The effect of elimination of macrophages on the tissue distribution of liposomes containing [3H]methotrexate

In the present study the tissue distribution of [³H]methotrexate was studied after intravenous injection of [³H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [³H]methotrexate liposomes in the blood, due to a decreased uptake of [³H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [³H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.     

Inhibition of rat liver retinyl palmitate hydrolase activity by ether analogs of cholesteryl esters and acylglycerides

In the present study the tissue distribution of [3H]methotrexate was studied after intravenous injection of [3H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [3H]methotrexate liposomes in the blood, due to a decreased uptake of [3H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [3H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.     

Inhibitory effect of cholesterol on the uptake of liposomes by liver and spleen

The effect of cholesterol content of small unilamellar (SUV) and reverse phase (REV) liposomes on blood clearance and tissue distribution has been studied. [¹⁴C]Inulin has been used as an aqueous marker of liposomes to represent the uptake of intact liposomes in tissues. The blood clearance of the intravenously-injected SUV and REV liposomes depends on the cholesterol content of liposomes. The cholesterol-free (0 mol%) liposomes are cleared more readily from the circulation than the cholesterol-poor liposomes (20 mol%) and the cholesterol-poor are cleared more rapidly than the cholesterol-rich (46.6 mol%) liposomes. This clearance pattern of liposomes from the circulation is not attributed to the change of size of liposomes due to the increase in cholesterol content of liposomes. However, poor stability of cholesterol-free or cholesterol-poor liposomes in the circulation is partly responsible, but the predominant factor responsible for the observed blood clearance pattern is the inhibitory effect of cholesterol on the uptake of liposomes by reticuloendothelial-rich tissues liver and spleen. Uptake of liposomes by these organs is decreased with increasing cholesterol content of vesicles. It is suggested that to produce liposome preparations with a long circulating half life in vivo it is necessary to inhibit their uptake by liver and spleen.     

Differential distribution of liposome-entrapped [3H]methotrexate and labelled lipids after intravenous injection in a primate

Positive liposomes consisting of phosphatidylcholine, cholesterol and stearylamine and negatively charged liposomes consisting of phosphatidylcholine, cholesterol and phosphatidylserine, were double labelled with either ³H-labelled dipalmitoyl phosphatidylcholine and [¹⁴C]cholesterol or with [¹⁴C]cholesterol and [³H]methotrexate entrapped in the aqueous phase. The plasma levels and urinary excretion of radioactivity from sonicated and non-sonicated liposomes were then compared with the levels of radioactivity from free [³H]methotrexate during a 4 h experimental period after an initial intravenous injection in cynomolgous monkeys. Tissue uptake at the completion of the 4 h experimental period was also measured.It was found that plasma radioactivity from [³H]methotrexate and [¹⁴C]cholesterol in sonicated positive liposomes was cleared more slowly than from comparable non-sonicated liposomes, and considerably slower than from free [³H]methotrexate. Radioactivity from sonicated negative liposomes was cleared more rapidly than from positive sonicated liposomes. Positive liposomes captured considerably more [³H]methotrexate than negative liposomes and showed very low permeability to [³H]methotrexate in in vitro studies, even in the presence of high concentrations of serum.[¹⁴C]Cholesterol radioactivity was cleared more rapidly from plasma than ³H-radioactivity from liposome-entrapped [³H]methotrexate for double-labelled sonicated liposomes and generally showed greater uptake into tissues and red blood cells. ³H-labelled dipalmitoyl phosphatidylcholine in sonicated positive liposomes was cleared faster than [¹⁴C]cholesterol during the first 3 h. The more rapid disappearance of [¹⁴C]cholesterol from the plasma was complemented by greater uptake into a number of tissues, and positive non-sonicated liposomes were taken up to a greater extent by the spleen than equivalent sonicated liposomes.Renal excretion of ³H from liposome-entrapped [³H]methotrexate was considerably less than that of ³H from free [³H]methotrexate. There was insignificant excretion, however, of ¹⁴C from cholesterol in the urine.Entrapment in liposomes completely prevented the otherwise considerable breakdown of free methotrexate to ³H-containing products in plasma and partially prevented its breakdown in tissues.These studies indicate marked differences in the distribution of liposomes in vivo due to surface charge and size, and some degree of exchange of the lipid components of the liposome bilayer independent of the distribution of the entrapped species. They also show that entrapment in liposomes can reduce metabolic degradation of a drug, maintain high plasma levels and reduce its renal excretion.     

