In the presence of ferritin, visible light induces lipid peroxidation of the porcine photoreceptor outer segment

We studied the synergistic effect of visible light and ferritin on the lipid peroxidation on a fraction of porcine photoreceptor outer segment (POS). Reaction mixtures containing the POS fraction and horse spleen ferritin were irradiated under white fluorescent light mainly at 17,000 lx or incubated under dark conditions at 37°C. The lipid peroxidation was evaluated by both the thiobarbituric acid method and the ferrous oxidation/xylenol orange method. The irradiation-induced lipid peroxidation was affected by some experimental factors such as the irradiation dose and acidity of the material. When the irradiation was stopped, the lipid peroxidation was also stopped; thereafter, the re-irradiation induced lipid peroxidation. Moreover, this lipid peroxidation was inhibited by desferrioxamine, an iron chelator, or by dimethylthiourea, a hydroxyl radical scavenger, suggesting that the lipid peroxidation involves hydroxyl radicals generated via the Fenton reaction by iron ion released from ferritin. The lipid peroxidation did not take place under dark conditions or in the absence of ferritin. This study suggested the possibility that the visible light-induced lipid peroxidation of the POS fraction in the presence of ferritin may participate in the etiology of human retinal degenerative diseases as the human retina is exposed to light for life.     

In the presence of ferritin,visible light induces lipid peroxidation of the porcine photoreceptor outer segment

We studied the synergistic effect of visible light and ferritin on the lipid peroxidation on a fraction of porcine photoreceptor outer segment (POS). Reaction mixtures containing the POS fraction and horse spleen ferritin were irradiated under white fluorescent light mainly at 17,000 lx or incubated under dark conditions at 37°C. The lipid peroxidation was evaluated by both the thiobarbituric acid method and the ferrous oxidation/xylenol orange method. The irradiation-induced lipid peroxidation was affected by some experimental factors such as the irradiation dose and acidity of the material. When the irradiation was stopped, the lipid peroxidation was also stopped; thereafter, the re-irradiation induced lipid peroxidation. Moreover, this lipid peroxidation was inhibited by desferrioxamine, an iron chelator, or by dimethylthiourea, a hydroxyl radical scavenger, suggesting that the lipid peroxidation involves hydroxyl radicals generated via the Fenton reaction by iron ion released from ferritin. The lipid peroxidation did not take place under dark conditions or in the absence of ferritin. This study suggested the possibility that the visible light-induced lipid peroxidation of the POS fraction in the presence of ferritin may participate in the etiology of human retinal degenerative diseases as the human retina is exposed to light for life.     

Lipid peroxidation of rat spleen and thymus lymphocytes treated with x-rays (0-5-1.0 Gy)

It is established, that low doses of X-ray irradiation have affected activation of lipid peroxidation (LPO) in immunocompetent cells of the spleen and thymus. The amount of malonic dialdehyde (MDA) in lymphocytes of spleen and thymocytes increases 2 times twenty-four hours after animals' irradiation by X-rays in a dose of 0.5 Gy; when a dose grows to 1.0 Gy, the MDA content in the spleen lymphocytes increases from the 1st to the 6th days and in thymocytes from the 1st to the 3d days reaching its maximum at the 3d day. MDA accumulation in the immunocompetent cells of irradiated animals varies depending on the method of lipid peroxidation initiation.     

Positively selected Lyt-2+ and Lyt-2- mouse T lymphocytes are comparable, after Con A stimulation, in release of IL 2 and of lymphokines acting on B cells, macrophages, and mast cells, but differ in interferon production

Purified mouse T lymphocytes were separated into Lyt-2+ and Lyt-2- populations by the procedure of panning, in which a monoclonal rat anti-Lyt-2 antibody and dishes coated with affinity-purified mouse anti-rat Ig antibodies were used. The populations obtained were 95 to 99% pure as determined by immunofluorescence. Graded doses of these T cells were cultured with optimal mitogenic doses of concanavalin A and the 0 to 24 and 24 to 48-hr culture supernatants were collected. The dose-curve assays of the supernatants of Lyt-2+ and Lyt-2- cells showed comparable activity in interleukin 2 (IL 2) and T cell-replacing factor (TRF), assayed on antigen-stimulated culture of T-depleted spleen cells. Limiting dilution assays of IL 2-secreting precursor cells stimulated by Con A showed a high frequency of precursors in both populations, slightly higher among Lyt-2- cells. The supernatants also contained comparable levels of IPA (inducer of plasminogen activator production by the macrophages), MAF (macrophage-activating factor, assayed by induction of their cytolytic function), and MCGF (mast cells growth factor, assayed on a mast cell line). IPA and MAF were not produced with the same kinetics and in the same T cell concentration conditions as IL 2 and TRF. In contrast, interferon was principally produced by the Lyt-2+ cells.     

