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BA10-IR transformed cells, obtained by treating Syrian hamster embryo fibroblasts (HEF) with 7-methylbenz(a)anthracene and cultivated for a long period, are highly tumorigenic and grow in suspension as aggregates (spheroids) (Levy et al., 1976). They also grow in attached form or as spheroids in serum-free (S-) synthetic medium, without insulin and transferrin, and form anchorage-independent (AI) colonies in this same, but semi-solid, medium. This exceptional phenotype was acquired stepwise, after other transformation parameters, and appears to be related to the capacity of the transformed cells to respond to a mitogenic growth factor which they secrete. The response to this autocrine factor is amplified by insulin and transferrin. Untransformed HEF, at late and early passages, and also mouse and rat embryo fibroblasts, secrete factors equally active on BA10-IR cells; but HEF do not respond, in S- medium, to their factor, or that of BA10-IR cells. Rat FR3T3 fibroblasts transformed by Kirsten murine sarcoma virus (FR3T3-Ki cells) also form AI colonies in semi-solid S- medium, secrete an autocrine factor potentiated by insulin and transferrin, and respond to the factors active on BA10-IR cells. However, they form far fewer colonies without additives, and respond as well to the mitogenic factors only in the presence of insulin and transferrin. BA10-IR cells and FR3T3-Ki cells also release beta-TGF, or a related factor, in an active and a latent form, activable by acidification, and HEF latent, activable beta-TGF. However, the factors shed by BA10-IR cells or HEF which stimulate AI growth of BA10-IR and FR3T3-Ki cells are proteins which seem unrelated to known transforming growth factors. Two major cellular alterations characteristic of the transformed phenotype in vitro are the ability to grow in the absence of anchorage, in semi-solid medium, and reduced dependence on serum growth factors (Hanafusa, 1977; Tooze, 1980). These alterations are often expressed together, and anchorage independence also appears to be the in vitro transformation parameter which correlates best with the tumorigenicity of the transformed cells (Pollack et al., 1975; Shin et al., 1975; Cifone and Fidler, 1980). However, this correlation is not constant (cf., Tooze, 1980). The cellular changes which confer anchorage independence remain unknown, but the culture conditions which allow anchorage-independent (AI) growth are better known. This growth occurs in the same media which permit the growth of attached cells, but generally requires serum.(ABSTRACT TRUNCATED AT 400 WORDS)

Transforming Growth Factors (TGFs) are a sub-group of a larger family of protein hormones. Two major types of TGF are currently known, alpha-TGF and beta-TGF. Biologically their most important property is to act synergistically to induce anchorage-independent growth of target cells otherwise incapable of such growth. Since this growth parameter is well correlated with tumorigenicity in vivo, these factors may play a role in cancer development. Biochemically both alpha-TGF and beta-TGF have been well characterised in some types of cells and tissues. Their role in normal and neoplastic growth is actively studied.

The humoral and cell-mediated immune responses of murine lymphocytes exposed to lead in vivo and in vitro were investigated. In vivo Pb was administered via the drinking water (0 to 10 mM) for 1 to 10 weeks. In vivo exposure of the mice to Pb did not alter significantly their plaque-forming cell response to sheep erythrocytes; however, their susceptibility to Listeria infection was reduced significantly with Pb dosages of greater than 0.4 mM. Although the in vivo plaque-forming cell responses did not appear to be altered, in vitro assessment of the reactivity of these in vivo Pb-exposed lymphocytes indicated that intermediate doses enhanced, but a high dose (10 mM) was suppressive. The 10 mM in vivo Pb dose suppressed the in vitro plaque-forming cell response, the mixed-lymphocyte culture response, and lipopolysaccharide-induced proliferation, but it did not affect concanavalin A- or phytohemagglutinin-induced proliferation. Interestingly, in vitro Pb exposure (10(-6) to 10(-4) M) of murine spleen cells caused an enhancement of most activities even though these in vitro concentrations of Pb were slightly above the in vivo concentrations. Direct in vitro Pb effects on the lymphocytes could be measured, and Pb consistently enhanced humoral and cell-mediated immunity.

Cultured bovine aortic endothelial cells are associated with an unusually stable fibrinolytic inhibitor (Loskutoff, D.J., van Mourik, J.A., Erickson, L.A., and Lawrence, D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2956-2960). This inhibitor was purified to apparent homogeneity from medium conditioned by these cells by a combination of concanavalin A affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a single-chain glycoprotein of apparent Mr 50,000 +/- 2,500 and isoelectric point of 4.5-5.0, and inhibits the ability of both urokinase and tissue-type plasminogen activator to cleave and active plasminogen. This inhibition of plasminogen activator activity is associated with the formation of an enzyme-inhibitor complex which can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified inhibitor retains full activity after incubation in the presence of 0.1% sodium dodecyl sulfate, or at pH 2.7, two treatments which rapidly destroy the activity of protease nexin, another cellular inhibitor of fibrinolysis. The inhibitor purified from cloned endothelial cells cultured in the presence of L-[3,4,5-3H]leucine represented 2.5-12% of the total radiolabeled protein released by the cells in a 24-h period. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a protein which inhibits plasminogen activators and is distinct from protease nexin. It is a major endothelial cell product, and, as such, probably plays an important role in regulating the fibrinolytic system of these cells.