Differential distribution of liposome-entrapped [H]methotrexate and labelled lipids after intravenous injection in a primate

Positive liposomes consisting of phosphatidylcholine, cholesterol and stearylamine and negatively charged liposomes consisting of phosphatidylcholine, cholesterol and phosphatidylserine, were double labelled with either ³H-labelled dipalmitoyl phosphatidylcholine and [¹⁴C]cholesterol or with [¹⁴C]cholesterol and [³H]methotrexate entrapped in the aqueous phase. The plasma levels and urinary excretion of radioactivity from sonicated and non-sonicated liposomes were then compared with the levels of radioactivity from free [³H]methotrexate during a 4 h experimental period after an initial intravenous injection in cynomolgous monkeys. Tissue uptake at the completion of the 4 h experimental period was also measured.It was found that plasma radioactivity from [³H]methotrexate and [¹⁴C]cholesterol in sonicated positive liposomes was cleared more slowly than from comparable non-sonicated liposomes, and considerably slower than from free [³H]methotrexate. Radioactivity from sonicated negative liposomes was cleared more rapidly than from positive sonicated liposomes. Positive liposomes captured considerably more [³H]methotrexate than negative liposomes and showed very low permeability to [³H]methotrexate in in vitro studies, even in the presence of high concentrations of serum.[¹⁴C]Cholesterol radioactivity was cleared more rapidly from plasma than ³H-radioactivity from liposome-entrapped [³H]methotrexate for double-labelled sonicated liposomes and generally showed greater uptake into tissues and red blood cells. ³H-labelled dipalmitoyl phosphatidylcholine in sonicated positive liposomes was cleared faster than [¹⁴C]cholesterol during the first 3 h. The more rapid disappearance of [¹⁴C]cholesterol from the plasma was complemented by greater uptake into a number of tissues, and positive non-sonicated liposomes were taken up to a greater extent by the spleen than equivalent sonicated liposomes.Renal excretion of ³H from liposome-entrapped [³H]methotrexate was considerably less than that of ³H from free [³H]methotrexate. There was insignificant excretion, however, of ¹⁴C from cholesterol in the urine.Entrapment in liposomes completely prevented the otherwise considerable breakdown of free methotrexate to ³H-containing products in plasma and partially prevented its breakdown in tissues.These studies indicate marked differences in the distribution of liposomes in vivo due to surface charge and size, and some degree of exchange of the lipid components of the liposome bilayer independent of the distribution of the entrapped species. They also show that entrapment in liposomes can reduce metabolic degradation of a drug, maintain high plasma levels and reduce its renal excretion.     

Intrahepatic Distribution of Long-Circulating Liposomes Containing Polyethylene Glycol) Distearoyl Phosphatidylethanolamine