Bactericidal activity of photocatalytic TiO(2) reaction: toward an understanding of its killing mechanism.

When titanium dioxide (TiO(2)) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of Escherichia coli K-12 cells that are irradiated in the presence of the TiO(2) photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol. mg (dry weight) of cells(-1) after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO(2). We concluded that TiO(2) photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the E. coli cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.     

Bactericidal Activity of Photocatalytic TiO2 Reaction: toward an Understanding of Its Killing Mechanism

When titanium dioxide (TiO₂) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of Escherichia coli K-12 cells that are irradiated in the presence of the TiO₂ photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol · mg (dry weight) of cells⁻¹ after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO₂. We concluded that TiO₂ photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the E. coli cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.     

A human myeloma cell line that does not express immunoglobulin but yields a high frequency of antibody-secreting hybridomas

We selected an 8-azaguanine-resistant variant of a human myeloma cell line (RPMI 8226) by cloning the parental cells on a feeder layer of mouse spleen cells in the presence of increasing concentrations of 8-azaguanine. Culture media and cellfree extracts of both the parental and variant (8226 AR/NIP4-1) cell lines were assayed for production of immunoglobulin heavy and light chains by double immunodiffusion and for lambda-chain by radioimmunoassay. Secretion of free lambda-chain by the parental cell line was confirmed. In contrast, no immunoglobulin heavy or light chains were detected in culture medium of the variant cell line by either immunodiffusion or radioimmunoassay. No intracellular lambda-chain could be detected in the variant cells by radioimmunoassay of cellfree extracts or by immunofluorescence of fixed cells. Hybridomas were produced by fusion of 8226AR/NIP4-1 cells with lymphocytes from a mesenteric lymph node recovered at surgery from a hypertransfused renal transplant recipient. Twenty hybrid culture supernatants were assayed for immunoglobulin by double immunodiffusion, and 15 contained either IgG (lambda) or IgG (kappa). None produced IgM or IgA. An IgG (kappa)-producing hybridoma was shown by immunofluorescence not to express lambda-chain. A second fusion between the variant cell line and spleen cells from a renal transplant patient produced a stable hybridoma secreting IgM (lambda) antibody specific for the I antigen.     

Melatonin Decreases Nitric oxide Production, Inducible Nitric oxide Synthase Expression and Lipid Peroxidation Induced by Venezuelan Encephalitis Equine Virus in Neuroblastoma Cell Cultures

Increased expression of inducible nitric oxide synthase has been shown in murine Venezuelan equine encephalitis (VEE) virus infection. In this experimental model, melatonin (MTL) treatment has shown to be beneficial. The aim of this study was to determine the effect of VEE virus on the nitric oxide (NO) production and lipid peroxidation in neuroblastoma cell cultures, and to investigate the role of MTL during cell-virus interaction. Neuroblastoma cells were co-cultured with VEE virus and treated with MTL at doses ranging from 0 to 1.8 mM, for 6, 12, 24 and 48 h. NO and lipid peroxidation were measured in culture supernatants and in the cellular content by nitrite concentration and thiobarbituric acid assay, respectively. Expression of inducible nitric oxide synthase (iNOS) was determined by indirect immunofluorescence. Increased production of NO and lipid peroxidation products were found in supernatants and cellular contents of VEE virus treated cultures. Both NO and lipid peroxidation were decreased by MTL treatment in a time dependent manner. Increased iNOS expression was observed in VEE virus infected cultures that was reduced by MTL treatment. These results could be related to the beneficial role of MTL in the VEE experimental disease and address the possible therapeutic potential of the hormone in human VEE virus infection.     

Effect of splenosid on lipid peroxidation process and glutathione antioxidant system in rats exposed to fractionated radiation

The dynamics of lipid peroxidation, antioxidant glutathione system condition in blood and viscerals (brain, heart, liver, spleen) of rats which were fractionally irradiated (10 fractions) in the total dose 1.0 Gy and oxidative homeostasis increase of primary and secondary lipid peroxidation products and glutathione system disturbances were established in the irradiated rats. The administration of splenosid diminished the disturbances of oxidative homeostasis but does not completely normalize the latter. The administration of splenosid during the irradiation course and after its finishing is more effective than only during the irradiation course.     

