The effects of activated macrophages on tumor target cells: Escape from cytostasis

In contrast to normal mouse peritoneal macrophages, activated macrophages almost totally inhibit [³H]TdR uptake by tumor target cells 24 hr after challenge. However, when the period of observation was extended to 48 or 72 hr, renewed [³H]TdR uptake by target cells was often, but not always, observed in the presence of activated macrophages. This apparent escape of target cells from the cytostatic effects of activated macrophages was not due to a subpopulation of resistant target cells, and autoradiographic studies revealed that target cells, inhibited from incorporating [³H]TdR by activated macrophages at 24 hr, were subsequently able to renew DNA synthesis and multiply. These results suggest that in the presence of activated macrophages, the almost total cytostasis of target cells does not necessarily mean that these cells are irreversibly damaged or killed.Escape from or maintenance of cytostasis was not peculiar to any of the target cells (L cells, EMT-6, Bladder 4934) or mouse strains (SW, C57BL, BALB/c) employed nor was it consistent with any of the forms of stimulation used for obtaining activated macrophages (Toxoplasma or Besnoitia infection; C. parvum treatment). However, the results suggest that when escape of target cells from the cytostatic effects of activated macrophages occurred, it may have been due to a qualitative or quantitative inadequacy of the population of macrophages employed.     

Effects of human peripheral blood monocytes,monocyte-derived macrophages,and spleen mononuclear phagocytes on Toxoplasma gondii

In vitro effects of human peripheral blood monocytes, peripheral blood monocyte-derived macrophages, and spleen mononuclear phagocytes on Toxoplasma gondii were studied. In almost all instances, over 80% of human monocytes and monocyte-derived macrophages infected with Toxoplasma in vitro destroyed the organism. Degeneration of intracellular Toxoplasma was not due to decreased viability of organisms in the challenge inoculum. Human monocytes did not elaborate into the culture medium substances which altered the capacity of Toxoplasma to survive and replicate within mouse macrophages. The early reduction in intracellular Toxoplasma was not affected by inhibitors of various intracellular processes or by diseases associated with altered cellular immunity (sarcoidosis, infectious mononucleosis, or lymphoma.) The Toxoplasma that remained after 6 hr within human monocytes and macrophages multiplied. This multiplication was observed both microscopically and in a radioassay which detects uptake of [³H]uracil or [³H]deoxyuridine into nucleic acids of intracellular Toxoplasma. Intracellular Toxoplasma in monocytes cultured with poly(I:C) or in monocyte-derived macrophages cultured with lymphokines showed decreased uptake of radiolabeled precursors into nucleic acids of intracellular Toxoplasma. Treatment of monocytes with endotoxin did not alter nucleic acid synthesis of surviving intracellular Toxoplasma. These results suggest that human mononuclear phagocytes in peripheral blood and in tissue (spleen) have the capacity to eliminate a large percentage of the Toxoplasma that they ingest or that invade them. The inhibition of nucleic acid synthesis of remaining Toxoplasma by exposure of monocyte-derived macrophages to lymphokines suggests that lymphocyte products may be important for elimination of the Toxoplasma that remain and multiply within a small proportion of mononuclear phagocytes.     

In vitro killing of tumor cells by soluble products of activated guinea pig peritoneal macrophages

A partially purified fraction from supernatants of macrophage cultures from BCG-infected guinea pigs, which is rich in anti-Listeria activity, was cytotoxic to malignant and not normal cells. The susceptibility of target cells to this material varied considerably. Concentrations of egg white lysozyme, equal to those of the macrophage product, had considerably less effect on line 1 hepatoma cells.     

