Pyran Copolymer Research Articles

Soluble–insoluble polyelectrolyte complex of quaternized chitosan with DIVEMA (pyran copolymer)

To endow chitosan with solubility in the whole pH-range without loss of functionality of the amino groups, the cationic polysaccharide was exhaustively alkylated yielding N-[(2-hydroxy-3-trimethylammonium) propyl]chitosan chloride (QCht). Each alkylated unit of QCht contained both quaternary amino group and secondary amino group. Recently we demonstrated that QCht forms with nucleic acids of soluble polyelectrolyte complexes stable at physiological conditions and capable of cell transfection in vitro. In the current work, the anionic counterpart of QCht was hydrolyzed copolymer of divinyl ether and maleic anhydride (DIVEMA) which is known to possess some anti-tumor and immune stimulating activity and use as a drug carrier in anti-tumor drug delivery systems. According to the potentiometry data and ζ-potential measurements, almost all carboxylic groups of DIVEMA were able to form ion pairs with QCht. In aqueous and water–salt solutions, formation of either soluble or insoluble complexes was controlled by pH, ionic strength, a ratio of the oppositely charged groups, and degree of polymerization of the chains following general rules revealed on studying polyelectrolyte complexes of polycarboxylic acids. These findings evidence plausible advantages of the complex formation as the non-covalent modification that imparts to both polyelectrolytes of the ability for reversible soluble–insoluble transformation under enzyme-friendly conditions.

Interferon and its inducers--a never-ending story: "old" and "new" data in a new perspective.

The discovery of interferon (IFN), as an antiviral substance, by Isaacs and Lindenmann in 1957 [1] attracted the attention of a number of investigators, including Pieter De Somer at the Rega Institute in Leuven (Belgium) and Tom Merigan at Stanford University (California). De Somer’s original attempts were aimed at determining the mode of action of IFN [2], whereas Merigan’s work focused on trying to understand the role of IFN in human viral diseases [3], by use of the mouse as an animal model. Of pioneering importance was the demonstration by Merigan [4] in 1967 that IFN could be induced in mice by a synthetic polyanion called maleic divinyl ether copolymer (or pyran copolymer). In further experiments, pyran copolymer was also shown to induce IFN in humans [5], although the serum IFN titers obtained in humans were considerably lower than those in mice and, moreover, were accompanied by a considerable

Biological Effects and Antitumor Activity Induced by Benzocaine Conjugated Anionic Polymers

Three maleic anhydride copolymers [poly(maleic anhydride-vinyl acetate), poly(maleic anhydride-styrene) and poly(maleic anhydride-methyl methacrylate)] were prepared as well as the corresponding benzocaine conjugates. The maleic anhydride moiety was hydrolyzed to the corresponding polycarboxylate anions and then biologically evaluated using pyran copolymer as the standard. All of the polymers showed internal organ toxicity of the liver, kidneys and spleen which increased with increasing molecular weight, however, the benzocaine derivatives decreased this effect in most cases. The biological evaluations included hemoglobin levels, hematocrit levels, red blood indices and mean cell volumes which were increased on an average of 50%. The blood protein profiles were not changes significantly; on average they were changed about ±50%. The unmodified copolymers decreased the major serum and cholesterol levels by ∼75%. The copolymers (with or without) benzocaine decreased both the blood sugar and enzyme profiles. The antitumour activity was evaluated against Walker carcinosarconoma; although the benzocaine enhanced these effects, only pyran and the 18,000 Mw benzocaine-modified poly(maleic anhydride-vinyl acetate) produced significant effects.

SMANCS and polymer-conjugated macromolecular drugs: advantages in cancer chemotherapy

