Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles.

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.

Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture.

We investigated the effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of large unilamellar liposomes by rat Kupffer cells in maintenance culture. The phospholipid vesicles were labeled in the lipid moiety with phosphatidyl[14C]choline and contained [3H]inulin or [125I]iodoalbumin as nondegradable and degradable markers of the aqueous vesicle content, respectively. Cytochalasin B and dihydrocytochalasin B, inhibitors of microfilament function, reduced inert inulin label uptake by 75% maximally, but residual uptake was not followed by release of lipid degradation products from the cells. By contrast, colchicine, an inhibitor of microtubule assembly, reduced uptake of liposomal inulin by maximally 55% but could not inhibit release of lipid degradation products from the cells. It is concluded that the cytochalasins partly inhibit uptake but fully prevent the arrival of internalized liposomes in the lysosomal compartment, while the action of colchicine is to slow down the overall process of uptake and subsequent transportation to the lysosomes. Monensin reduced inulin uptake to an extent similar to that found with colchicine, but reversibly blocked degradation of liposomal lipid and encapsulated protein. The kinetics of degradation of liposomal constituents suggests that residual uptake in the presence of monensin represents accumulation in an intracellular compartment. Trifluoperazine did not affect binding, internalization or degradation of encapsulated protein at low concentration (6 microM), but completely inhibited release of liposomal lipid degradation products under these conditions. At intermediate concentration (14 microM), the drug also reduced the internalization, while a high concentration (22 microM) was required to inhibit protein degradation as well. We conclude that trifluoperazine has multiple sites of action in the uptake and processing of liposomal constituents by Kupffer cells.

Effects of ammonium chloride and chloroquine on endocytic uptake of liposomes by Kupffer cells in vitro.

In this study we investigated the interaction of liposomes with rat Kupffer cells in maintenance culture by using the lysosomotropic amines ammonium chloride and chloroquine as inhibitors of intralysosomal degradation. The liposomes (large unilamellar vesicles) contained either the metabolically inert 3H-labeled inulin or the degradable 125I-labeled bovine serum albumin. In control incubations, the cells released nearly all accumulated protein label and about 30% of the lipid label when they were incubated in the absence of liposomes, after an initial uptake period of 1 h in the presence of liposomes. This release of label was, for the greater part, suppressed in the presence of ammonia or chloroquine. When the inhibitors were present during the initial uptake period, a several-fold increase in the amount of protein label accumulating in the cells and a smaller, but still marked, increase in lipid label accumulation were observed. The effect of ammonia when present during uptake was readily reversible in contrast to that of chloroquine. Experiments with encapsulated inulin revealed that both lysosomotropic agents also affected the uptake process per se to some extent, probably as a result of impaired membrane/receptor recycling. Labeled liposomes adsorbed to the cells at 4 degrees C were effectively internalized and processed intracellulary after shifting the temperature to 37 degrees C, even when a 500-fold excess of unlabeled liposomes was present in the medium during the 37 degrees C incubation. The observed effects of ammonia and chloroquine indicate that, after uptake, the liposomes are degraded within lysosomes, thus confirming our previous conclusion that endocytosis is the major uptake mechanism at 37 degrees C. From the temperature-change experiments we conclude that, at 4 degrees C, the liposomes are bound with high affinity to the cells, remaining firmly attached to the cell-surface structures which initiate their internalization when the temperature is raised to 37 degrees C.

Gadolinium chloride-induced shifts in intrahepatic distributions of liposomes.

Intravenously administered gadolinium chloride caused only a slight decrease in the rate of elimination of small unilamellar liposomes from the blood and had no influence on the total hepatic uptake of these vesicles, but did alter their intrahepatic distribution substantially. Uptake by the non-parenchymal cells was substantially decreased, whereas uptake by the parenchymal cells showed a concomitant increase. Our earlier observations (Roerdink et al. (1981) Biochim. Biophys. Acta 677, 79-89) on the effect of lanthanides on the in vivo distribution of multilamellar liposomes have been extended, in that we demonstrate, in addition to the drop in elimination rate from the blood and in the over-all hepatic uptake, a shift of liposome distribution within the Kupffer cell population. While the larger Kupffer cells, which normally take up a major fraction of an injected liposome dose, were strongly inhibited in liposome uptake, the more numerous small macrophages showed a 3-4-fold increase in uptake.

In-vitro stability and cytostatic activity of liposomal formulations of 5-fluoro-2'-deoxyuridine and its diacylated derivatives.