Abstract

We investigated the intrahepatic distribution in rats of liposomes of 85 or 130 nm diameter, which were sterically stabilized with a polyethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) so as to increase their circulation time in blood. Various times after intravenous injection of radiolabeled ([³H-]cholesterylether) liposomes, parenchymal and non-parenchymal cells of the liver were isolated and their radioactivity content was determined. Control liposomes of 85 nm without PEG-PE distributed in an approximately 80:20 ratio to hepatocytes (H) and macrophages (M), respectively; the 130-nm control liposomes showed a 50:50 H/M distribution. Incorporation of PEG-PE reduced the rate of total liver uptake about 4-fold for liposomes of either size and shifted the H/M ratio to 60:40 for the smaller vesicles and to 40:60 for the larger ones. For both liposome sizes, PEG-PE apparently causes a shift in intrahepatic distribution in favor of the macrophages. It is concluded that PEG-PE has a stronger inhibitory effect on liposome uptake by hepatocytes than on uptake by macrophages. Attempts to shift liposome uptake more in favor of hepatocytes, by incorporation of lactosylceramide, failed. This compound, although causing an increase in hepatic uptake, particularly for the 130-nm liposomes, shifted the H/M ratio further towards the macrophages. We conclude that the galactose moiety of the glycolipid is sufficiently exposed on the surface of (PEG-PE)-containing liposomes to allow interaction with the galactose-binding lectin at the surface of the liver macrophage and that the extent of exposure is dependent on vesicle size.     

Liposome-Mediated transfer of bacterial RNA into carrot protoplasts

The uptake of liposome-encapsulated E. coli [³H]RNA by carrot (Daucus carota L.) protoplasts was examined. [³H]RNA extracted from protoplasts that had been incubated with [³H]RNA-containing, large, unilamellar lipid vesicles (liposomes) obtained by ether infusion, and examined by sucrose gradient centrifugation and formamide-polyacrylamide gel electrophoresis, appeared substantially degraded, with a total elimination of 23S RNA and a partial loss of 16S RNA. In contrast, no breakdown of the [³H]RNA was apparent in the liposomes after sequestration, even in the presence of externally added ribonuclease, or in the unfused liposomes remaining after incubation of protoplasts with liposomes. Thus, the degradation of the [³H]RNA extracted from the protoplasts must have occurred within the protoplasts and represents evidence for liposome-mediated RNA uptake. Naked RNA added to the protoplast culture was found to be totally degraded after incubation with the protoplasts. The uptake of liposome-sequestered RNA by protoplasts was demonstrated to be a function both of the lipid composition of the liposomal membrane and of the temperature of incubation of the liposomeprotoplast mixture. Furthermore, the mode of this uptake (fusion versus endocytosis) could be manipulated by adjusting the cholesterol content of the liposomal membrane. The implications of the ability to insert RNA into protoplasts without degradation by extracellular nucleases are discussed.     

Tissue distribution of 99mic-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues

Abstract

The tissue distribution of ^99mTc-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues was investigated for application to radiopharmaceuticals. The amphiphiles used were N,N-didodecyl-N α-[6-(trimethylammoniohexanoyl]-L-ala-ninamide bromide (N+C₅Ala2C₁₂), N,N-didodecyl-N_α-{6-[dimethyl(2-carboxyethyl)ammonio]hexanoyl}-L-alaninamide bromide (CAC₂N⁺C₅Ala2C₁₂) and S-{l-carboxy-2-([2,3-bis (he xadecyloxy)propoxy]carbony1)ethyl}homocy ste ine. These liposomes were stable in saline and 50% serum at 37° for at least 24h in comparison with the liposomes prepared from phosphatidylcholine and cholesterol (1:1). Most of the radioactivity of N+C₅Ala2C₁₂ and CAC₂N+C₅Ala2C₁₂ liposomes was firmly bound to Ehrlich ascites tumor cells in vitro. But the accumulation of three liposomes into the tumor of Ehrlich solid tumor-bearing mice after intravenous injection was low and most of the liposomes was taken up highly in liver and spleen which belong to the reticuloendothelial system (RES). Some approaches were made to reduce the RES uptake of N+C5Ala2C12 liposomes as follows: (1) the pretreatment of dextran sulfate depressed the uptake of the liposomes in the liver accompanied by increasing uptake in tumor and other tissues except stomach, (2) the modification of the liposomes with n-dodecyl glucoside or n-dodecyl sucrose depressed the uptake in liver and spleen, resulting in an increase in blood and other tissues such as tumor, duodenum and kidney, (3) the modification of the liposomes with ganglioside GM3 or GM1 reduced the uptake in liver and spleen, but increased scarcely the uptake in blood and tumor because of the rapid excretion into urine, (4) the intraperitoneal injection reduced the uptake of the liposomes in liver and increased significantly the accumulation in pancreas.     