In situ control of cell adhesion using photoresponsive culture surface

A photoresponsive culture surface (PRCS) allowing photocontrol of cell adhesion was prepared with a novel polymer material composed of poly(N-isopropylacrylamide) having spiropyran chromophores as side chains. Cell adhesion of the surface was drastically enhanced by the irradiation with ultraviolet (UV) light (wavelength: 365 nm); after subsequent cooling and washing on ice, many cells remained in the irradiated region, whereas most cells were removed from the nonirradiated region. The cell adhesion of the PRCS, which had been enhanced by previous UV irradiation, was reset by the visible light irradiation (wavelength 400-440 nm) and the annealing at 37 degrees C for 2 h. Also it was confirmed that the regional control of cell adhesion was induced several times by repeating the same series of operations. Further, living cell patterning with the 200 microm line width was produced readily by projecting UV light along a micropattern on the PRCS on which the living cells had been seeded uniformly in advance. By using a fluorescent probe that stains living cells only, it was confirmed that the cells maintained sufficient viability even after UV light irradiation followed by cooling and washing.     

Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection.

Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals.     

Induction of murine B cell proliferation and immunoglobulin synthesis by some bacterial ribosomes

Ribosomal preparations from Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, and Streptococcus pneumoniae were investigated with respect to their activating capacity towards murine lymphoid cells. The proliferation of BALB/c spleen cells was induced in a dose-dependent fashion (from 1 to 100 micrograms/ml) by ribosomes of K. pneumoniae, H. influenzae, and S. pyogenes with a peak activity at 48 or 72 hr of culture. The majority of the blast cells induced by these ribosomal preparations were positive for surface-immunoglobulin (S-Ig) and negative for Thy 1.2. Furthermore, K. pneumoniae, H. influenzae, and S. pyogenes ribosomes induced the synthesis of IgM and some IgA. Cell proliferation and induction of IgM production were also demonstrated with the 3 ribosomal preparations using spleen cells from athymic nude (nu+/nu+) mice, Lyb-5-defective CBA/N spleen cells, B cell-enriched and T cell-depleted BALB/c spleen cell suspensions, as well as spleen cells from the Ips gene-deficient C3H/HeJ strain. Cell culture supernatants contained specific anti-ribosome IgM antibodies. Antibodies of other specificities (anti-sheep erythrocytes) were also demonstrated in supernatants from K. pneumoniae-stimulated cultures. Evidence against a possible role of contamination of K. pneumoniae and H. influenzae ribosomes by lipopolysaccharide- or lipid A-associated proteins in this effect is discussed. Ribosomes from S. pneumoniae did not induce 3H-thymidine incorporation nor Ig production. None of the 4 ribosomal preparations was found to stimulate T cell blastogenesis or to induce interleukin-2 production by naive BALB/c spleen cells. Finally, ribosomes from H. influenzae, S. pyogenes, S. pneumoniae but not those of K. pneumoniae stimulated interleukin-1 production by adherent spleen cells, from BALB/c mice.     

Protective Effect of Carvacrol on Oxidative Stress and Cellular DNA Damage Induced by UVB Irradiation in Human Peripheral Lymphocytes

Exposure to ultraviolet B (UVB; 280‐320 nm) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of carvacrol on UVB‐induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular an‐tioxidant status in human lymphocytes. A series of in vitro assays (hydroxyl radical, superoxide, nitric oxide, DPPH (2,2‐Diphenyl‐1‐picryl hydrazyl), and ABTS (2,2‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid) radical scavenging assays) demonstrate antioxidant property of carvacrol in our study. UVB exposure significantly increased thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LHPs), % tail DNA and tail moment; decreased % cell viability and antioxidant status in UVB‐irradiated lymphocytes. Treatment with carvacrol 30 min prior to UVB‐exposure resulted in a significant decline of TBARS, LHP, % tail DNA, and tail moment and increased % cell viability as carvacrol concentration increased. UVB irradiated lymphocytes with carvacrol alone (at 10 μg/mL) gave no significant change in cell viability, TBARS, LHP, % tail DNA, and tail moment when compared with normal lymphocytes. On the basis of our results, we conclude that carvacrol, a dietary antioxidant, mediates its protective effect through modulation of UVB‐induced ROS.     