This review discusses the development and therapeutic potential of prototype macromolecular drugs for use in cancer chemotherapy, in particular the development and use of SMANCS, a conjugate of neocarzinostatin and poly(styrene-comaleic acid). The various topics covered include a brief description of the chemistry and polymer conjugation, the binding of the conjugate to albumin and the biological behaviour in vitro and in vivo after arterial injection in animals, including plasma half-life, and the lipid solubility of SMANCS in medium chain triglycerides and Lipiodol, a lipid contrast medium suitable for use in X-ray-computed tomography. The biological response-modifying effects and the tumor-targeting mechanism of SMANCS and other macromolecular drugs are also discussed. The latter mechanism is accounted for in terms of a tumor ‘enhanced permeability and retention’ (or EPR) effect. A principal advantage in the use of SMANCS or other macromolecular drugs is the potential for a reduction or elimination of toxicity. Macromolecular drugs such as a pyran copolymer–NCS conjugate show a marked reduction in bone marrow toxicity normally associated with the use of NCS. This is believed to be due to a hypothetical blood–bone marrow ‘barrier’ which, relative to NCS, restricts or limits access of the macromolecular drug to the bone marrow. In addition, the clinical possibilities for SMANCS are discussed, including the suggestion that angiotensin II-induced hypertension has clinical potential in improving the selective delivery of macromolecular drugs (i.e. SMANCS) to tumors. Aqueous SMANCS formulations have been tested in pilot studies in patients with solid tumors of the ovary, esophagus, lung, stomach, adrenal gland and in the brain. Formulations based on SMANCS/Lipiodol have been shown to be effective both as a diagnostic tool and for therapeutic use in solid tumors where the formulations are given arterially via a catheter. In a pilot study in primary unresectable hepatoma, an objective reduction in tumor size was observed for about 90% of cases when an adequate amount of the macromolecular drug was administered. A patient receiving such treatment with no active liver cirrhosis and tumor nodules/lesion confined within one liver segment might expect to have a 90% chance of survival after treatment for at least 5 years.

Nonspecific stimulation of the maternal immune system. I. Effects on teratogen‐induced fetal malformations

Background Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. Methods Teratogenic chemicals (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freund's complete adjuvant (FCA). Results Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. Conclusions For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation. Teratology 62:413–419, 2000. © 2000 Wiley-Liss, Inc.

Nonspecific stimulation of the maternal immune system. I. Effects On teratogen-induced fetal malformations.

Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freund's complete adjuvant (FCA). Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.

Anti-inflammatory effect of the conjugate of superoxide dismutase with the copolymer of divinyl ether and maleic anhydride against rat re-expansion pulmonary edema

Bovine erythrocyte superoxide dismutase (SOD) was conjugated with the copolymer of divinyl ether and maleic anhydride, known as DIVEMA or pyran copolymer. The DM–SOD conjugate was resistant against the proteolytic enzymes in serum, and showed a prolonged half-life in vivo after intravenous injection. DM–SOD was applied for the treatment of rat lung edema caused by the re-expansion after the left bronchus has been clipped for 3 days. The anti-inflammatory effect was evaluated by the inhibition rate of the water content due to the edema. Neither SOD nor DIVEMA reduced the increase in water content in the lung, whereas DM–SOD could effectively protect against the increase in water content such that the water content of the re-expanded lung was the same as that of the control right lung. Using electron microscopic observation the difference between the SOD and DM–SOD treatment in the water swelling of the interalveolar septum was clearly indicated. Observation of the surface of the endothelial wall in the blood vessel of the lung showed more a drastic difference. The leukocyte attachment on the endothelial layer observed following the treatment with SOD was not observed following that with DM–SOD. This observation indicates that DM–SOD had stopped the inflammation process at the first step of leukocyte adhesion, which should be followed by the rolling and expansion of the leukocytes to initiate the normal inflammation process.

Polymer Conjugation to Cu,Zn-SOD and Suppression of Hydroxyl Radical Generation on Exposure to H2O2: Improved Stability of SOD in Vitro and in Vivo

Highly reactive hydroxyl radical (OH) is generated during reaction of Cu,Zn-SOD (SOD)1 with H202, its own enzymatic reaction product (SOD plus 0;-). During inactivation of Cu,Zn-SOD with H202, Cu2+ liberated from the SOD catalyzes the formation of OH from H202. The OH thus generated will react with a,-protease inhibitor (a,-PI) if it is available in the milieu. To circumvent OH generation, we prepared eight different polymer-SOD conjugates and compared their stability against H202 treatment. Furthermore, the effect of each polymer-SOD conjugate on a,-PI in the presence of H202 was tested. Most polymer-SOD conjugates, except poly(styrene-co-maleic acid butylate; SMA)-SOD and poly(ethylene glycol; PEG)-SOD, showed higher stability than native SOD against inactivation by H202. The residual activity of divinylether maleic acid/anhydride copolymer (pyran copolymer)-conjugated SOD was 87% of the initial activity after incubation with 0.1 mM H202 for 2 h, whereas that of native SOD was 49%. Antiprotease activity of ca,-PI was completely abrogated when the inhibitor was treated with native SOD and 3.0 mM H202, but there was no change with pyran copolymer-SOD after 3 h of incubation with H202. Our results suggest that pyran copolymer captured loosely bound Cu2+ during the reaction of SOD with H202, thus resulting in suppression of both 0OH-mediated inactivation of SOD and suppression of activity of a,-PI. PEG showed little Cu2+-binding activity; no appreciable protection of SOD and a,-PI against OH generation was observed when Cu,Zn-SOD was conjugated with PEG. Circular dichroism and electron spin resonance studies of Cu,Zn-SOD treated with H202 indicated that the peptide conformation as well as the copper ligand of Cu,Zn-SOD can be stabilized not only by pyran copolymer per se but also by conjugation of Cu,Zn-SOD with some copolymers such as pyran.