The water-soluble antineoplastic agent 5-fluoro-2'-deoxyuridine (FUdR) was encapsulated in the water phase of liposomes of different lipid compositions. The retention of this drug upon storage and during contact with plasma was assessed. It was found that, upon refrigeration, diffusion of FUdR across the liposome bilayer was considerably faster when the drug was encapsulated in fluid-type liposomes (egg PC/PS/CHOL) than in solid-type liposomes (DSPC/DPPG/CHOL). With either composition, leakage of the drug from the liposomes was accelerated upon contact with plasma. To achieve improved liposomal retention of the drug, FUdR was converted to a lipophilic prodrug by esterifying the free hydroxyl groups in the deoxyribose moiety with fatty acids of different chain lengths. Thus FUdR-dipalmitate (C-16) and FUdR-dioctanoate (C-8) were synthesized and incorporated in liposomes. The dipalmitoyl derivative could be incorporated upto 13 mol% in solid-type liposomes but to only 2 mol% in fluid-type liposomes. Freeze-fracture electron microscopy revealed no major differences between control liposomes and those containing the prodrug. FUdR-dipalmitate was found to be firmly associated with the liposomal bilayer in both liposome-types: no exchange of the pro-drug with blood constituents or hydrolysis by serum esterases could be registered when the liposomes were incubated with serum. On the other hand, liposome-incorporated FUdR-dioctanoate was found to be readily extracted from the liposomes by serum components (predominantly albumin) and was found to be degraded rapidly by serum esterase activity. The antitumor activity of FUdR-prodrugs was determined using C26 colon adenocarcinoma cells. This cell line was found to be highly sensitive to FUdR. Liposomal FUdR-dioctanoate inhibited cell growth in the same concentration range as unesterified FUdR. FUdR-dipalmitate, however, was more than two orders of magnitude less potent in inhibiting cell proliferation. Its antiproliferative activity was dependent on the liposome-type used: when incorporated in fluid-type liposomes, antiproliferative activity of FUdR-dipalmitate was several-fold higher than in solid-type liposomes. The difference in antitumor activity between FUdR-dipalmitate and FUdR-dioctanoate and between FUdR-dipalmitate in the fluid- and solid-type liposomes could be explained by differences in the rate of hydrolysis of the prodrugs to FUdR by esterase activity in the tumor cells or in the growth medium.

Intrahepatic distribution of small unilamellar liposomes as a function of liposomal lipid composition.

We investigated the intrahepatic distribution of small unilamellar liposomes injected intravenously into rats at a dose of 0.10 mmol of lipid per kg body weight. Sonicated liposomes consisting of cholesterol/sphingomyelin (1:1), (A); cholesterol/egg phosphatidylcholine (1:1), (B); cholesterol/sphingomyelin/phosphatidylserine (5:4:1), (C) or cholesterol/egg-phosphatidylcholine/phosphatidylserine (5:4:1), (D) were labeled by encapsulation of [3H]inulin. The observed differences in rate of blood elimination and hepatic accumulation (A much less than B approximately equal to C less than D) confirmed earlier observations and reflected the rates of uptake of the four liposome formulations by isolated liver macrophages in monolayer culture. Fractionation of the liver into a parenchymal and a non-parenchymal cell fraction revealed that 80-90% of the slowly clearing type-A liposomes were taken up by the parenchymal cells while of the more rapidly eliminated type-B liposomes even more than 95% was associated with the parenchymal cells. Incorporation of phosphatidylserine into the sphingomyelin-based liposomes caused a significant increase in hepatocyte uptake but a much more substantial increase in non-parenchymal cell uptake, resulting in a major shift of the intrahepatic distribution towards the non-parenchymal cell fraction. For the phosphatidylcholine-based liposomes incorporation of phosphatidylserine did not increase the already high uptake by the parenchymal cells while uptake by the non-parenchymal cells was only moderately elevated; this resulted in only a small shift in distribution towards the non-parenchymal cells. The phosphatidylserine-induced increase in liposome uptake by non-parenchymal liver cells was paralleled by an increase in uptake by the spleen. Fractionation of the non-parenchymal liver cells in a Kupffer cell fraction and an endothelial cell fraction showed that even for the slowly eliminated liposomes of type A endothelial cells do not participate to a measurable extent in the elimination process, thus excluding involvement of fluid-phase pinocytosis in the uptake process.

Expression and fate of the nuclearly encoded subunits of cytochrome-c oxidase in cultured human cells depleted of mitochondrial gene products.