Augmentation of mouse natural killer cell activity by muramyl dipeptide and its analogs

The ability of muramyl dipeptide (MDP) and its structural analogs (des-MDP, abu-MDP, and des-abu-MDP) to influence mouse natural killer (NK) cells in two different strains of mice was examined. In CBA/J mice, administration of MDP by both intraperitoneal (ip) and intravenous (iv) routes enhanced splenic NK cell activity. Maximum augmentation of NK cell activity was observed 3 days after MDP treatment. NK cell activity was also stimulated upon in vitro culture of CBA/J mouse spleen cells with MDP. Only iv inoculation of MDP to C57BL/6 mice 7 days previously enhanced NK cell activity of spleen cells. Peritoneal NK cell activity was not affected in either strain of mice, regardless of the route of inoculation of MDP. Two structural analogs of MDP, abu-MDP and des-abu-MDP, enhanced peritoneal NK cell activity, whereas des-MDP had no effect when tested 3 days after ip treatment of CBA/J mice with these compounds. Peritoneal NK cell activity of C57BL/6 mice was not modulated by des-MDP, abu-MDP, or des-abu-MDP. A synergistic effect on peritoneal NK cell activity was observed in both CBA/J and C57BL/6 mice treated first with MDP and then with lipopolysaccharide (LPS) or Bacillus Calmette-Guerin (BCG).     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. IV. Role of energy metabolism

The role of energy metabolism on tumor cell killing by in vitro activated macrophages was studied. Depletion of extracellular glucose had little effect on the cytotoxic capacity of mediator-activated macrophages. Respiratory antagonists did not inhibit cytotoxicity regardless of whether or not the assays were carried out in low-glucose-containing medium. Sodium fluoride, a known inhibitor of glycolysis, inhibited the killing of tumor cells by activated macrophages. 2-Deoxyglucose, an analog of glucose, was found to be an effective inhibitor of cytotoxicity. Three other analogs, 5-thio-d-glucose, 3-O-methylglucose, and 2-deoxy-d-galactose, were without effect. The concentrations of 2-DG that inhibited cytotoxicity did not lower cellular ATP levels to an appreciable extent. The combined addition of inhibitors of glycolysis and respiration resulted in a marked reduction in ATP levels. Under these experimental conditions, macrophage-mediated cytotoxicity was also significantly inhibited.     

Enhancement of human natural killer cell activity by subcellular components of Toxoplasma gondii

The ability of sonicates and subcellular fractions of the intracellular parasite Toxoplasma gondii to enhance in vitro human natural killer (NK) cell activity was examined. Incubation of nylon-wool-non-adherent human peripheral blood lymphocytes (PBL) with sonicates of T. gondii for 18-72 hr resulted in increased NK activity against an NK-sensitive, as well as an insensitive, target cell. Single-cell assays revealed that augmentation of NK activity was not due to an increased binding of K562 target cells to effector cells. Differential centrifugation studies indicated that NK-augmenting activity was distributed in membrane-enriched and cytoplasmic fractions. This activity was found to be resistant to treatment with ribonuclease (RNase) and deoxyribonuclease (DNase), but susceptible to proteolysis. Antibodies present in the serum of humans infected with Toxoplasma blocked the NK cell-augmenting effect of the membrane-enriched fractions. Enhancement of NK activity by PBL incubated with Toxoplasma sonicate was accompanied by a concomitant increase in interferon (IFN), but not of interleukin 2 (IL-2), levels in supernatants of the cell cultures.     

Natural cell-mediated cytotoxicity in mice treated with total lymphoid irradiation (TLI)

Fractionated total lymphoid irradiation (TLI) of adult (BALB/c × C57BL/6)F₁ mice resulted in transiently augmented natural killer (NK) and natural cytotoxic (NC) cell activities. Thus, 1 day after completion of TLI, NK and NC activities in the spleens of treated mice were lower than controls but values increased and reached a maximum level of 23- to 190-fold above control at 6 days after irradiation, returning to normal levels 9 days later. Cytotoxicity was enhanced after removal of the plastic adherent population. No cytotoxicity was observed against P 815 target cells, which are sensitive to activated macrophages but not to NK. The significance of this modulation of natural cell-mediated cytotoxicity following TLI is discussed.     