A new technique, reverse fibrin autography, was developed to detect protease inhibitors previously fractionated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Exogenous proteases were incorporated into fibrin-agar indicator films, eventually causing the fibrin to lyse. When an acrylamide gel containing inhibitors was placed on top of such an indicator, the positions of the inhibitors were revealed by the formation of opaque, lysis-resistant zones in the otherwise cleared fibrin film. The technique was versatile in that a variety of inhibitors were revealed, and semiquantitative since the size of the lysis-resistant zone in the indicator increased in proportion to the amount of inhibitor subjected to electrophoresis. This approach could be used not only to detect inhibitors having different protease specificities, but also to distinguish between the inhibitor activities of antibodies directed against urokinase or tissue-type plasminogen activator. Thus, reverse fibrin autography offers a convenient new approach to rapidly screen and partially characterize inhibitors present in complex biological samples.

Glutathione (GSH) the most abundant nonprotein thiol, is involved in the maintenance of the cellular redox state. In this capacity it may influence lymphocyte responsiveness to various stimuli. We have investigated the requirement of GSH during the activation and proliferation of PBMC. The intracellular GSH content of PBMC was altered by continuous culture or pretreatment with buthionine-S,R-sulfoximine (BSO), a specific and irreversible inhibitor of GSH synthesis. Initial experiments demonstrated that the addition of BSO at the initiation of culture, or shortly thereafter (6 hr), inhibited DNA synthesis and produced a simultaneous decrease in intracellular GSH. It was necessary that the BSO be present in the culture for at least 24 hr prior to the initiation of DNA synthesis for maximal inhibition. Cell cycle analysis revealed that BSO did not affect the entry and progression of PBMC through G1 of the cell cycle, however, entry into S-phase was inhibited in a dose-dependent fashion. These results were further substantiated by the inability of BSO to inhibit IL-2 production and expression of the IL-2R. In addition the timely expression of the transferrin receptor by BSO-treated cells indicated that the block occurred at the G1/S transition. The influence of GSH on early activation events was determined by BSO pretreatments. Lowering the intracellular GSH level of PBMC to less than 10% of the initial content prior to mitogenic stimulation did not impair the ability of these cells to produce IL-2 and express IL-2R, indicating that GSH may not be involved in the generation and response to early activation signals. Furthermore, the removal of BSO from these cultures rapidly reversed its inhibitory effects on DNA and GSH synthesis. In the course of these studies we also observed a modest (17%) albeit consistent increase during activation in the total thiol levels of GSH-depleted PBMC. These thiols may have a key role in the activation process. These data support our hypothesis that GSH is required for lymphocyte proliferation and that additional thiols are involved during the activation process.

The rapidly acting plasminogen activator inhibitor (PAI) purified from cultured bovine aortic endothelial cells (BAEs) was inactivated during iodination with chloramine T and other oxidizing iodination systems. Inactivation was observed in the absence of iodine, suggesting that the loss of activity resulted from the oxidizing conditions employed. In an attempt to further study the nature of this inactivation, the PAI was treated with chloramine T under conditions that specifically oxidize methionine and cysteine residues. Both PAI inhibitory activity and the ability of the PAI to form complexes with tissue-type PA were decreased in a dose-dependent manner by such treatment. The PAI was more sensitive to oxidative inactivation than urokinase, elastase, and alpha 1-protease inhibitor. Incubation of the chloramine T inactivated PAI with methionine sulfoxide peptide reductase in the presence of dithiothreitol (DTT) restored more than 90% of the PAI activity. The reductase is a DTT-dependent enzyme that specifically converts methionine sulfoxide to methionine. Little activity was restored by either the reductase or DTT alone. These results indicate that the oxidation of at least one critical methionine residue is responsible for the loss of PAI activity upon iodination. In this respect, the BAE PAI resembles alpha 1-protease inhibitor, a well-characterized elastase inhibitor that also is inactivated by oxidants. Both inhibitors are members of the serine protease inhibitor superfamily (Serpins), and both have a methionine residue in their reactive center.

Transformation of rat NRK-49F cells (49F) by Kirsten murine sarcoma virus (Ki-MSV) renders these cells (Ki-49F cells) capable of autonomous anchorage independent (AI) growth. As compared to nontransformed 49F cells, the transformation by Ki-MSV does not modify the cell response to transforming growth factor-beta (TGF-beta) in monolayer conditions, but alters it in A I growth conditions. The growth of nontransformed or Ki-MSV-transformed adherent 49F cells is slowed down by porcine TGF-beta, and this effect is reversed by epidermal growth factor (EGF). This decrease in the cell growth rate, induced by TGF-beta, does not affect the cloning efficiency of untransformed and transformed adherent 49F cells. Contrarily, porcine TGF-beta decreases the A I cloning efficiency of Ki-49F cells in agar-gelled medium; this effect is only partly reversed by EGF, which does not synergise with TGF-beta to enhance the A I growth as in the case of untransformed 49F cells. Media conditioned by 49F cells, Ki-49F cells, and chicken embryo fibroblasts contain a latent TGF-beta whose capacity to promote the A I growth of 49F cells and to inhibit that of Ki-49F cells is unmasked by acidification. The same situation exists concerning TGF-beta from human platelets. Neutral extracts are inefficient in both tests of promotion and inhibition of A I growth and contain an acid-activable component with an apparent molecular weight of 600 kd. In acid extracts, a 5-9 kd apparent molecular weight component is responsible for the A I growth enhancement of 49F cells and the A I growth inhibition of Ki-49F cells. Further purification by reverse phase chromatography shows that both activities strictly coelute at the same point (32%) of an acetonitrile gradient. These results indicate that TGF-beta is present in physiological conditions as a latent form which requires activation for inhibiting the A I growth of transformed cells as well as for enhancing that of 49F cells.

