Macrophage-mediated fungistasis: requirement for a macromolecular component in serum

Peritoneal macrophages from Mycobacterium bovis- or Toxoplasma gondii-infected mice cultured in vitro in Dulbecco's medium containing 10% fetal bovine serum (FBS) and endotoxin stopped replication of Cryptococcus neoformans for 30 hr, whereas yeast cells cultured alone reproduced with a 3.0-hr doubling time. Without at least 5% FBS, macrophage fungistasis was poor. FBS without macrophages enhanced the growth rate of cryptococci. Macrophages preincubated in vitro for 24 hr without serum became fungistatic when challenged with cryptococci in medium with FBS but were not fungistatic without FBS. Macrophages preincubated in medium with FBS were never subsequently fungistatic. Dialyzed, heated (56 degrees C, 30 min), or delipidated FBS supported macrophage fungistasis, whereas FBS heated at 70 degrees C for 30 min did not. FBS contained no measurable opsonic activity for C. neoformans. Inclusion of endotoxin and/or murine IFN-gamma over wide concentration ranges did not substitute for FBS. Ultrafiltration estimation of FBS activity localized to 50 to 150 Kd. By gel filtration chromatography, FBS activity ran in the 25 to 100 Kd range. Dye-ligand affinity chromatography on Cibacron blue agarose gel dissociated the FBS activity from the albumin and lipoprotein fractions. Anion-exchange chromatography on DEAE-Sephacel revealed activity in the first fraction eluting at low ionic strength, pointing to a protein(s) with an isoelectric point toward neutral. Activated macrophages can prevent microbial replication within host tissues; the local environment is critical for fulfillment of this important physiologic function. These results point to a macromolecular factor(s) present in serum that is essential for full fungistatic capability of activated macrophages.     

Macrophage-mediated in vitro sensitization of lymphocytes

Summary Antigen-fed macrophages were able to induce specific sensitization of unprimed syngeneic lymphocytes in vitro. The sensitized lymphocytes caused specific injury to target cells that carried the relevant antigens. In the present study, we investigated the in vivo activity of lymphocytes sensitized by antigen-fed macrophages. Mouse spleen cells were sensitized by macrophages that had been exposed to the radiation leukemia virus (RadLV). The sensitized lymphocytes, which were enriched for T-cells, were injected to syngeneic normal recipients and 4 days later the mice were challenged with RadLV-induced lymphoma cells. By following tumor growth and survival of mice, we have found that the sensitized lymphocytes protected the recipient mice against lymphoma development if injected 4 days before the tumor cells. The protective activity of the sensitized lymphocytes was radioresistant, but they could not protect irradiated hosts. It is suggested that macrophagemediated in vitro sensitization of lymphocytes induces initiator cells that can protect the recipient host by recruitment of a defensive immune response.     

Age-related and carcinogen-induced alterations of the extracellular growth factor requirements for cell proliferation in vitro

Cells from thigh muscles of fetal rats proliferate readily in vitro in medium containing homologous adult rat "plasma". As the donor animals mature, these cells become unable to make DNA and proliferate in "plasma" medium, but retain an ability to proliferate in medium containing fetal bovine serum (FBS). This age-related loss of the ability to proliferate in "plasma"-medium is due to an increasing need by the cells for an exogenous prereplicative promoter which is found in FBS and a crude preparation of bovine luteinizing hormone. Adult cells (and possibly fetal cells) also require a "cycle-completion" factor which is found in FBS and adult rat "plasma". The requirements for such external proliferative promoters is completely eliminated by neoplastic transformation in vivo, and neoplastic adult cells isolated from a nickel sulfide-induced mixed rhabdomyo-fibro-sarcoma can make DNA and proliferate in vitro in the complete absence of exogenous growth factors.     

Soil fungistasis: elevation of the exogenous carbon and nitrogen requirements for spore germination by fungistatic volatiles in soils.