14 C-acetate incorporation and nucleic acid synthesis in human lymphocytes stimulated by PHA and tuberculin in vitro

Studies on the timing of incorporation of labeled acetate in relationship to other cellular events in phytohemagglutinin (PHA)-treated lymphocytes have suggested that acetylation of nuclear histones may constitute an important regulatory mechanism for gene activation. In the present investigation, it was shown that PHA stimulation of lymphocytes from a tuberculin-positive patient caused an early increased incorporation of ¹⁴C-acetate prior to RNA and DNA synthesis. Lymphocytes from the same patient, however, repeatedly showed no increased incorporation of ¹⁴C-acetate following exposure to the sensitizing antigen, tuberculin (PPD), even though RNA and DNA synthesis were markedly stimulated. These results suggest that regulatory mechanisms of DNA template activity other than acetylation may be operative in sensitized lymphocytes responding to specific antigen. One possible explanation for the differences in ¹⁴C-acetate incorporation is that the increased uptake of acetate exhibited by PHA-treated cells is an effect related to nonspecific membrane changes caused by the PHA. If this is the case, then template regulation in PHA and antigen-stimulated lymphocytes may be achieved via similar but yet to be defined mechanisms.     

Reversible inhibition by interferon of the maturation of human peripheral blood monocytes to macrophages

We demonstrated that interferon delays the maturation of human monocytes to macrophages in vitro as assessed by morphologic, histochemical, and biochemical parameters. After exposure to interferon, monocytes were slightly smaller, less stretched out, and had a delayed loss of granules with peroxidase positive reactivity, as compared with control, noninterferon-treated cells. Also, interferon prevented the increase of the specific activity of three lysosomal enzymes (β-galactosidase, β-glucuronidase, and β-N-acetylglucosaminidase) in the monocytes, and enzyme activities were 30–40% of that observed in untreated cells. Both human leukocyte and human fibroblast interferons were effective in delaying the maturation process. The effects of the interferon were species specific and reversible and were neutralized by antiinterferon serum. These studies describe a new nonantiviral effect of interferon, unique in that it involves the delay of maturation of cells in a system which is not associated with cell proliferation. Thus interferon could potentially effect host defense mechanisms and aspects of the immune response which are dependent on the maturation of monocytes to macrophages.     

Estimation of branched-chain α-keto acids in blood by gas chromatography

Plasma or whole blood is treated with o-phenylenediamine dihydrochloride in phosphoric acid under conditions found spectrophotometrically to give maximum yields of the quinoxalinols. The quinoxalinols are extracted and, after removing phosphoric acid, etc., silylated with bis-trimethylsilyltrifluoracetamide in acetonitrile. Other solvents caused instability of the trimethylsilyl(TMS)-quinoxalinols. Gas chromatography on a packed column of trifluoropropyl silicone gave good separation of the TMS-quinoxalinols from one another and from other substances derived from blood. Some representative values for normal arterial and venous human and canine plasma are reported.     

Differential effects of ouabain on human cell-mediated cytotoxicity. I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets.

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed ⁵¹Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10^?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.     

Activation of the contractile system in crustacean muscle: Ultrastructural evidence for the role of the T system

Freeze-fracture and thin sections of lobster abdominal fast flexor muscle were used to study the morphology of the sarcoplasmic reticulum (SR) and T system of crustacean muscle. Tannic acid mordanting, which can result in a dense black deposit in the T system lumen, was used to distinguish T system from SR membranes. Ferritin was also used as an extracellular tracer to confirm the tannic acid method. The T system consists of an extensive network of flattened sacs which fills most of the space between the mycfibrils and is in close contact with them. The SR also appears as flattened sacs, sometimes with fenestrations. There is extensive junctional contact between the SR and T system. Quantitative estimates of the volume and surface area of the membranes show that the T system has about 50 % more surface area than the SR. The intramembrane particle (IMP) density of the PF face of the T system is about 1100/ μm² membrane, while the IMP density of the PF face of the SR is about 4800/ μm² membrane. In morphology, extent, and IMP density, the T system of lobster abdominal fast flexor muscle appears (AFF) adapted to provide at least part of the Ca²⁺ for muscle activation and the transport system for relaxation.     

Fluorescence and conformational changes caused by proton binding to troponin C

The binding of H⁺ to troponin C induces a large conformational change and an enhancement of the tyrosyl fluorescence. Carboxyl groups with abnormal pK' values of 6.0 appear to be controlling these changes.     