Suppression of developmental anomalies by maternal macrophages in mice.

We tested whether nonspecific tumoricidal immune cells can suppress congenital malformations by killing precursor cells destined to cause such defects. Pretreatment of pregnant ICR mice with synthetic (Pyran copolymer) and biological (Bacillus Calmette-Guérin) agents significantly suppressed radiation- and chemical-induced congenital malformations (cleft palate, digit anomalies, tail anomalies, etc.). Such suppressive effects were associated with the activation of maternal macrophages by these agents, but were lost either after the disruption of activated macrophages by supersonic waves or by inhibition of their lysosomal enzyme activity with trypan blue. These results indicate that a live activated macrophage with active lysosomal enzymes can be an effector cell to suppress maldevelopment. A similar reduction by activated macrophages was observed in strain CL/Fr, which has a high spontaneous frequency of cleft lips and palates. Furthermore, Pyran-activated maternal macrophages could pass through the placenta, and enhanced urethane-induced cell killing (but not somatic mutation) in the embryo. It is likely that a maternal immunosurveillance system eliminating preteratogenic cells allows for the replacement with normal totipotent blast cells during the pregnancy to protect abnormal development.

MTT colorimetric assay for testing macrophage cytotoxic activity in vitro

The MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) cleavage,assay, originally described by Mosmann (1983, J. Immunol. Methods 65, 55) for measuring cell survival/proliferation, has been used successfully to quantitate macrophage-mediated cytotoxicity. Peritoneal macrophages, from control or pyran copolymer MVE 2-treated mice and from TU5 and L929 murine cell lines, were used as effectors and targets respectively in a 48 h cytotoxicity test, at three effector: target ratios (10:1, 5:1, 2:1). The amount of MTT reduced by cells to its blue formazan derivative during an additional 4 h of culture was quantified spectrophotometrically at 570 nm using an ELISA reader. A linear relationship between the formazan generated and the number of viable TU5 and L929 cells was demonstrated, together with time-dependent growth characteristics for these cells. The formazan produced by macrophages was independent of their functional state and did not interfere with the target cell signal. MVE 2-activated macrophages strongly inhibited the survival/growth of target cells in a dose-related way, whereas the cytotoxic activity of control macrophages was very low. Finally, the MTT method compared favorably with the 3H-TdR uptake method in evaluating macrophage cytotoxicity, and both of them were more sensitive than the 3H-TdR release assay. The MTT cleavage method is a useful alternative to radioisotopic methods for quantitating macrophage cytotoxicity for actively growing in vitro targets. Its main advantages are: (a) sensitivity and reproducibility; (b) elimination of the need for radioactive compounds; (c) ease with which it can be performed and quantified; (d) rapidity.

Reduced bone marrow toxicity of neocarzinostatin by conjugation with divinyl ether-maleic acid copolymer

Neocarzinostatin (NCS) was conjugated with divinyl ether-maleic acid/anhydride copolymer (pyran copolymer), and its therapeutic effect was compared with that of NCS. The conjugated NCS (pyran-NCS) with a molecular weight of about 23,000, exhibited in vitro cytotoxic activity against eight cell lines and bone marrow cells that was similar to the cytotoxic activity of NCS on a molar basis. Furthermore, both drugs had similar effects against a multidrug-resistant Chinese hamster ovary cell line (CH RC 5) and its parent cell line (AUXB 1) in vitro. However, pharmacological analysis showed that pyran-NCS had reduced accumulation in the spleen, and most important was three times less hematotoxic in vivo compared with NCS. Also, pyran-NCS had a 1.7-fold higher 50% lethal dose ( ld 50 ). Antitumor activity of pyran-NCS and NCS was tested against two different forms of Meth A tumor. In a solid tumor model, pyran-NCS and NCS suppressed tumor growth at three-fourths of the ld 50 to 12.8 and 19.0% of the control tumor as evaluated on day 28, respectively ( P < 0.025). In an ascitic tumor model, the percentage increase in the median life span caused by pyran-NCS and NCS was more than 400 and 150% on day 60, respectively. Pyran-NCS is more effective than NCS because the reduced acute toxicity permits an increased drug dosage.