An increase in the concentration of cytosolic Ca+2 ([Ca-2]i) is among the earliest changes seen in mitogen-stimulated lymphocytes and is a consequence of signal transduction which usually results in the initiation of cell cycle progression. However, increased [Ca+2]i has also been correlated with cytotoxicity. We have determined whether modulations of [Ca+2]i are involved in the functional inactivation of cells observed with sublethal concentrations of oxidants. Specifically, [Ca+2]i was measured in mouse splenic lymphocytes that were treated with different oxidants in order to determine if oxidative stress interferes with mitogen-stimulated increases in [Ca+2]i, if oxidants themselves modulated [Ca+2]i, and, if so, whether such Ca+2 modulations by oxidants had stimulatory or inhibitory effects on the response of lymphocytes to mitogens. The oxidants employed were copper phenanthroline (CuP; surface thiol oxidizer), N-ethyl maleimide (NEM; permeant thiol alkylator), hydrogen peroxide (H2O2; generates hydroxyl radical within the cell), and radiation (Cs137; generates hydroxyl radical by radiolysis). Growth of all treated cells was equally inhibited upon stimulation with Con A or PMA/A23187, suggesting that all the oxidants inhibited cell functions required distal in activation to the transduction pathway utilized by Con A but bypassed by PMA/A23187. Doses of CuP, NEM, and radiation which fully inhibited Con A-stimulated proliferation had little effect on resting or mitogen-stimulated changes of [Ca+2]i, but H2O2 doses which fully inhibited proliferation increased [Ca+2]i in unstimulated cells and prevented the increase normally caused by Con A. Both intra- and extracellular Ca+2 contributed to the increased [Ca+2]i seen in unstimulated cells. An elevated [Ca+2]i was sufficient to reduce responsiveness, since pharmacologically increasing the [Ca+2]i with the ionophore A23187 rendered lymphocytes less responsive to Con A. Unlike A23187, H2O2 was unable to synergize with PMA, suggesting that the H2O2-induced increase of [Ca+2]i delivered predominantly negative signals to the cell. The results also suggest that [Ca+2]i utilization by Con A versus PMA-activated lymphocytes must be different. When cells were treated with H2O2 under conditions where intracellular and extracellular Ca+2 were chelated with BAPTA and EGTA, respectively, the response to Con A was restored. Under these conditions, higher concentrations of H2O2 were required to inhibit the response to Con A. Our results indicate that signal transduction may be compromised in cells treated with H2O2, but not in cells treated with CuP, NEM, or radiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Normal rat kidney cells of the clone 49F and their Ki-MSV-transformed counterparts spontaneously release the same transforming growth factor (TGF) activity in an inactive form. By acidification followed by neutralization prior to assay, this TGF activity is unmasked and promotes anchorage-independent growth of the NRK-49F indicator cells in the presence of epidermal growth factor. The TGF activity released by both cell types has an apparent molecular weight of 9,000 under acidic conditions, does not compete for binding to epidermal growth factor receptors, is heat resistant but dithiothreitol and trypsin sensitive, and therefore is of the beta-TGF class.

The immunotoxicity of hydroxyethyl starch (HES), a reagent used in leukocytapheresis or as a plasma expander, was assessed. HES did not significantly alter host resistance to Listeria monocytogenes or Streptococcus pneumoniae. HES (4-32 ml/kg), as well as a physiological saline solution (32 ml/kg), did inhibit the in vitro lymphoproliferation of spleen cells from mice intravenously injected 1 hour prior to removal of the spleens; the proliferation induced by a T-cell and B-cell mitogen was suppressed. However, this suppression was transient, in that, HES and saline injections given 4 and 24 hours prior to removal of the spleens produced no significant inhibition. Unlike the HES effects on lymphoproliferation, HES did suppress the in vivo humoral immune response to sheep erythrocytes (SRBC) when given 24 hours prior to antigen, but this inhibition was obtained only with the 32 ml per kg dose. Interestingly, a similar dose of mouse albumin significantly enhanced the response. In vitro analysis of humoral and cell-mediated immune responsiveness with in vivo treated spleen cells produced results that were not dose dependent. Although HES was more suppressive than saline, both saline and HES were inhibitory. The lack of a dose-dependent effect suggests that the in vitro analysis of in vivo treated cells was not a good index of their in vivo reactivities. The greater variability and apparent sensitivity of the in vitro analysis probably reflect the transient effects of in vivo dilution of serum factors by relatively large intravenous injections and/or the transient effects of injection trauma.