Differential effects of ouabain on human cell-mediated cytotoxicity. II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets.

We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10^?5M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10^?9M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.     

Mechanism of action of agents which uncouple oxidative phosphorylation: Direct correlation between protoncarrying and respiratory-releasing properties using rat liver mitochondria

The proton-carrying properties of uncoupling agents were investigated by measuring passive mitochondrial swelling under conditions where electrogenic proton transport was rate limiting. The ability of uncoupling agents to transport protons into mitochondria, measured in this way, was compared with respiratory stimulation. The results show that with the single exception of arsenate, all agents tested which uncouple oxidative phosphorylation demonstrate a very close correlation between release of respiration and proton transport. These findings are in support of Mitchell's original proposal that uncoupling agents act by promoting electrogenic hydrogen ion transport across the mitochondrial inner membrane.     

Synthesis of immunoglobulin by rabbit lymphocytes in vitro in response to anti-immunoglobulin antiserum

Stimulation of synthesis of immunoglobulin (Ig) in vitro by Con A and anti-Ig in cultures of rabbit lymphoid cells has been analyzed qualitatively using an assay that measures the incorporation of [³H]leucine into newly synthesized proteins, followed by the specific absorption of tritiated immunoglobulin by staphylococcal protein A. Whereas Con A stimulates Ig production by spleen cells only if T lymphocytes are present, anti-immunoglobulin serum enhances Ig synthesis in the absence of T lymphocytes. In contrast, neither Con A nor anti-immunoglobulin serum stimulates peripheral blood lymphocytes to produce enhanced levels of Ig. It is concluded that both Con A and anti-immunoglobulin serum do not activate resting B cells but drive differentiation of B cells which are already synthesizing Ig. Anti-Ig acts directly whereas stimulation of B-cell Ig synthesis by Con A occurs indirectly through stimulation of T cells.     

Developmental patterns of copper and zinc concentrations in mouse liver and brain: evidence that the gene crinkled (cr) is associated with an abnormality in copper metabolism

An abnormality in copper metabolism during both the prenatal and postnatal (preweaning) periods was found to be associated with the autosomal recessive gene ”crinkled“ (cr) in mice. Liver copper concentration was significantly lower in crinkled mice (cr/cr) than in littermate controls (+/?) from 18 days of gestation to 20 days after birth. Crinkled mice older than 20 days of age had liver copper concentrations similar to those of littermate controls. Liver zinc and brain copper and zinc were similar in crinkled and noncrinkled mice at all times tested. In both crinkled and noncrinkled mice, brain copper concentration increased during the suckling period, and liver copper concentration decreased.     

Nucleotide sequence of genes coding for dicyclohexylcarbodiimide-binding protein and the alpha subunit of proton-translocating ATPase of Escherichia coli

A liquid membrane electrode has been made which is selective for ethidium ion. The membrane is formed in a capillary by a 3-nitro-o-xylene solution of an ethidium-tetraphenyl borate complex. The electrode emf (vs saturated KCl-calomel reference) has a linear dependence upon the logarithm of ethidium concentration from 2 μM to 0.5 mM. The electrode is used here to measure free ethidium ion in mixtures with calf thymus DNA. The binding isotherms obtained are in general agreement with a control photometric titration and with literature results. Direct measurement of free ethidium concentration by convenient potentiometric methods is useful in the study of ligand binding to nucleic acids and to related compounds.     

What is “language” that can facilitate the flow of information? a contribution to a fundamental theory of language and communication

A theory of language is derived for complex systems by contrasting the difference between “hard” systems and “soft” systems. Complexity in a field system made up of atomistic entities is defined by the capability of the atomistic-like entities in soft systems to absorb energy internally into fluid-like, gel-like, dissipative processes rather than to equipartition energy rapidly among all degrees of atomistic freedom. Language then emerges as the “mechanistic” linkages that can catalytically switch or evoke changed atomistic states in such “soft” systems. A note on the source of syntax is also provided.