Differentiation in hepatic and splenic phagocytic activity during reticuloendothelial blockade with cholesterol-free and cholesterol-rich liposomes

We have shown earlier that liver and spleen reticuloendothelial cells have low affinity to phagocyte liposomes containing cholesterol. In the present study, we predosed mice with cholesterol-rich (identical to = 46.6 mol% cholesterol content) and cholesterol-free (identical to 0 mol%) liposomes to saturate the reticuloendothelial cells and examined the tissue distribution of the second dose of the test liposomes containing an aqueous marker, 125I-labelled poly(vinylpyrrolidone). The result shows that both preparations of the predosed liposomes caused suppression in hepatic uptake and delay in the blood clearance of the test liposomes, but the cholesterol-free liposomes were more effective in producing these effects than the cholesterol-rich liposomes. The suppression in hepatic phagocytic function, in accordance with the 'spillover' phenomenon [16, 17], caused an enhancement in spleen and lung uptake. The increase in lung uptake was proportionally related to the degree of suppression in the hepatic uptake, but the results of the splenic uptake showed some discrepancy. The predosed cholesterol-free liposomes which caused the maximum spillover of the test liposomes from the liver did not achieve maximum enhancement in the splenic uptake. Instead, the maximum enhancement was recorded with the predosed cholesterol-rich liposomes. This discrepancy in splenic uptake suggests that the predosed liposomes caused saturation of not only liver also the spleen reticuloendothelial system. However, instead of suppression in the splenic uptake due to the saturation, enhancement in uptake of the test liposomes was observed. We suggest the cause of this apparent increase the splenic phagocytic activity may be due to stimulation, by some unknown mechanism of splenic macrophages endothelial cells and/or lymphocytes, to phagocyte the excess of the test liposomes spillover from the liver with impaired phagocytic function.     

Pharmacokinetics,Tissue Distribution and Metabolism of Intravenously Administered Digalactosyldiacylglycerol and Monogalactosyldiacylglycerol in the Rat

Abstract

A bilayer forming galactolipid, digalactosyldiacylglycerol (DGalDG) has been identified as a tool with suitable physicochemichal properties for pharmaceutical formulation work. One possible application is as a carrier for liposome entrapped drugs for intravenous administration. The fate of intravenously administered galactolipids is not known. In this study liposomal dispersions of galactolipids, containing [³H]fatty acid labelled DGalDG or monogalactosyldiacylglycerol (MGalDG) were injected intravenously in the rat and the disappearance from blood and uptake by tissues were examined. The T1/2 of [³H]DGalDG in plasma was 3 to 5 minutes. Of the tissues examined (liver, spleen, kidneys, lung, heart, stomach, upper and lower small intestine and colon), the liver contained the highest radioactivity per g tissue after both 15 min. and 4 h. Autoradiographic examinations after 15 min, 1 h and 4 h showed that the uptake of radiolabeled DGalDG and MGalDG occurred mainly to the hepatocytes. Less than 6 % of the injected [³H]DGalDG remained in liver and plasma as [³H]DGalDG after 4 h. [³H]MGalDG exhibited a similar pattern of metabolism although the initial disappearance rate was faster than for [³H]DGalDG. The study thus shows that the hepatocytes take up and hydrolyse galactolipids after intravenous administration.     