Transdifferentiated embryonic quail neuroretina cells synthesize in vitro crystallins (the lens-specific proteins) and form lentoid bodies (structures that mimic lens fiber cells) which also contain crystallins. A comparative study on the size of crystallins is reported in 7-day-old embryonic quail lenses, in 7-day-old embryonic quail transdifferentiated neuroretina cells (normal and MH2 transformed), and in isolated lentoid bodies. Analyses are performed using Superose FPLC in combination with SDS-PAGE and Western blot procedures. In quail lenses, an apparent 560-580-kDa alpha crystallin homopolymer is found and delta crystallin, the major avian lens protein, is detected as a 180-kDa tetramer. beta Crystallins, present in low amount within the 180-kDa peak, are a heterogeneous population composed of subunits of molecular weight identical to those found in chick lenses. In addition, an apparent 46-kDa monomeric delta crystallin is found. Normal and MH2-transformed neuroretina cultures produce an alpha crystallin polymer of lower molecular weight (450 kDa) and delta crystallin in a monomeric or dimeric form. The Western blot pattern of beta crystallins from MH2-transformed neuroretina cultures is strictly identical to that of quail lens beta crystallins. In particular, the beta B1 crystallin, which is specific to lens fiber cell differentiation, and the major beta 25-kDa crystallin are present. However, analysis of isolated lentoid bodies from normal transdifferentiated quail neuroretina cultures showed alpha and delta crystallins of comparable size to those found in lens extract, in particular the delta crystallin in tetrameric form. The lentoid body lens-like structure could favour the crystallin aggregation process.

Conditioned media from Rous sarcoma virus transformed chicken embryo fibroblasts stimulate the uptake of 2-deoxyglucose in normal chicken fibroblasts. The factor responsible for this effect, which is also shed in very low amount by non-transformed fibroblasts, is destroyed by trypsin and not linked to the protease and plasminogen activator activities present in the media. Its apparent molecular weight, determined by gel filtration, is about 20,000 daltons. The factor released by transformed cells might be related to the monomeric form of a family of glucose binding and transport proteins recently reported by Lee and Lipmann ('78) to be detached by detergents from normal and transformed cells.

Four thiol-modifying compounds were used to inhibit murine lymphocyte mitogenesis. The compounds were a copper sulfate/O-phenanthroline complex (CuP) to oxidize surface thiols, N-ethyl maleimide (NEM) to alkylate surface and intracellular thiols, D,L-buthionine-S,R-sulfoximine (BSO) to prevent synthesis of glutathione, and hydrogen peroxide, which reacts with various cellular constituents, including sulfhydryls. Splenic lymphocytes were incubated with one of the four compounds, washed, and then stimulated with the B cell mitogen, LPS, or the T cell mitogen, Con A. In spite of their differing chemical reactivities and differing effects on cell viability, lipids, and total, protein, and non-protein thiols, the four sulfhydryl-modifying compounds had very similar effects on the kinetics and inhibition of lymphocyte growth. All compounds had complex effects on mitogenesis, causing enhanced, delayed, or inhibited tritiated thymidine incorporation. Although the total thiol contents of untreated T cells and B cells were found to be equivalent, the LPS response consistently was inhibited by lower concentrations than the Con A response, suggesting that B cells were more sensitive than T cells to thiol modification. To compare compounds the efficiency of inhibition was determined by functionally relating reductions in mitogenesis with reductions in thiol content of the cells. The compounds differed in inhibitory efficiency; thus, damage to some thiols must be more important than damage to others. CuP ablated mitogenesis with the least change in thiol content. Therefore, surface sulfhydryls appear critical in lymphocyte mitogenesis. With all compounds inhibition of mitogenesis occurred over a very narrow range of thiol content, suggesting that the thiols important in inhibition were few in number relative to the total thiol content of the cell.

Chicken embryo fibroblasts sensitized by ts RSV respond to TGFs present in the media of non-transformed FR3T3 and NRK-4 rat cells and of the same cells transformed by KiMSV or RSV. They also respond to TGFs present in the media of BHK hamster cells transformed by MoMSV, PyV or RSV. Two other indicator rat cell lines, untransformed NRK-4 and FR3T3, sensitized by ts KiMSV, respond to the same TGF-containing media, and this response is increased by exogenous EGF. Normal FR3T3 cells failed to respond to any of the media. The most sensitive target cells were the ts KiMSV-FR3T3 cells at the restrictive temperature (39.5 degrees C). All the media tested on NRK-49F target cells required EGF for their TGF activity which was essentially dependent on prior activation by acidification. These data show that the above media from non-transformed or transformed cells contain beta-TGFs, with no detectable accompanying alpha-TGF activity. The release of and the response to these TGFs are not interdependent. A function of ts mutant src and k-ras viral oncogenes, still expressed at the restrictive temperature, can sensitize non-responsive cells, without there being any specificity towards the TGF producer cells.