Effects of activation on lipid and lipoprotein metabolism in murine macrophages.

The effects of activation on lipid and lipoprotein metabolism were examined in resident murine macrophages, inflammatory cells elicited by thioglycolate, primed cells elicited by pyran copolymer, and activated cells elicited by Corynebacterium parvum. Low density lipoprotein receptors were reduced by 70%, while scavenger receptors were reduced 60% in activated cells. Basal cholesteryl ester and triglyceride synthesis were increased fourfold in activated cells, whereas the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase was high in resident cells and progressively declined by greater than 80% in activated cells. Activities of neutral cholesteryl esterase and neutral triglyceride lipase were increased two- to fourfold in inflammatory, primed, and activated macrophages. These results demonstrate the diverse changes in lipid and lipoprotein metabolism that occur with activation and emphasize how the behavior of macrophages in atherosclerotic lesions can be altered by activation.

Free radical generation during brief period of cerebral ischemia may trigger delayed neuronal death

We investigated the pathogenic role of free radical formation in ischemic neuronal death using radical scavenger, Superoxide dismutase. Cerebral ischemia was produced in the gerbil by bilateral common carotid occlusion for 5 min, which consistently resulted in delayed neuronal death in the CA1 region of the hippocampus. The effects of free Superoxide dismutase and a derivatized Superoxide dismutase, pyran copolymer conjugated Superoxide dismutase, on early ischémie damages, detected sensitively by the immunohistochemical reaction for microtubule associated protein 2, and a subsequent delayed neuronal death after restoration of blood flow were investigated. Preischemic treatment by pyran conjugated Superoxide dismutase showed clear protective effects against both the neuronal damages detected by immunohistochemistry after 5 min ischemia and the delayed neuronal necrosis after one week of recovery, although no clear beneficial effects were observed when this drug was administered just before the recirculation or free Superoxide dismutase was used. These results strongly suggest that free radical generation during brief period of ischemia plays a pivotal role in triggering the ischémie neuronal damages causing delayed neuronal death at the selectively vulnerable areas of the brain.

Poly(carboxylic acid) polymers as carriers for anthracyclines

Anthracycline-poly(carboxylic acid) polymer conjugates can be easily prepared in alkalinized aqueous media by a process involving a reaction of 14-bromo derivatives of the drug carrying the methylketone side chain. The method can be applied to a variety of poly(carboxylic acid) polymers. Following i.v. administration, the drug potency and efficacy was found to depend on the molecular weight of the polymer. Using pyran copolymer as carrier, a critical influence of the polymer itself on the overall efficacy of the conjugate was observed. The understanding of the in vivo interaction between drug and polymer and of the mechanism of action of drug-polymer conjugates should establish guidelines for future attempts to optimize antitumor efficacy. Although an increased selectivity in drug targeting can be achieved, in theory, by the attachment of specific targeting moieties, the preclinical evaluation of drug-poly(carboxylic acid) polymer conjugates themselves indicated therapeutic advantages over free drug. Reduction of systemic drug toxicity and, at least in selected tumor systems, increased efficacy implies improved drug delivery. In particular, the toxicologie and pharmacologie profile of doxorubicin-poly( l-aspartic acid) conjugate warrants a clinical evaluation.

Augmentation of mouse liver-associated natural killer activity by biologic response modifiers occurs largely via rapid recruitment of large granular lymphocytes from the bone marrow.