Axenic, washed conidia of Fusarium solani f. sp. phaseoli, Aspergillus flavus, and Verticillium albo-atrum were placed on washed Difco purified agar discs along with an inorganic salt solution containing various levels of carbon and nitrogen substrates. These discs were exposed to volatiles from six soils (pH 5.1-8.6). Fusarium solani macroconidial germination was inhibited mostly by volatiles from soils of pH 5.1, 6.1, 7.0, and 7.5, but high levels of glucose and NH4Cl reversed this inhibition, raising germination to that of no-soil, no-carbon or nitrogen controls. Conidial germination of A. flavus was inhibited mainly by volatiles from high pH (7.0, 7.8, and 8.6) soils, and increased levels of glucose plus an amino acid mixture nullified this inhibition. Volatiles from soils of pH 5.1, 6.1, and 7.5 stimulated A. flavus conidial germination. Assays after the removal of CO2 from the air above soil of pH 5.1 demonstrated that volatiles inhibitory to A. flavus were produced by this soil. Assays indicated that a KOH-soluble compound was a fungistatic soil volatile to F. solani macroconidial germination. The nullification by carbon and nitrogen substrates of F. solani and A. flavus inhibition caused by soil volatiles parallels that for soil fungistasis. Conidial germination of V. albo-atrum was markedly stimulated by volatiles in all soils tested, and was not affected by removal of CO2. Inhibitory soil volatiles may increase the nutritional requirements for spore germination of certain fungi.     

Different selenium-containing proteins in the extracellular and intracellular media of leucocytes cultivated in vitro

The purpose of this communication is to elucidate if selenium plays a role in the function of granulocytes and lymphocytes. Thus, the incorpo ration of selenium in proteins from granulocytes and lymphocytes cultured with 1ΜCi/mL radioactive Na₂ ⁷⁵SeO₃ was studied. The protein peaks containing⁷⁵Se from two columns of Heparin Sepharose CL-6B and Sephacryl S-200 HR were separated further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results showed that the incorporation of⁷⁵Se into granulocytes was about six times higher than that of lymphocytes during a 96-h cultivation, however, the GSH-Px activity in granulocytes did not change significantly. On the other hand, the GSH-Px activity of lymphocytes rose significantly after three days cultivation. These data indicated that the main chemical form of selenium in granulocytes was not GSH-Px. Results from SDS-PAGE revealed a strongly⁷⁵Se-labeled protein band with subunit molecular weight of 15 kDa in the supernatant of granulocyte homogenate. However, the main chemical forms of selenium in the culture media of granulocytes and lymphocytes were found to be selenoprotein P. The different forms of selenium-containing proteins in the intracellular and extracellular media of granulocytes indicated the different functions of these proteins.     

Magnesium deficiency in vitro enhances free radical-induced intracellular oxidation and cytotoxicity in endothelial cells.

The effect of magnesium (Mg)-deficient culture on endothelial cell susceptibility to oxidative stress was examined. Bovine endothelial cells were cultured in either control sufficient (0.8 mM) or deficient (0.4 mM) levels of MgCl2. Oxygen radicals were produced extracellularly by the addition of dihydroxyfumarate and Fe(3+)-ADP. Isolated Mg-deficient endothelial cells produced 2- to 3-fold higher levels of thiobarbituric acid (TBA)-reactive materials when incubated with this free radical system. Additional studies were performed using digitized video microscopy and 2',7'-dichlorofluorescein diacetate (DCFDA) as an intracellular indicator for oxidative events at the single cell level. In response to the exogenous oxidative stress, endothelial cells exhibited a time-dependent increase in fluorescence, suggestive of intracellular lipid peroxidation. The increase in cellular fluorescence began within 1 min of free radical addition; the Mg-deficient cells exhibited a more rapid increase in fluorescence than that of Mg-sufficient cells. In separate experiments, cellular viability was assessed using the Trypan blue exclusion assay. Mg deficiency increased cytotoxicity of the added oxyradicals, but the loss of cellular viability began to occur only after 15 min of free radical exposure, lagging behind the detection of intracellular oxidation products. These results suggest that increased oxidative endothelial cell injury may contribute to vascular injury during Mg deficiency.     