The kinetics of allohelp mediated by diffusable factors revealed that help by nonirradiated T cells (TOR) peaked at 48 to 72 hr, followed by a sharp decline if the T cells remained in the cultures. The temporal decrease in help after 72 hr was not mediated by suppressor lymphokines because mixtures of early (24 to 48 hr) and late (120-hr) allogeneic supernatants enhanced help synergistically. Lyt-1, Ia- T cells mediated the temporal decline in help and suppressed allogeneic B cell activation in co-cultures, and this "down-regulatory" activity (allosuppression) was radiosensitive. Help by irradiated T cells (T1000R) increased gradually until it plateaued between 96 and 120 hr. The helper activities of the allogeneic supernatants were directly proportional to their T cell growth factor (TCGF) activities. In addition, their kinetics were identical, and the removal of TCGF from 48-hr allogeneic supernatants by adsorption with TCGF-dependent HT-2 cells depleted both helper and TCGF activities. Help was restored to depleted 48-hr and 120-hr allogeneic supernatants by preparations of TCGF obtained from concanavalin A (Con A)-stimulated FS6-14.13 hybridoma cells that were adsorbed with lipopolysaccharide (LPS)-activated B cells or normal spleen cells (NS), but not with HT-2 cells. These results indicate that allohelp is dependent on TCGF. Moreover, help was dependent on at least one factor in addition to TCGF, because a high level of synergy occurred between TCGF and the "help-deficient" 120-hr allogeneic supernatant. In conclusion, the mechanism whereby Lyt-1, Ia- T cells regulated B cell activation with positive and negative allogeneic effects was through the production and subsequent exhaustion of TCGF, respectively. The production of TCGF and help was radioresistant, but exhaustion of TCGF and suppression was radiosensitive.

Chicken embryo fibroblasts and hamster BHK cells transformed by Rous sarcoma virus (RSV) release in their culture media growth factors which enhance markedly anchorage-independent colony formation in gelified medium, at the restrictive temperature (41 degrees 5 C), of chicken embryo fibroblasts (CEF) infected by RSV mutants with a ts mutation of the src gene. This action is not observed with uninfected CEF, and, therefore, appears to require some expression of the viral src gene in the target cells. The enhancing factors are proteins related to the family of the transforming growth factors (TGFs) by their molecular weight (about 20 kd), their heat and acid resistance, and their sensitivity to dithiothreitol. They do not compete with 125I EGF for binding on the EGF receptors of the membrane of A431 cells. As chicken embryo fibroblasts are devoid of EGF receptors, their activity is not potentiated by EGF.

Transforming growth factor-beta (TGF-beta) is a multifunctional protein involved in the control of proliferation, differentiation and other functions in many cell types. The anchorage-independent growth of some established lines of untransformed fibroblasts in soft agar is induced by TGF-beta and requires in addition exogenous EGF for certain target cells, notably rat NRK-49 cells. The formation of colonies of NRK-49F cells is completely inhibited by the synthetic 11-beta substituted nor-steroid RU38486 added at a final concentration of 1.3 X 10(-5) M. We also explored the effect of TGF-beta on Daudi and Raji lymphoma cells by measuring the production of Epstein-Barr Virus (EBV) early antigens (EA). In Daudi cells an induction capacity giving rise to 10-16% positive EA-cells was observed; in Raji cells the induction only reached between 6 and 8%. The induction was partially inhibited by the anti-steroid RU38486 in both systems. Thus, RU38486 not only antagonizes the glucocorticoid hormone action but also interferes with the effects of TGF-beta in fibroblasts and in lymphoma cells. The molecular basis of the interactions observed was investigated by considering (1) the binding to specific receptors, (2) transfection experiments, in order to examine if the interference of the anti-steroid with TGF-beta activities occurs at the transcriptional level as in the case of glucocorticoid induction. The results suggest that the blocking by antiglucocorticoids of the effects of TGF-beta and glucocorticoids, in fibroblasts and lymphoma cells, occurs by different mechanisms.

The ability of lipopolysaccharide (LPS) to influence lymphocyte mitogen responsiveness to 2-mercaptoethanol (2-ME) was investigated. 2-ME was mitogenic for splenocytes only when fetal bovine serum lots containing elevated endotoxin levels were used to supplement (5%) the culture medium. 2-ME did not stimulate mitosis of nylon-wool-fractionated splenocytes (NWSC); however, 2-ME induced significant proliferation in the NWSC cell preparations when endotoxin-deficient culture medium was supplemented with doses of LPS which alone did not stimulate significant proliferation. The sIg+ and Thy-1-/Lyt-1- fractions of NWSC, which represented less than 5% of the cells in the 'T-cell preparations', were almost completely dependent on 2-ME for proliferation in response to LPS stimulation. These data indicate that nylon-wool-fractionated preparations contain splenic B cells that incorporate significant amounts of [3H]thymidine when stimulated with LPS and 2-ME.

Methionyl adenylate (Met-AMP) inhibits protein synthesis by interacting with methionyl-tRNA synthetase. Addition of 1--3 mM inhibitor to chick embryo fibroblasts rapidly stops protein synthesis and DNA synthesis but not RNA synthesis. These effects can be reversed by renewal of the medium. The extent and reversibility of protein and DNA syntheses depend on the concentration of MetAMP in the cultures, the length of exposure and the cellular density. MetAMP is recognised by several enzymes as substrate and/or as inhibitor. MetAmp is degraded to methionol plus 5'-adenylic acid by 5'-phosphodiesterase. Adenosine deaminase, adenylic acid deaminase and 3':5'-phosphodiesterase cannot use MetAMP as substrate but the last enzyme is inhibited. The presence of MetAMP in cultures provokes a small but reproducible increase in the level of methionyl-tRNA synthetase and 5'-phosphodiesterase.