A variety of biologic response modifiers (BRM) can potently augment NK activity in nonlymphoid organs. By using the liver as a model organ, we have shown that this augmentation of organ-associated NK activity is coincident with a 10- to 15-fold increase in the number of large granular lymphocytes (LGL) which can be isolated. The present study was designed to investigate the mechanism by which BRM induce this increase in liver-associated LGL and the coincident increase in hepatic NK activity. Initial studies confirm that a single dose of the pyran copolymer, maleic anhydride divinyl ether (MVE-2), augmented hepatic NK activity and increased the number of liver-associated LGL from 3 x 10(4)/liver to 5 x 10(5)/liver (a 17-fold increase). Multiple injections of MVE-2 further augmented total liver-associated NK activity and LGL number (to 13 x 10(5)/liver). As expected, both the NK activity and detectable LGL were eliminated by treatment of the mice with antiasialo GM1 (asGM1) serum. Three possible mechanisms for the BRM-induced increase in liver-associated LGL have been investigated, including 1) the rapid proliferation of resident hepatic LGL, 2) the redistribution of mature LGL from peripheral sites such as the spleen, or by 3) a rapid output and subsequent hepatic localization of LGL or their precursors recently derived from the bone marrow (BM). Our results demonstrated that the contribution of in situ proliferation to the BRM-induced increase in liver-LGL was relatively small, since the number of cells expressing NK-associated markers (i.e., asGM1, Thy-1.2, and NK1.1) and in G2/M phase (as assessed by propidium iodide uptake) was only 4 to 8%. Further experiments demonstrated that splenectomy before the administration of MVE-2 did not inhibit the augmentation of liver-associated NK activity. This result argued against a recruitment of mature LGL from the spleen. In contrast, selective depletion of the BM following administration of 89Sr decreased the ability of MVE-2 to augment liver-associated NK activity by greater than 80%. This procedure also significantly decreased the ability of Propionibacterium acnes (85%) and multiple doses of IL-2 (49%) to augment liver-associated NK activity. These results demonstrate that the rapid augmentation of liver-associated NK activity by BRM is largely due to localization and accumulation in the liver of LGL recently derived from the BM.

Effects of Pyran Copolymer on Host Resistance of Mice to Plasmodium yoelii

Female B6C3F1 mice treated with 25 mg/kg pyran intravenously (i.v.) on days -4 and -3 were more susceptible to nonlethal Plasmodium yoelii 17XNL or lethal Plasmodium berghei ATCC-30090 than untreated mice or mice treated intraperitoneally (i.p.). Female B6C3F1 mice treated with pyran i.p. displayed enhanced resistance to Listeria monocytogenes as compared to untreated mice or mice given pyran i.v. Peritoneal exudate cells (PEC) primed by pyran i.p. possessed enhanced ability to kill Listeria but impaired ability to destroy Plasmodium. Phagocytosis of Covaspheres by PEC was greater for mice given pyran i.p. than those given pyran i.v. Chemiluminescence evoked by zymosan was less for PEC from mice given pyran i.v. than for those from untreated mice or those given pyran i.p. Chemiluminescence was greater for adherent splenocytes from mice treated with pyran i.p. than for those from untreated mice or those from mice treated i.v. Pyran administered i.v. is less effective in modulating the host immune response than pyran administered i.p. Immunomodulatory agents such as pyran have adverse as well as beneficial effects depending upon the route of administration.

Oxygen Radicals in Influenza-Induced Pathogenesis and Treatment with Pyran Polymer-Conjugated SOD

The pathogenicity of influenza virus infection in the mice involves, at least in part, overreaction of the immune responses of the host rather than a direct effect of virus multiplication. Xanthine oxidase, which is responsible for the generation of oxygen free radicals, was elevated in serum and lung tissue of mice infected with influenza virus. To test the theory that oxygen-free radicals are involved in pathogenesis, free radicals were removed by injecting superoxide dismutase (SOD), a specific superoxide radical scavenger, which was conjugated with a pyran copolymer. The conjugate protected mice against a potentially lethal influenza virus infection if administered 5 to 8 days after infection. These findings indicate that oxygen radicals are important in the pathogenesis of influenza virus infection, and that a polymer-conjugated SOD has therapeutic potential for this virus infection and other diseases associated with free radicals.

Anticancer Potential of Viva-Natural, a Dietary Seaweed Extract, on Lewis Lung Carcinoma in Comparison with Chemical Immunomodulators and on Cyclosporine-Accelerated AKR Leukemia

Viva-Natural, extracted from a dietary seaweed, containing a macrophage-activating polysaccharide, has been confirmed to be active against intraperitoneally implanted Lewis lung carcinoma (LLC) and spontaneous AKR T cell leukemia. The antitumor potential against LLC has been evaluated in comparison with standard synthetic immunomodulators such as pyran copolymer (MVE-2), isoprinosine, levamisole, and tilorone while manipulating the immune systems by immunosuppressive agents, cyclophosphamide (CY) and 2-chloroadenosine. The anti-LLC activity of Viva-Natural has been found to be superior to that of isoprinosine but inferior to that of MVE-2. LLC-enhancing effect of CY could be partially reserved by the subsequent administration of Viva-Natural or MVE-2 but not by isoprinosine. 2-Chloroadenosine, a specific macrophage inhibitor, abrogated the anti-LLC activity of Viva-Natural and isoprinosine but not the activity of MVE-2. Levamisole and tilorone showed no anti-LLC activity. Ethanol-precipitable fraction of water-soluble part of Viva-Natural (crude polysaccharide) demonstrated curative activity similar to that of MVE-2. Viva-Natural reversed the potentiation effect of ciclosporin on the development of leukemia in AKR mice at preleukemic stage.