Comparative studies of the cytotoxic T lymphocyte-mediated cytotoxicity and of extracellular ATP-induced cell lysis. Different requirements in extracellular Mg2+ and pH

We recently proposed that extracellular ATP (ATPo) may be involved in CTL-mediated cytotoxicity by acting in concert with yet unidentified cellular components (ATPo receptors/ATPo-binding proteins, ectoprotein kinases). The TCR-triggered ATPo accumulation by CTL has been demonstrated, whereas the resistance of CTL to ATPo was explained by the action of highly active ecto-ATPases or by the absence of relevant ATP-binding proteins. However, no data were available to discriminate between the possibilities of: i) ATPo acting alone as a "hit" molecule because of the cell-permeabilizing properties of ATP4- or ii) ATPo acting as a "messenger" (as MgATP2-) in concert with other molecules. Comparing ATPo-induced and CTL-mediated cell lysis, we found that ATPo-induced lysis of some target cells is greatly decreased at neutral and acidic pH, whereas Ca(2+)-dependent CTL-mediated lysis of the same cells is barely affected. In agreement with the observed pH dependency, at low Mg2+ concentrations, which favor ATP4- over MgATP2-, maximal ATPo-induced lysis was observed. However, CTL-mediated cytotoxicity in both Ag-specific and retargeting assays was markedly reduced at low Mg2+ concentrations. These results suggest that ATPo acting alone as a "hit" molecule cannot fully account for the extracellular Ca(2+)-dependent lethal hit delivery by CTL or that ATP4- is active at very low concentrations. This conclusion was further supported by studying the lytic effect of ATPo and CTL on the anti-TCR mAb-coupled SRBC. CTL were efficient in the SRBC lysis, whereas no lysis of SRBC by ATPo was detected. The resistance of SRBC to ATPo is not caused by a high ATPo degradation, because the ecto-ATPase activity of SRBC was much lower than in ATPo-resistant CTL OE4 cells and comparable with EL4 tumor cells, which were easily lysed by ATPo. These data suggested the need for careful consideration of the pH and cation composition of the media used for studying ATPo effects. The caveats in the use of ATP-degrading enzymes to implicate the role of extracellular ATPo in the CTL-mediated cytotoxicity are described here. A clarification of the previously described cytotoxicity inhibition by hexokinase, which is caused by an inhibitory salt effect, is presented. It is suggested that if Ca(2+)-dependent lysis of SRBC and of other target cells by CTL does involve extracellular ATP, it may function as a "messenger" in concert with other extracellular molecules.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methylation of erythrocyte membrane proteins at extracellular and intracellular D-aspartyl sites in vitro. Saturation of intracellular sites in vivo

A cytosolic protein carboxyl methyltransferase (S-adenosyl-L-methionine:protein O-methyltransferase, E.C. 2.1.1.24) purified from human erythrocytes catalyzes the methylation of erythrocyte membrane proteins in vitro using S-adenosyl-L-[methyl-3H]methionine as the methyl group donor. The principal methyl-accepting proteins have been identified by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 and fluorography as the anion transport protein (band 3), ankyrin (band 2.1), and integral membrane proteins with molecular weights of 45,000, 28,000, and 21,000. Many of the methylation sites associated with intrinsic membrane proteins may reside in their extracellular portions, since these same proteins are methylated when intact cells are used as the substrate. The maximal number of methyl groups transferred in these experiments is approximately 30 pmol/mg of membrane protein, a value which represents less than one methyl group/50 polypeptide chains of any methyl-accepting species. The number of methylation sites associated with the membranes is increased, but not to stoichiometric levels, by prior demethylation of the membranes. The additional sites are associated primarily with bands 2.1 and 4.1, the principal methyl acceptors in vivo, suggesting that most methylation sites are fully modified in vivo. Extracellular methylation sites are not increased by demethylation of membranes. The aspartic acid beta-methyl ester which can be isolated from carboxypeptidase Y digests of [3H]methylated membranes is in the unusual D-stereoconfiguration. Similar results have been obtained with [3H]methylated membranes isolated from intact cells (McFadden, P.N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464). It is proposed that the methyltransferase recognizes D-aspartyl residues in proteins and is involved with the metabolism of damaged proteins in vivo.     

