chicken lymphocytes which suggests that normally B cells transform in vitro in response to this antigen. In contrast, cultured peripheral blood leucocytes from both immunized normal and agammaglobulinemic chickens produce significantly more monocyte chemotactic factor in response to Brucella than leucocytes from nonimmune chickens. This indicates that the production of this mediator is a B cell independent function and suggests that T cells are the producers of this lymphokine.     

Effects of chelating agents on the initial interaction of phytohemagglutinin with lymphocytes and the subsequent stimulation of amino acid uptake

The chelating agents, ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) and EDTA, had no effect on the initial interaction of phytohemagglutinin with lymphocytes at concentrations which have been shown previously to inhibit the development of the phytohemagglutinin response completely. However, they had a marked inhibitory effect on uptake of the amino acid analog, α-aminoisobutyric acid in both unstimulated and phytohemagglutinin-stimulated cells. The inhibition of amino acid uptake by EGTA could be reversed by adding Ca²⁺ but not Mg²⁺. These results demonstrated that Ca²⁺ is not essential to the initial interaction of phytohemagglutinin with the cell, but does influence amino acid transport which may be a critical preparatory event for later increased protein synthesis.     

T cell-mediated immunoregulation of Epstein Barr virus- (EBV) induced B lymphocyte activation in EBV-seropositive and EBV-seronegative individuals

Infection with Epstein Barr virus (EBV) is accompanied by seroconversion and life-long persistence of the virus in B lymphocytes. During acute EBV-induced infectious mononucleosis, suppressor T cells become activated, which may provide an additional mechanism of host defense against the causative agent. When cultures of lymphocytes from normal adults seropositive for EBV were stimulated with the B95-8 strain of EBV, purified B cells produced increasingly higher numbers of immunoglobulin- (Ig) secreting cells, whereas in co-cultures of autologous B and T cells a profound suppressor T cell activity inhibited further B cell activation after 10 to 12 days in culture. No such T cell-mediated inhibitory effect was seen in cultures of lymphocytes obtained from normal adults seronegative for EBV, indicating a correlation between the suppressor effect with evidence of prior immunity to this virus. The T cell-mediated suppression in patients with infectious mononucleosis is characterized by an early-acting inhibitory effect on B cell differentiation that is not specific in that all polyclonal B cell activators are inhibited, whereas in EBV-seropositive normal subjects suppression is delayed in time and affects only EBV-activated cultures. These data indicate that after infection with EBV, immunoregulatory T cells are generated that are capable of inhibiting further EBV-induced activation of autologous B cells and thus may provide an additional unique mechanism of host defense against persisting EBV-infected B cells.     

Oncogenic Transformation of Hamster Cells after Exposure to Herpes Simplex Virus Type 2

THE possibility of a relationship between herpes simplex viruses (HSV) and human cancer has been suggested^1–4 chiefly on the basis of studies of the epidemiology of cervical cancer, but so far it has not been possible to demonstrate that human herpes viruses can induce primary transformation of normal cells. Injection of herpes simplex virus type 1 (ref. 5) or type 2 (ref. 6) into Syrian hamsters rarely leads to the production of a tumour and it has been difficult to demonstrate herpes viral antigens in tumour cells. Human herpes simplex viruses grown in vitro are characterized by the rapidity with which the infected cell is destroyed, so that cell transformation is impossible, but this effect can be mitigated by inactivation of the herpes virus by ultraviolet irradiation. Indeed, this procedure may have the additional advantage that viral infectivity is removed more quickly than the viral transforming potential⁷.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Unmasking a killer: DNA O6-methylguanine and the cytotoxicity of methylating agents

Methylating agents are potent carcinogens that are mutagenic and cytotoxic towards bacteria and mammalian cells. Their effects can be ascribed to an ability to modify DNA covalently. Pioneering studies of the chemical reactivity of methylating agents towards DNA components and their effectiveness as animal carcinogens identified O⁶-methylguanine (O⁶meG) as a potentially important DNA lesion. Subsequent analysis of the effects of methylating carcinogens in bacteria and cultured mammalian cells — including the discovery of the inducible adaptive response to alkylating agents in Escherichia coli — have defined the contributions of O⁶meG and other methylated DNA bases to the biological effects of these chemicals. More recently, the role of O⁶meG in killing mammalian cells has been revealed by the lethal interaction between persistent DNA O⁶meG and the mismatch repair pathway. Here, we briefly review the results which led to the identification of the biological consequences of persistent DNA O⁶meG. We consider the possible consequences for a human cell of chronic exposure to low levels of a methylating agent. Such exposure may increase the probability that the cell's mismatch repair pathway becomes inactive. Loss of mismatch repair predisposes the cell to mutation induction, not only through uncorrected replication errors but also by methylating agents and other mutagens.     

Nanog-induced dedifferentiation of p53-deficient mouse astrocytes into brain cancer stem-like cells

Self-renewal, differentiation, and tumorigenicity characterize cancer stem cells (CSCs), which are rare and maintained by specific cell fate regulators. CSCs are isolated from glioblastoma multiforme (GBM) and may be responsible for the lethality of incurable brain tumors. Brain CSCs may arise from the transformation of undifferentiated, nestin-positive neural stem or progenitor cells and GFAP-expressing astrocytes. Here, we report a role of Nanog in the genesis of cancer stem-like cells. Using primary murine p53-knockout astrocytes (p53^−/− astrocytes), we provide evidence that enforced Nanog expression can increase the cellular growth rate and transform phenotypes in vitro and in vivo. In addition, Nanog drives p53^−/− astrocytes toward a dedifferentiated, CSC-like phenotype with characteristic neural stem cell/progenitor marker expression, neurosphere formation, self-renewal activity, and tumor development. These findings suggest that Nanog promotes dedifferentiation of p53-deficient mouse astrocytes into cancer stem-like cells by changing the cell fate and transforming cell properties.     