Effects of assay medium composition on macrophage-mediated tumor cell binding and lysis

Activated macrophages express selective, high avidity tumor binding and cytotoxicity for tumor targets. The reported macrophage-mediated tumor binding and killing activities vary considerably among studies. Whereas, some of these studies utilized identical tumor target cells and similar sources of macrophages, the composition of the assay media used by different groups varied with respect to: type of medium (Eagle's minimal essential medium (EMEM), RPMI-1640, Dulbecco's modified Eagle's medium with 1.0 g/l glucose (DMEMLG), or with 4.5 g/l, glucose (DMEMHG)), concentration of serum (0–20%), and the addition of certain reagents (amphotericin B, pyruvate, and Hepes). The purpose of this study was to evaluate the effects of varying these parameters of medium composition on macrophage-mediated high (HA) and low avidity tumor binding and cytolysis. Tumor cytolysis was measured with an 18 h 51Cr release assay using peritoneal macrophages from C3H/HeN mice primed in vivo with pyran copolymer and further stimulated in vitro with lipopolysaccharide (10 ng/ml). P815 tumor cells were used as targets at a 10:1 effector: target ratio. Binding of targets to macrophages was termined by a method utilizing inverted centrifugation. Under optimal conditions, 2 × 10 5 macrophages bound as many as 17600 ± 3565 tumor cells and caused up to 50.4 ± 3.6% cytolysis. Assays performed in DMEMHG compared with the other three media tested resulted in HA tumor binding and cytolysis which were decreased by up to 42.5% ( P < 0.05) and 64.3% ( P < 0.01), respectively, compared with the three other types of medium. The addition of pyruvate (1 mM) to EMEM with 5% fetal calf serum (FCS) stimulated 98.8% ( P < 0.01) and 50.6% ( P < 0.1) increases in tumor binding and cytolysis, respectively compared with EMEM/5% FCS alone, while Hepes (25 mM) stimulated 58.3% ( P < 0.01) and 37.5% ( P < 0.1) increases in these activities. Amphotericin B (2.5 μg/ml) completely abrogated tumor cytolysis, but it caused no change in tumor binding. Serum produced variable effects on macrophage-mediated tumor killing. Five of six lots of FCS inhibited tumor lysis, by 16 to 98% (32.6 ± 28.6%; mean ± SD). However, the same lot of FCS which inhibited cytolysis by 98% enhanced HA binding by 152% ( P < 0.05). Finally, several commercially available serumless medium preparations supported macrophage-mediated tumor binding; however, none of the serumless media tested supported macrophage-mediated tumor cytolysis. We conclude that common differences in assay medium composition can markedly alter macrophage-mediated tumor cell binding and cytolysis.

Liver-associated macrophage precursor cells proliferate under impairment of regular hemopoiesis.

We reported previously that immature macrophage precursor cells can be isolated from spleen and liver of cyclophosphamide or pyran copolymer-pretreated mice. We now extended our investigations to livers of normal, untreated specific pathogen-free mice. Using the response to the macrophage growth factor colony-stimulating factor-1 (CSF-1) and the presence of the mouse macrophage-specific F4/80 antigen as criteria of definition, in the liver of normal mice we could demonstrate macrophage precursor (M phi P) cells by means of proliferation assays and flow cytometric analysis. The amount of M phi P present in the normal liver was significantly increased after administration of pyran copolymer. Also an enhanced proliferative response to CSF-1 as well as augmented natural killer activity and cytostasis of Candida albicans was noted in liver nonparenychymal cells (LNPC) after treatment of bone marrow (BM)-irradiated, splenectomized mice with pyran copolymer. Since the irradiated BM was actually proven to be silent by assessment of BM number and proliferative capacity and by scoring white blood cells, our findings suggest a response of endogenous liver M phi P under the applied conditions. Further evidence for the presence of endogenous liver hemopoietic cells was obtained from transplantation experiments in which LNPC brought about the survival of lethally irradiated mice. The data point towards a significance of the liver in disposing hemopoietic cells to the organism under impairment of regular hemopoiesis.