Minimum and maximum extracellular Ca2+ requirements during mouse sperm capacitation and fertilization in vitro

The minimum and maximum extracellular Ca2+ concentrations required to promote capacitation, the acrosome reaction, hyperactivated motility, zona penetration and gamete fusion in the mouse have been established. The traces of free calcium in Ca2+-deficient medium were shown not to enhance capacitation since the inclusion of EGTA to chelate free ions during a 120 min preincubation failed to alter the kinetics of capacitation from those observed in the absence of EGTA; 1 h after addition of 1.80 mM-Ca2+, both suspensions were highly fertile. Complete capacitation, when suspensions were immediately functional upon the addition of 1.80 mM-Ca2+, required the presence of greater than or equal to 90 microM-Ca2. Considerably higher concentrations were required to initiate optimal sperm responses: acrosome reaction, 900 microM; gamete fusion, 900 microM; hyperactivated motility, 1.80 mM; zona penetration, 1.80 mM. None of these changes was effected when Ca2+ was less than 450 microM. The responses to elevated Ca2+ were dependent on the length of incubation, being initially positive and then negative. A short (30 min) exposure to 3.40 mM-Ca2+ (x 2 the standard) accelerated capacitation, as evidenced by significantly increased acrosome loss, precocious expression of hyperactivated motility and enhanced fertilizing ability when Ca2+ was reduced to 1.80 mM. However, extended (120 min) preincubation irreversibly damaged sperm function. In the presence of 7.20 mM-Ca2+ (x 4), fertilizing ability was inhibited at both 30 and 120 min, despite a high incidence of acrosome loss. The primary deleterious effect appeared to be on motility which was judged to be more erratic than in 1.80 mM-Ca2+, possibly due to elevated intracellular Ca2+. Because of the considerable difference in threshold Ca2+ concentrations, it is now possible to dissociate the Ca2+-dependent events of capacitation from those of the acrosome reaction and motility changes.     

Self-organized nanogels responding to tumor extracellular pH: pH-dependent drug release and in vitro cytotoxicity against MCF-7 cells

The principal objective of this study was to fabricate doxorubicin-loaded self-organized nanogels composed of hydrophobized pullulan (PUL)-Nalpha-Boc-L-histidine (bHis) conjugates. Their responses to tumor extracellular pH (pHe) were determined, and they were also evaluated with regard to their anticancer efficacy against breast cancer cell lines (MCF-7). bHis was grafted to a PUL-deoxycholic acid (DO) conjugate (PUL-DO) via an ester linkage. PUL-DO/bHis conjugates with two different degrees of bHis substitutions (PUL-DO/bHis36 and PUL-DO/bHis78) were synthesized. PUL-DO/bHis nanogels formed via dialysis at a pH of 8.5 evidenced larger particle sizes (<140 nm) and lower critical aggregation concentrations (CACs) than did the PUL-DO nanogels (90 nm). The pH-dependent CAC of PUL-DO/bHis78 changed dramatically, from 1.2 microg/mL at pH 8.5, to 10 at 7.0, and to 660 at 6.2. A similar tendency in pH-dependent size was also noted. The ionization of the imidazole ring in bHis is principally responsible for pH dependency. The bHis moieties function as a switching tool responding to external pH. Doxorubicin (DOX)-loaded nanogels were assessed for pH-dependent releasing kinetics. The release rate of DOX from the PUL-DO/bHis78 nanogels increased significantly with reductions in pH. This resulted in increased cytotoxicity (30% cell viability at a dose of 10 microg/mL DOX equivalent) against sensitive MCF-7 cells at a pH of 6.8 and low cytotoxicity at pH 7.4 (65% cell viability at an identical dose). The results show that PUL-DO/bHis nanogels may potentially be employed as anti-tumor drug carriers.     