Cytotoxic cells induced in vitro by Epstein-Barr Virus antigens

Lymphocyte blastogenesis and cytotoxicity for lymphoid cells lines (LCL) were induced in cells from immune donors by stimulation in vitro with irradiated Epstein-Barr Virus (EBV). Velocity sedimentation of blastogenic populations indicated that much of the killing was associated with large E-rosetting cells; it was reduced by [³H]thymidine suicide immediately prior to sedimentation. Target cells were killed irrespective of EBV content or sensitivity to natural killer (NK cells), though the pattern of lysis varied with repeated use of individual donors. Up to 9% of the normal resting lymphocytes became blastogenic though fewer than 30% of the dividing cells at termination of culture had undergone more than one division. It is suggested that EBV antigens induce a polyclonal activation of cytotoxic T cells. The possible significance of this response is discussed with particular reference to the origin of non-EBV-specific cytotoxic blast cells in infectious mononucleosis.     

Epstein Barr Virus (EBV) Fusion with Hepathoma Cell Line (Li7A): A Possibility to Use Ebv Virosohes for Drugs Delivery to Liver Cells

Abstract

EBV a causative agent of mononucleosis and several human cancers, infects cell via complement receptor type 2 (CR2). Expression of this receptor is restricted to B lymphocytes, some epithelial cells and immature thymocytes, expression of CR2 like proteins has been also found on T cells. In the present report we identified the presence on the membrane of Li7A cells of a novel EBV receptor distinct from CR2 capable to trigger fusion with EBV virions with a kinetics faster than that found with lymphoblastoid cells (Raji).     

Quantitative analysis of the 51Cr release cytotoxicity assay for cytotoxic lymphocytes

Using ⁵¹Cr-labelled P-815 mastocytoma cells as target cells and CS7BL/6 spleen cells sensitized against DBA/2 antigens as effector cells, it is shown that the variation in the observed specific ⁵¹Cr release over a broad range of experimental conditions can be explained on the basis of a simple physical model of the interaction process. The model assumes that a target cell can be destroyed only after contact with an effector cell, contact takes place on a random basis, one contact is sufficient, and that one effector cell can kill several targets with unchanged efficiency. The fraction of target cells destroyed (f) depends only on the incubation time (t), the number of effector cells (n) and a constant interaction probability (δ). Thus f = 1 ? e^?nδt. However, the experimental measurement, the fraction of ⁵¹Cr specifically released into the supernatant during the assay, may not be the same as the fraction of target cells destroyed because it takes considerable time for the releasable ⁵¹Cr to be released from a damaged target cell. This can be overcome experimentally by following the standard 37 °C incubation with a further incubation at 45 °C during which there are no new lytic events but all previously damaged target cells release the remainder of their releasable ⁵¹Cr. The model enables one to obtain accurate measurements of relative effector cell frequency over a broad range of experimental conditions.     

Quantitative studies on RNA accumulation in human PHA-stimulated lymphocytes during blast transformation

The net RNA accumulation in PHA-stimulated individual lymphocytes during blast transformation was assayed. The RNA content of the cells was measured by UV microspectrophotometry and the concentration of ribosomes in the cytoplasm of non-stimulated and transforming lymphocytes was determined by counting RNP particles in electronmicrographs.The mean RNA content of non-stimulated lymphocytes was 1.9 ± 0.8 × 10⁻¹²g/cell. During transformation the RNA content increased considerably, the maximal increase being about ten-fold. This increase was proportional to the increase in cellular dry mass. In fact, the RNA concentration was constant during transformation and approximately the same in non-stimulated and transforming lymphocytes. Furthermore the ribosome concentration was approximately the same in the cytoplasm of non-stimulated and transforming lymphocytes. However, a pronounced transition of single ribosomes to ribosomal clusters occurred during transformation and was one of the earliest structural signs of stimulation observed.     

DNA repair replication,DNA breaks and sister-chromatid exchange in human cells treated with adriamycin in vitro

The effects of adriamycin (AM) on DNA repair replication, the frequency of sister-chromatid exchange (SCE), the rate of cell proliferation and the frequency of DNA strand breaks were studied in human cells in vitro. No repair replication was observed in lymphocytes exposed to AM in concentrations up to 10^?3 moles/1. DNA repair replication induced by UV and alkylating agents was not affected by a concentration of AM that completely inhibited cell proliferation (10^?6 moles/1).Fibroblasts exposed to AM at 10^?4 moles/1 in the presence of hydroxyurea showed an increase of strand breaks and cross-links in DNA. When AM was added to UV-irradiated fibroblasts, there was an increase of DNA strand breaks in addition to the breaks caused by UV alone. Similar effects were observed in lymphocytes.A dose-dependent increase of SCE was observed in lymphocytes exposed to low concentrations of AM (<10^?7 moles/1). At higher concentrations the increase of SCE levelled off, and cell proliferation became severely inhibited. There was no evidence of removal of SCE-inducing damage in cells exposed to AM during G₀ or G₁. The level of SCE induced in the third cell cycle after treatment with AM was not different from that induced during the first two cell cycles.These results suggest that the various genotoxic and cytotoxic effects of AM are caused by different types of cellular damage. Moreover, AM-induced DNA damage persists for several cell cycles in human cells in vitro and seems to be resistant to repair activity.