Immunostimulatory effect of<Emphasis Type="Italic">Bacillus firmus</Emphasis> on mouse lymphocytes

Bacillus firmus (a Gram-positive nonpathogenic and harmless bacterium), was shown to be a strong polyclonal activator of mouse B lymphocytes as estimated by ELISA testing of Ig concentrations in culture supernatants after incubation of BALB/c mouse splenocytes with inactivated bacillus. Synthesis of all main Ig classes and all IgG subclasses was stimulatedin vitro, the considerable effect on IgA formation being the most interesting feature. B cell stimulation was T cell dependent, as was demonstrated by the effect ofB. firmus on all Ig isotypes and by comparison of lymphocyte response of nu/nu mice and heterozygous nu/+mice. The effect ofB. firmus on splenocyte proliferation was stimulatory or suppressive depending on the dose of the bacterium. Increased synthesis of IFN-γ and IL-10 (detected by ELISA in splenocyte culture supernatants) showed probable stimulation of Th1 and Th2 subpopulations. Considering the stimulatory effect on IgA formation and macrophage stimulation,B. firmus seems to be a prospective mucosal adjuvant and/or probiotic.     

Effect of ursolic acid, a triterpenoid antioxidant, on ultraviolet-B radiation-induced cytotoxicity, lipid peroxidation and DNA damage in human lymphocytes

Exposure to ultraviolet B (UVB, 280-320) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of ursolic acid on UVB-induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular antioxidant status in human lymphocytes. Series of in vitro tests (hydroxyl radical, superoxide, nitric oxide, DPPH and ABTS radical scavenging assays) demonstrates antioxidant property of ursolic acid in our study. Treatment of lymphocytes with ursolic acid alone (at 10 microg/mL) gave no significant change in cell viability, thiobarbituric acid reactive substances (TBARSs), lipid hydroperoxides (LHPs), % tail DNA and tail moment when compared with normal lymphocytes. UVB-exposure significantly increased TBARS, LHP and % tail DNA, tail moment; decreased % cell viability and antioxidant status in irradiated lymphocytes. Treatment with ursolic acid 30 min prior to UVB-exposure resulted in a significant decline of TBARS, LHP, % tail DNA and tail moment and increased % cell viability as ursolic acid concentration increased. Based on our results we conclude that ursolic acid, a dietary antioxidant, mediates its protective effect through modulation of UVB-induced reactive oxygen species.     

Enhancement of murine B cell responses to lipopolysaccharide by supernatants of irradiated peripheral blood leukocytes

Summary At low cell density, the proliferative response of B cells to lipopolysaccharide (LPS) is not detectable. We investigated under these experimental conditions the role of several cell populations on the LPS-induced B-cell proliferation. The addition to murine B cells of irradiated peripheral blood leukocytes (PBL) from the C3H/ HeJ mouse strain, or of culture supernatants of these cells, efficiently restored a response to LPS. Similar results were also obtained with irradiated PBL from other mouse strains and from rabbits. The activities of the culture supernatants were not significantly modified when the PBL were depleted of adherent cells or of Thy-1.2 positive cells, thus suggesting that the active factors were secreted neither by T cells, nor by monocytes.Abbreviations BSS balanced salt solution - ConA concanavalin A - EBMR enhancement of B-cell mitogenic response - J-B, J-T, J-Th, J-MØ, J-PBL, J-RBC splenic bone marrow-derived lymphocytes, splenic thymus-derived lymphocytes, thymocytes, splenic macrophages, peripheral blood leukocytes, red blood cells, obtained from the LPS-non-responding C3H/ HeJ-Pas mouse strain - R-PBL peripheral blood leukocytes obtained from the LPS-responding C3H/ He-Pas mouse strain - LPS lipopolysaccharide - MO macrophages - PBL peripheral blood leukocytes     

Lipopolysaccharide induction of immunoglobulin synthesis in irradiated lymphocytes.