We have studied the effect of addition of 30 ml cod liver oil (FO) daily to a highly atherogenic butter (BT) diet for swine on lesion development in the coronary arteries and aorta, plasma lipoprotein (LP) patterns, plasma levels of thiobarbituric acid-reactive substances (TBARS) and on tritiated thymidine-labeling indices ([3H]TdR LI) of smooth muscle cells (SMC) and monocyte/macrophages (M/M phi) in the atherosclerotic lesions. Seventeen male Yorkshire swine (11.1 +/- 0.4 kg) were divided into 3 groups: BT (n = 6), BT + FO (n = 6) and mash (n = 5). They were fed the respective diets for 4 months. Terminally, fasting plasma was obtained and cholesterol contents were determined in various fractions of lipoproteins separated by density gradient ultracentrifugation, Pevikon block electrophoresis and immunoelectrophoresis. Apoprotein (B, A-I, E and C) contents of the plasma and lipoprotein fractions were determined by polyacrylamide gel electrophoresis and densitometry of gels stained with Coomassie blue. Swine were injected intramuscularly with 0.5 mCi/kg of [3H]TdR 2 h before death. The aorta and coronary arteries were perfusion fixed in situ under anesthesia. Samples were obtained for microscopic morphometry, autoradiography and immunohistochemistry from distal abdominal aorta, thoracic aorta, and proximal coronary arteries; left main (LM), left anterior descending (LAD), left circumflex (LCX), right main (RM), and right coronary artery (RCA). On the BT diet without FO there was extensive atherosclerotic (AS) lesion development, which was drastically reduced by the addition of FO to the BT diet in all sites by from 71 to 94%. The overall plasma cholesterol (CH) levels were reduced only modestly by the FO (816 +/- 64 to 629 +/- 14 mg/dl) but the distribution of CH in the various lipoprotein classes was remarkably altered. The CH in the large lipoprotein molecules containing both B and E apoproteins was reduced from 488 +/- 84 to 204 +/- 17 mg/dl by the FO with an almost corresponding increase in the conventional LDL molecules containing apo B only (158 +/- 29 to 344 +/- 15 mg/dl). We offer the hypothesis that the large apo B,E containing molecules are much more atherogenic than the smaller apo B containing molecules. This hypothesis is supported by a highly significant correlation between extent of lesion development in all arterial sites and plasma levels of CH in apo B,E containing lipoproteins. Plasma TBARS were elevated by the BT + FO diet but seemed to have no significant effect on the lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Normal chicken, mouse, and human embryo fibroblasts release into their culture media transforming growth factors (TGFs) in a latent form. Their soft agar colony-forming activity on two widely used target cells, rat NRK-49F and mouse AKR-2B, is essentially revealed only after prior acidification of cell-conditioned media. These TGFs are EGF-dependent when assayed on NRK-49F cells and EGF-independent on AKR-2B cells. The TGF activity from the chicken source is released in three (apparent) molecular weight forms of 500 kd, 125 kd, and 20 kd.

Lupus-prone (NZB X NZW)F1 (B X W) mice and MRL-lpr and BXSB mice were examined for the prevalence of hypertension and levels of plasma renin activity (PRA). Hypertension (greater than 145 mmHg) was observed only in female and male B X W mice with severe nephritis; in female MRL-lpr and male BXSB mice severe nephritis developed without blood pressure elevation (80-135 mmHg). The B X W parental strains, NZB and NZW, and the MRL-lpr congenic partners, MRL- +, did not become hypertensive as they aged. Other strains of mice, aged 3-32 months (A/HeN, BALB/cJ, BALB/cByJ, B10.S/Sg, B10.D2/ oSn , CBA/J, C3H/HeJ, SJL/J and [SJL X NZW]F1), also had normal blood pressure (98-122 mmHg). All mice with lupus nephritis had low PRA, even those with hypertension; furthermore, the MRL-lpr strain had low or undetectable PRA (2 +/- 1 ng/ml/hr), even when kidneys were normal. NZB, NZW, and MRL- + mice had normal PRA (10-16 ng/ml/hr). Thus, B X W mice frequently developed low renin hypertension during the last phase of their renal disease; whereas MRL-lpr and BXSB mice died from renal disease without observable increases in blood pressure.

The cytophilic activity of human myeloma proteins of different classes and subclasses for lymphocytes, monocytes, and neutrophils was investigated. Binding of both unaggregated immunoglobulins (Ig) and Ig aggregated with rabbit F(ab)2 anti-Fab fragment sera was determined. Lymphocytes bound unaggregated IgG1 and IgG3 proteins, but none of the proteins of the other classes. In contrast, after aggregation, IgG of all subclasses and IgE proteins bound to lymphocytes; aggregated proteins of the other classes did not bind. Monocytes bound unaggregated IgG1 and Ig3 better than Ig4 whereas the binding of proteins of other classes was insignificant. Neutrophils bound unaggregated IgG1 and IgG3 proteins and, in addition, IgA1, IgA2, secretory IgA, and IgG4 proteins. After aggregation, the neutrophils bound more Ig of all classes; however, the differences between the amounts bound remained similar to the amounts of unaggregated proteins. The native structure of the Ig molecule is necessary for the maintenance of complete activity, because Fc fragments bound less than intact Ig, and reduction and alkylation abolished cytophilia. The Fc receptors on all cell types tested showed no specificity for any of the respective cytophilic IgG subclasses; however, neutrophils appear to have separate receptors for IgG and IgA proteins.