Antigenic requirements for the generation of secondary cytotoxicity.

The in vitro role of products of the H-2 complex in restimulation of secondary proliferative and cytotoxic responses has been studied. In this paper we have studied in particular the role of CD antigens, the target antigens for cytotoxic T lymphocytes. We have used UV-treated cells that express their CD antigens but no LD. Like others, we demonstrate that CD antigens alone are able to generate a secondary cytotoxic response and furthermore that these CD antigens must be the same as those used for primary sensitization. However, in contrast to the results of others, in some cases UV-treated cells were unable to restimulate such a response even when appropriate controls showed that the CD antigens on UV-treated cells were functional. Furthermore, in these cases the addition of an LD stimulus in the presence of UV-treated cells results in a secondary cytotoxic response that is significantly greater than that elicited by LD restimulation alone. Such potentiation is not observed if the CD antigens used for restimulation are different from those used for the primary sensitization. In addition, a non-H 2 differences(s), presumably the M1s locus, appears to be able to generate a secondary response. The implication of these results are discussed in terms of "memory" T lymphocytes in cell-mediated immunity.     

Evidence for frequency-dependent extracellular impedance from the transfer function between extracellular and intracellular potentials

Dopaminergic (DA) neurons of the mammalian midbrain exhibit unusually low firing frequencies in vitro. Furthermore, injection of depolarizing current induces depolarization block before high frequencies are achieved. The maximum steady and transient rates are about 10 and 20 Hz, respectively, despite the ability of these neurons to generate bursts at higher frequencies in vivo. We use a three-compartment model calibrated to reproduce DA neuron responses to several pharmacological manipulations to uncover mechanisms of frequency limitation. The model exhibits a slow oscillatory potential (SOP) dependent on the interplay between the L-type Ca²⁺ current and the small conductance K⁺ (SK) current that is unmasked by fast Na⁺ current block. Contrary to previous theoretical work, the SOP does not pace the steady spiking frequency in our model. The main currents that determine the spontaneous firing frequency are the subthreshold L-type Ca²⁺ and the A-type K⁺ currents. The model identifies the channel densities for the fast Na⁺ and the delayed rectifier K⁺ currents as critical parameters limiting the maximal steady frequency evoked by a depolarizing pulse. We hypothesize that the low maximal steady frequencies result from a low safety factor for action potential generation. In the model, the rate of Ca²⁺ accumulation in the distal dendrites controls the transient initial frequency in response to a depolarizing pulse. Similar results are obtained when the same model parameters are used in a multi-compartmental model with a realistic reconstructed morphology, indicating that the salient contributions of the dendritic architecture have been captured by the simpler model.     

Heterologous sera: a target for in vitro cell-mediated cytotoxicity.

During the course of studies in inbred Strain 2 Guinea pigs on in vitro cell-mediated cytotoxicity to viral induced fibrosarcomas, it was observed that fetal calf serum in the medium in which the target cells were grown was responsible in some instances for much of the cytotoxicity observed. This fetal calf serum-specific cytotoxicity seemed mainly mediated by T cells and appeared to be independent on histocompatibility between target and killer cells.     

Comparison of intracellular and extracellular mitogenic activity

Endothelial cell-derived growth factor (ECDGF) is a soluble mitogen secreted in vitro by bovine aortic endothelium. ECDGF is a mixture of at least two distinct heat-stable and trypsin-sensitive mitogens. Large amounts of mitogenic activity were found in lysates prepared from cultured endothelial cells. Other nonmitogen-secreting cells in culture, including bovine dermal fibroblasts and vascular smooth muscle cells, also contained a similar activity. In contrast to ECDGF, the lysate mitogenic activities were sensitive to heat (56 degrees C) and were not inactivated by trypsin. Similar to platelet-derived growth factor (PDGF), ECDGF and cell lysate mitogens promoted cell proliferation in the absence of other defined mitogens when added to culture medium and after exposure to plastic. The cytoplasmic mitogens, however, were distinct from PDGF by receptor competition assays and other criteria.