Normal or heavily irradiated mouse spleen cells were stimulated to immunoglobulin synthesis by lipopolysaccharides (LPS). Total immunoglobulin synthesis was measured at the cellular level by plaque formation. It was found that only a subpopulation of lymphocytes is inducible after irradiation. Lymphocytes in that subpopulation are larger than small lymphocytes and do not adhere to plastic; their resistance to irradiation appears to be related to their cycling state, not to their differentiation stage.     

p53-Mediated gene activation in mice at high doses of chronic low-dose-rate γ radiation

The time course of the changes in the expression of p53-mediated genes in vivo after high doses of chronic low-dose-rate γ radiation remains unclear. Here we analyzed peripheral blood cell counts and the expression of p53-mediated genes in the spleens of mice chronically irradiated at low dose rate (0.0167 Gy/h) for 1-40 days. Low-dose-rate irradiation induced p53-dependent chronic decreases in white blood cell (WBC) counts in p53 wild-type mice. Upregulation of p53-mediated genes by low-dose-rate radiation was confirmed in the whole spleen cells from the p53 wild-type mice, while suppressed gene expression was observed in the spleen cells of p53-deficient mice. The expression of p21 and Bax in radiosensitive cells such as T and B lymphocytes from low-dose-rate irradiated mice at 10, 20, and 40 days were increased, although that of Mdm2 in both the lymphocytes was decreased at 20 and 40 days. Moreover, spleen weights for low-dose-rate irradiated mice were decreased at 20 and 40 days. Thus downregulation of Mdm2 in both T and B lymphocytes by low-dose-rate radiation may cause higher p53 activation; further, higher p53 expression may determine the radiosensitivity and cause a reduction in the spleen weights in low-dose-rate irradiated mice. These results indicate that p53 may be chronically activated by low-dose-rate radiation.     

Induction of interleukin-3 by interleukin-1 in the absence of other exogenous stimuli

We have previously reported that lipopolysaccharide (LPS) could induce the production of interleukin-3 (IL-3) by mouse spleen cells. In the present study, we show that recombinant human interleukin-1, in the absence of other stimuli, is able to induce the production of IL-3. IL-3 was detected in the supernatants of adult, although neither in young nor in nude mouse splenocytes and was assessed by its capacity to support the growth of the IL-3-dependent FDC-P2 cell line. The presence of IL-3 was antigenically confirmed with a monoclonal anti-IL-3 antibody. Both recombinant IL-1 alpha and IL-1 beta had similar potential for inducing IL-3 production. IL-3 activity was detected in the supernatants of cells cultured in the presence of 100 pg/ml IL-1; maximal IL-3 levels were obtained with 10-30 ng/ml IL-1. Kinetic studies of IL-1-induced IL-3 production indicated that 4-6 days of culture were required for optimal production, whereas 1-2 days were sufficient in cultures stimulated with concanavalin A. Recombinant IL-6 failed to induce significant amounts of IL-3, and TNF alpha induced only weak IL-3 production. GM-CSF but not M-CSF could lead to the appearance of IL-3 in spleen cell culture supernatants. Removal of macrophages decreased the production of IL-3 induced by LPS and GMF-CSF though did not affect the IL-3 production induced by IL-1. This observation suggests that IL-1 production might be an intermediate event in IL-3 production induced by LPS and GM-CSF through the activation of macrophages. IL-3 was detected in culture supernatants of B-cell-depleted splenocytes indicating that T-cells were the source of IL-3. Surprisingly T-cell-depleted populations could also produce IL-3 upon IL-1 stimulation. Preliminary experiments with an autoreactive CD4- CD8- V beta 8+ clone suggested that these cells might also be involved in the described IL-3 production.     

T cell growth factor from human splenic cell cultures: conditions for its production, and its utilization for maintenance of cytotoxic T cell lines

We prepared the T cell growth factor (TCGF) from human spleen cell cultures stimulated with phytohemagglutinin (PHA). Various cell culture conditions and agents supporting the active TCGF production of the spleen cells were examined. The highest TCGF activity was obtained in the supernatants under the conditions that 2 x 10(6)/ml spleen cells were stimulated with PHA for 48 hr. Production of TCGF from spleen cells depended markedly on their individual sources. Addition of indomethacin to the culture or irradiation of the responding spleen cells increased TCGF activity in the supernatant of the culture. Further, addition of irradiated cells of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) to spleen cell cultures stimulated with PHA greatly enhanced TCGF production. Human splenic TCGF facilitated the establishment of human cytotoxic T cell (Tc) lines specific for EBV-transformed LCL cells when those Tc line cells were stimulated periodically with irradiated autologous LCL cells but not with the other two types (K-562 or Molt-4) of cells. Allogeneic LCL stimulators allowed the Tc line cells to proliferate. However, Tc line cells cocultured once with allogeneic LCL stimulators no longer exhibited EBV-specificity in their cytotoxicities.