A microfluorometric assay for thiols has been developed using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM). The technique may be used to quantitate either cellular or plasma thiols over a range of 0.01 to 3.0 nmol and may be used with as few as 1-3 X 10(5) cells giving highly proportional and reproducible results. Values for nonprotein thiols obtained with this assay agree well with previous reports on glutathione (GSH) levels for both lymphocytes and plasma. Readings are determined with the aid of an automated fluorescence microplate reader which allows up to 96 samples, including standards, to be read at the same time. Cellular thiols accessible after lysis were also quantitated before and after treatment of intact cells with various thiol-reactive chemicals. Interestingly, HgCl2, bromoethanesulfonic acid, and N-ethylmaleimide differentially modified protein and nonprotein thiol levels.

Lead (Pb) has been shown to alter various parameters of immune function such as host resistance and antibody formation. In addition, various heavy metals have been implicated as inducers of autoimmunity. In these experiments, macrophages, isolated from the peritoneal cavity of mice exposed to various doses of lead in vivo as well as cells exposed in vitro were tested for the following immunologic parameters: phagocytosis, antigen presentation, interleukin 1 production, and their ability to stimulate the autologous mixed lymphocyte reaction (AMLR). The results obtained indicate that Pb appears to alter the ability of macrophages to present antigen by enhancing the AMLR while having no effect on phagocytosis or IL-1 production. These data suggest that Pb may interfere with antigen-specific interactions between macrophages and T cells.

To determine the relative efficacy of several dosage regimens of testosterone enanthate in the treatment of male hypogonadism, we treated men who had primary hypogonadism with the following dosage regimens: 100 mg once a week, 200 mg every 2 weeks, 300 mg every 3 weeks, and 400 mg every 4 weeks, each for 12--16 weeks. Twenty-three men completed 37 dosage regimens. The 100-, 200-, and 300-mg dosages all suppressed the initially elevated serum LH concentrations to normal, but the 400-mg dosage did not. The 100- and 200-mg regimens suppressed the initially elevated serum FSH concentrations to normal, and the 300-mg regimen almost did not so. All four regimens produced serum testosterone concentrations that fluctuated largely within the normal range, the average concentration between doses was highest with 100 mg and lowest with 400 mg. The regimens of 200 mg every 2 weeks and 300 mg every 3 weeks appeared to be the most effective of those tested in terms of suppression of the serum LH concentration to normal and infrequency of administration. The close parallel of the FSH response to that of LH suggests that testosterone is the major physiological inhibitor of FSH as well as of LH.

The cytophilic binding of immune complexes by peritoneal exudate cells (PEC) from adjuvant-stimulated guinea-pigs was studied using 125I-labelled guinea-pig IgG1, IgG2 and IgM antibodies to the dinitrophenyl (DNP) group. The influence of hapten density upon cytophilic activity was studied by the addition of DNP-conjugated antigens to antibody in 2-200 molar ratios of DNP:antibody. Only IgG2 binding was enhanced by immune complex formation, and the increased binding of IgG2 anti-DNP was dependent on the number of DNP determinants per antigen molecule. Cytophilic activity with epsilon-DNP-L-lysine (DNP-LYS), alpha,epsilon-di-DNP-L-lysine (DNP-LYS-DNP), or DNP1-8-BSA was no greater than that seen in the absence of hapten. Increased cytophilic binding was noted only with DNP20-41-BSA. The binding of IgG2 and IgG2 anti-DNP:DNP-bovine serum albumin (BSA) complexes was inhibited by monomeric IgG2. The relative cytophilic capacities of guinea-pig immunoglobulins appeared as follows: IgG greater than IgG1 greater than IgM. IgG1 and IgM binding of DNP conjugates did not enhance their cytophilic activity; therefore, IgG1 and IgM cytophilic binding to PEC was considered biologically insignificant. This investigation provides further evidence that cytophilic binding of immune complexes to macrophages is due to the co-operative action of multiple Fc sites rather than a conformational change in the IgG2 antibodies, and serum proteins, notably complement components, can alter the binding and/or phagocytosis of IgG2 anti-DNP:DNP-BSA complexes.

Bone marrow cell responsiveness to hematopoietic growth factors is an integral part of immune responsiveness. Since host resistance is often dependent on bone marrow cell responsiveness and Pb alters host resistance, the influence of Pb on bone marrow cell responsiveness to the hematopoietic growth factor CSF-1 was evaluated. Cell number, soft agar colony formation, cell cycle analysis, as well as 3H-thymidine incorporation were utilized to determine if CSF-1 driven bone marrow-derived macrophage (BMDM) proliferation in vitro is altered in the presence of PbCl2. Data obtained indicate that Pb potentiates the ability of CSF-1 to stimulate thymidine incorporation by BMDM; however, colony formation is inhibited reversibly, and the absolute number of cells in culture is adversely affected by Pb. Propagation of BMDM appears to be more sensitive to Pb (100 nM) than other immune parameters. The decrease in bone marrow cell responsiveness to CSF-1 in the presence of Pb observed in this system may contribute to the decrease in host resistance observed in Pb-exposed animals.