Effect of cholesterol on the uptake and intracellular degradation of liposomes by liver and spleen; a combined biochemical and gamma-ray perturbed angular correlation study

We investigated the effect of cholesterol on the uptake and intracellular degradation of liposomes by rat liver and spleen macrophages. Multilamellar vesicles (MLV) consisting of distearoylphosphatidylcholine/phosphatidylserine (molar ratio 9:1) or distearoylphosphatidylcholine/cholesterol/phosphatidylserine (molar ratio 4:5:1) were labeled with [3H]cholesteryl hexadecyl ether and/or cholesteryl [14C]oleate. After i.v. injection the cholesterol-containing liposomes were eliminated less rapidly from the bloodstream and taken up to a lesser extent by the liver (macrophages) than the cholesterol-free liposomes. Assessment of the 3H/14C ratios in liver and spleen cells revealed that the cholesterol-containing liposomes are substantially more resistant towards intracellular degradation than the cholesterol-free liposomes. These results could be confirmed by measuring the release of 111In from liposomes after uptake by liver and spleen by means of gamma-ray perturbed angular correlation spectroscopy. Experiments with cultured Kupffer cells in monolayer also revealed that incorporation of cholesterol results in a decrease of the uptake and an increase of the intracellular stability of cholesteryl [14C]oleate-labeled liposomes. Finally, incubation of both types of liposomes with lysosomal fractions prepared from rat liver demonstrated a difference in susceptibility to lysosomal degradation: the cholesterol-free vesicles were much more sensitive to lysosomal esterase than the cholesterol-containing liposomes. These results may be relevant to the application of liposomes as a drug carrier system to liver and spleen (macrophages).     

Transport of folates at maternal and fetal sides of the placenta: lack of inhibition by methotrexate

Folate (pteroylglutamate) and methotrexate rapid (seconds) uptake by the trophoblast was investigated from either the maternal or fetal circulations of the isolated dually-perfused guinea-pig placenta. Tissue uptake was measured by using a single-circulation paired-tracer (³H-test and ¹⁴C-extracellular marker) technique. [³H]Folate uptakes were 80 and 52% (mean) in perfusates without unlabelled folate, on maternal and fetal sides, respectively. There was negligible ³H-tracer backflux into the circulation up to 6 min probably due to metabolic sequestration. [³H]Methotrexate uptakes were about 85 and 22% on maternal and fetal sides, respectively; however these uptakes were followed by rapid and complete backflux of the label. Specific transplacental transfer of [³H]folate or [³H]methotrexate in either direction was not detectable within 5–6 min. At the brush-border side (maternal) uptake of [³H]folate was highly inhibited by 100 nM unlabelled folate or its reduced form, methyltetrahydrofolate (the main form in plasma); however, equimolar methotrexate (an antifolate chemotherapeutic agent) failed to produce any inhibition of folate uptake. Our findings demonstrate that on both sides of the placenta a high-affinity transport system exists for trophoblast uptake of folate compounds. For methotrexate, either a separate transport system may exist or methotrexate may have a very low affinity for the folate system. These results are distinct from the findings reported in mouse L1210 leukemia cells.     

Uptake and biodistribution of free and liposomally incorporated lipopolysaccharide of aeromonas salmonicida administered via different routes to rainbow trout: (Oncorhynchus mykiss)

Abstract

The effects on uptake and biodistribution of radiolabelled lipopolysaccharide (LPS) due to changing routes of administration, encapsulation of LPS within liposomes and altering liposomal surface charge were examined in rainbow trout (Oncorhynchus mykiss). ³H-labelled LPS, positively- and negatively-charged (¹⁴C-labelled) liposomes or ¹⁴C-labelled liposomes containing ³H-LPS were administered to trout via intravenous, intraperitoneal, intramuscular, or oral routes. Twenty-four hours following administration, relative uptake of LPS and multilamellar vesicles (MLV) based on detection of ³H and ^1AC, respectively, was determined in samples taken from the kidney, spleen, liver, plasma, blood cells and skeletal muscle. In general, regardless of the route of administration, ³H-LPS, ^1AC-MLV and liposomally encapsulated LPS were recovered primarily in the kidney and spleen. Intravenous administration resulted in the greatest uptake of radiolabel by the kidney and spleen, followed by the intraperitoneal and intramuscular routes. Although oral administration yielded the lowest overall uptake of labelled material, detection of ³H and ¹⁴C in the liver was enhanced when compared with the other routes. Negatively-charged MLV were delivered more efficiently to the kidney and spleen than positively-charged MLV; but negatively- and positively-charged MLV containing LPS demonstrated the opposite relationship between charge and distribution among the kidney and spleen. These results suggest that liposomal encapsulation (particularly within positively-charged MLV) enhances delivery of LPS to the primary hemopoietic organs in rainbow trout.     