The capacity of the heavy cations Pb, Ni, and Zn to modulate murine in vitro lymphocyte responses was examined. Pb and Ni (100 microM) were shown to enhance the in vitro plaque-forming cell (PFC) response to sheep red blood cells while 100 microM Zn had inhibitory effects. Each metal was able to stimulate the proliferation of murine splenocytes as determined by [3H]thymidine incorporation and autoradiography. The enhancing effect of the metals on the PFC response was observed whether the results were expressed on a per culture or a per cell basis, indicating an actual increase in B-cell differentiation. Both the PFC response and the proliferative response were shown to be sensitive to the type of medium employed (M-199 gave optimum results) and to the presence or absence of 2-mercaptoethanol. As in autologous mixed-lymphocyte responses peak proliferation occurred after Day 5 in culture, was cell density dependent, and required the presence of both T cells and Ia+ cells. Treatment of splenocytes with anti-Thy-1.2, anti-Lyt-1, or anti-L3T4 plus complement completely abrogated the proliferative response, indicating that a Lyt-1+, Lyt-2-, L3T4+ T-cell was required for the induction of proliferation. The data are consistent with the hypothesis that the metals are capable of modifying the immune response directed at self either by directly altering self constituents (class II) or by modulating the autologous T-cell response.

The heavy metals Pb, Ni and Zn have previously been demonstrated to stimulate the proliferation of an Lyt1+2-, L3T4+T-cell. This proliferation required the presence of Ia+ cells and was blocked by monoclonal antibodies directed against self I-A, self I-E and L3T4a. The work reported here examined the role of T-cell factors (IL-2 and gamma-IFN) and their receptors in the metal-induced lymphoproliferation. The metals Pb, Ni and Zn, at concentrations (100 microM) which stimulate T-cell proliferation, had little effect on the ability of the IL-2-dependent cell line HT-2 to respond to exogenous IL-2. This suggests that Pb, Ni and Zn do not modulate the ability of IL-2 to interact with the IL-2 receptor. Ni and Zn significantly enhanced the synthesis/secretion of IL-2 by cultured splenocytes and the expression of the receptor for IL-2; however, Pb produced only slight enhancement. Likewise, anti-IL-2 receptor (7D4) antibodies were able to inhibit a significant portion of the Ni- and Zn-, but not Pb-, induced lymphoproliferation. The residual 3H-thymidine incorporation observed in the presence of anti-IL-2 and anti-IL-2R may represent cycling B-cells induced to proliferate by activated, but non-cyclin, T-cells. Monoclonal anti-gamma-IFN (R4/6A2) equally inhibited all metal-induced lymphoproliferation, suggesting that metal-induced lymphoproliferation is dependent on the induction of gamma-IFN as well as IL-2 synthesis. The Pb-induced response being the least dependent on IL-2 lends support to the hypothesis that Pb, Ni and Zn may activate T-cells and/or B-cells by different mechanisms.

Human blood platelets, the richest known source of beta-transforming Growth Factor extractable under acid conditions, release in neutral extracts (pH 7.2) a latent form of this growth factor with an apparent molecular weight of 400 Kd. This latent form, poorly active on rat NRK-49F indicator cells in soft agar assays can be activated by exposure to acid pH or 8 molar urea. The acid activated beta-Transforming Growth Factor from neutral extracts elutes on Biogel P60, in 1 molar acetic acid, as a broad peak of apparent molecular weight 15-30 Kd, like when this factor is extracted from platelets by the usual acid-ethanol procedure. Moreover, beta-Transforming Growth Factor from both acid activated neutral extracts and from acid-ethanol extracts elutes on reverse phase at 30% acetonitrile. We suggest that beta-Transforming Growth Factor is stored in human blood platelets as a poorly active high molecular weight complex which may be dissociated and activated in appropriate in vivo microenvironments.

A latent beta-TGF activity is spontaneously released into serum-free culture medium by chicken embryo fibroblasts. Anchorage-independent growth activity measured on NRK-49F indicator cells, of this latent beta-TGF can be revealed by four different treatments: acidification, alkalinisation, exposure to urea, and heating to 100 degrees C for 3 minutes. This lact activating treatment indicates that latent beta-TGF activation in vitro is non-enzymatic. Active beta-TGF exists in a low molecular weight form 16 Kd (apparent) in 1M acetic acid, which elutes on reverse phase (FPLC) between 33-35% acetonitrile. Under neutral conditions only a high molecular weight form excluded on Biogel P60 is observed. This form is poorly active on NRK-49F for anchorage independent growth but can be fully activated by prior acidification. Rechromatography of the latent beta-TGF-containing fractions under acidic conditions converts the high molecular weight form to an apparent 16 Kd active form. We suggest that the high molecular weight form may correspond to a complex of a beta-TGF associated with a carrier or binding protein.