Apolipoprotein B stimulates formation of monocyte-macrophage surface-connected compartments and mediates uptake of low density lipoprotein-derived liposomes into these compartments

Much of the cholesterol that accumulates in atherosclerotic plaques is found within monocyte-macrophages transforming these cells into "foam cells." Native low density lipoprotein (LDL) does not cause foam cell formation. Treatment of LDL with cholesterol esterase converts LDL into cholesterol-rich liposomes having >90% cholesterol in unesterified form. Similar cholesterol-rich liposomes are found in early developing atherosclerotic plaques surrounding foam cells. We now show that cholesterol-rich liposomes produced from cholesterol esterase-treated LDL can cause human monocyte-macrophage foam cell formation inducing a 3-5-fold increase in macrophage cholesterol content of which >60% is esterified. Although cytochalasin D inhibited LDL liposome-induced macrophage cholesteryl ester accumulation, LDL liposomes did not enter macrophages by phagocytosis. Rather, the LDL liposomes induced and entered surface-connected compartments within the macrophages, a unique endocytic pathway in these cells that we call patocytosis. LDL liposome apoB rather than LDL liposome lipid mediated LDL liposome uptake by macrophages. This was shown by the findings that: 1) protease treatment of the LDL liposomes prevented macrophage cholesterol accumulation; 2) liposomes prepared from LDL lipid extracts did not cause macrophage cholesterol accumulation; and 3) purified apoB induced and accumulated within macrophage surface-connected compartments. Although apoB mediated the macrophage uptake of LDL liposomes, this uptake did not occur through LDL, LDL receptor-related protein, or scavenger receptors. Also, LDL liposome uptake was not sensitive to treatment of macrophages with trypsin or heparinase. Cholesterol esterase-mediated transformation of LDL into cholesterol-rich liposomes is an LDL modification that: 1) stimulates uptake of LDL cholesterol by apoB-dependent endocytosis into surface-connected compartments, and 2) causes human monocyte-macrophage foam cell formation.     

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.     

Macrophage regulation of DNA synthesis in lymphoid cells: effects of a soluble factor from macrophages

Addition of rat peritoneal macrophages to nonadherent rat spleen cells in culture results in enhancement or suppression of DNA synthesis depending on the ratio of macrophages to lymphocytes. At high ratios of macrophages to lymphocytes (1:5), suppression can be observed as early as four hours. Macrophages suppress incorporation of thymidine (TdR) by nonadherent spleen, thymus and bone marrow cells, in most instances, to less than 5% of that observed in culture to which macrophages were not added. In the presence of macrophages, incorporation of [³H]uridine and [¹⁴C] amino acids by spleen cells was also moderately suppressed. Based on ⁵¹Chromium release and dye exclusion assays, it appears that suppression is not due to cytotoxicity. Furthermore, suppression of [³H]TdR incorporation by nonadherent spleen cells is reversible, in the presence of an antigenic stimulus, following removal of the macrophages from the cultures. The suppressive effects are not elicited by extracts of macrophages, freeze-thawed or heated macrophages, but appear to be due to a low molecular weight, heat stable factor released into the macrophage culture fluid.     

Targeting of lactosylceramide-containing liposomes to hepatocytes in vivo

Incorporation of 8 mol% lactosylceramide in small unilamellar vesicles consisting of cholesterol, dimyristoylphosphatidylcholine and phosphatidylserine in a molar ratio of 5:4:1 and containing [³H]inulin as an aqueous-space marker resulted in a 3-fold decreased half-life of the vesicles in blood and a corresponding increase in liver uptake after intracardial injection into rats. The increase in liver uptake was mostly accounted for by an enhanced uptake in the parenchymal cells, while the uptake by the non-parenchymal cells was only slightly increased. The uptake of both the control and the glycolipid-containing vesicles by the non-parenchymal cell fraction could be attributed completely to the Kupffer cells; no radioactivity was found in the endothelial cells. The effect of lactosylceramide on liver uptake and blood disappearance of the liposomes was effectively counteracted by desialylated fetuin, injected shortly before the liposome dose. This observation supports the notion that a galactose-specific receptor is involved in the liver uptake of lactosylceramide liposomes.     

On the uptake and storage of 5-hydroxytryptamine, 5-hydroxytryptophan and catecholamines by adrenal chromaffin cells and nerve endings

Summary Light-microscopic autoradiographs of the adrenal medulla at various intervals after the intravenous injection of [³H] 5-HTP, [³H] 5-HT, [³H] noradrenaline and [³H] adrenaline have been studied. The distribution of silver grains following [³H] 5-HTP uptake was found to be uniform over each of the two main cell populations, adrenaline-storing (A) cells and noradrenaline-storing (NA) cells in the adrenal medulla, but A cells were twice as active as NA cells in incorporating the isotope, a situation very similar to that found after [³H] dopa uptake. 5-HT administration resulted in a pattern resembling the distribution of [³H] noradrenaline uptake, with A cells being 4 or 5 times more active than NA cells and a gradient of activity from the periphery of the medulla inwards. However, the time-course for the loss of radioactivity was not the same for both amines: levels of 5-HT activity were not significantly reduced after one week whereas the degree of [³H] noradrenaline labelling after one week was less than 10% of that at one hour. Thus 5-HT may be bound to sites in the adrenal medulla normally occupied by noradrenaline but it would appear that the release mechanism is different. There was no evidence of 5-HT uptake by adrenal nerve endings.     

Processing of different liposome markers after in vitro uptake of immunoglobulin-coated liposomes by rat liver macrophages

We compared the metabolic fate of [³H]cholesteryl[¹⁴C]oleate, [³H]cholesteryl hexadecylether, ¹²⁵I-labeled bovine serum albumin and [³H]inulin as constituents of large immunoglobulin-coupled unilamellar lipid vesicles following their internalization by rat liver macrophages (Kupffer cells) in monolayer culture. Under serum-free conditions, the cholesteryl oleate that is taken up is hydrolyzed, for the greater part, within 2 h. This occurs in the lysosomal compartment as judged by the inhibitory effect of the lysosomotropic agents monensin and chloroquin. After hydrolysis, the cholesterol moiety is accommodated in the cellular pool of free cholesterol and the oleate is reutilized for the synthesis mainly of phospholipids and, to a lesser extent of triacylglycerols. During incubation in plasma, however, substantial proportions of both the cholesterol and the oleate are shed from the cells, predominantly in the unesterified form. When the liposomes are labeled with the cholesteryl ester analog [³H]cholesteryl hexadecylether only a very small fraction of the label is released from the cells, even in the presence of plasma. Similar to the label remaining associated with the cells, the released label is identified in that case as unchanged cholesteryl ether. The liposomal aqueous phase marker ¹²⁵I-labeled bovine serum albumin is also readily degraded intralysosomally and the radioactive label is rapidly released from the cells in a trichloroacetic acid-soluble form. Also, as much as 20% of the aqueous phase marker [³H]inulin that becomes cell-associated during a 2-h incubation with inulin-containing liposomes, is released from the cells during a subsequent 4-h incubation period in medium or rat plasma. The usefulness of the various liposomal labels as parameters of liposome uptake and intracellular processing is discussed.