Synthesis, import, assembly and turnover of the nuclearly encoded subunits of cytochrome-c oxidase were investigated in cultured human cells depleted of mitochondrial gene products by continuous inhibition of mitochondrial protein synthesis (OP- cells). Immunoprecipitation after pulse labeling demonstrated that the synthesis of the nuclear subunits was not preferentially inhibited, implying that there is no tight regulation in the synthesis of mitochondrial and nuclear subunits of mitochondrial enzyme complexes. Quantitative analysis of the mitochondrial membrane potential in OP- cells indicated that its magnitude was about 30% of that in control cells. This explains the normal import of the nuclearly encoded subunits of cytochrome-c oxidase and other nuclearly encoded mitochondrial proteins into the mitochondria that was found in OP- cells. The turnover rate of nuclear subunits of cytochrome-c oxidase, determined in pulse-chase experiments, showed a specific increase in OP- cells. Moreover, immunoblotting demonstrated that the steady-state levels of nuclear subunits of cytochrome-c oxidase were severely reduced in these cells, in contrast to those of the F1 part of complex V. Native electrophoresis of mitochondrial enzyme complexes showed that assembly of the nuclear subunits of cytochrome-c oxidase did not occur in OP- cells, whereas the (nuclear) subunits of F1 were assembled. The increased turnover of the nuclear subunits of cytochrome-c oxidase in OP- cells is, therefore, most likely due to an increased susceptibility of unassembled subunits to intra-mitochondrial degradation.

Regulation of the expression of mitochondrial proteins: relationship between mtDNA copy number and cytochrome-c oxidase activity in human cells and tissues.

The relationship between the relative amounts of nuclear and mitochondrial genes for cytochrome-c oxidase subunits and their transcripts and cytochrome-c oxidase activity was investigated in several human tissues and cell lines to get more insight into the regulation of the expression of this mitochondrial enzyme complex. The results show: (1) a wide range of mtDNA copy numbers; (2) constant ratios between the steady-state levels of the transcripts for the various cytochrome-c oxidase subunits, and (3) large variations in cytochrome-c oxidase activity in different tissues and cell lines that could not be related to the differences in mtDNA copy number. We conclude that the transcription of genes for both mitochondrial and nuclear cytochrome-c oxidase subunits is regulated coordinatedly, but also that the mtDNA copy number plays a minor role in determining differences in cytochrome-c oxidase activity between different cell and tissue types.

Altered kinetics of cytochrome c oxidase in a patient with severe mitochondrial encephalomyopathy.

Deficiency of cytochrome c oxidase activity was established in a girl born to consanguineous parents. She showed symptoms of dysmaturity, generalized hypotonia, myoclonic seizures and progressive respiratory failure, leading to death on the seventh day of life. Structural abnormalities of the central nervous system consisted of severe cerebellar hypoplasia and optic nerve atrophy. Biochemical analysis of a muscle biopsy specimen demonstrated deficiency of cytochrome c oxidase activity. Cultured fibroblasts from this patient also showed a selective decrease in the activity of cytochrome c oxidase, excluding a muscle-specific type of deficiency. Further investigations in cultured fibroblasts revealed that synthesis, assembly and stability of both the mitochondrial and the nuclear subunits of the enzyme were entirely normal. The steady-state concentration of cytochrome c oxidase in the fibroblasts of the patient was also normal, suggesting that the kinetic properties of the enzyme were altered. Analysis of the kinetic parameters of cytochrome c oxidase demonstrated an aberrant interaction between cytochrome c oxidase and its substrate, cytochrome c, most likely because of a mutation in one of the nuclear subunits of the enzyme.

Mitochondria in cultured human muscle cells depleted of mitochondrial DNA.

Cultured human muscle cells were depleted of mitochondrial DNA (mtDNA) by prolonged treatment with ethidium bromide (EB). In these respiration-deficient muscle cells neither cytochrome c oxidase activity nor mtDNA were detectable. However, mitochondrial matrix enzymes remained present and were localized in mitochondria-like organelles, as shown by subcellular fractionation. Metabolic labeling showed synthesis of cytochrome c oxidase subunits coded by nuclear DNA (nDNA). These results indicate that depletion of mtDNA in cultured human myoblasts does not inhibit expression of nDNA-coded mitochondrial proteins. The characteristic thread-like pattern of mitochondria was lost in mtDNA-depleted myoblasts, as shown by immunofluorescence with antibodies against cytochrome c oxidase and the F1 part of the mitochondrial ATP synthase (F1-ATPase) and by fluorescence of the carbocyanine dye, 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)). The organelles visualized by these methods were round and swollen and had a localization different from lysosomes as shown by double-labeling with mitochondrial and lysosomal antibodies. These results indicate that not only synthesis, but also import of mitochondrial proteins into mitochondria-like organelles remains possible in respiration-deficient cells.

The suitability of cells from different tissues for use in tissue-engineered skin substitutes.

Tissue-engineered skin substitutes may be a future remedy for burn wounds and chronic wounds, as wound contraction and scar formation cannot be prevented with the current standard treatment. The aim of this study therefore was to identify readily available sources of fibroblasts suitable for dermal substitution. Three different tissues were studied: dermal tissue from split-skin graft, subcutaneous fat tissue and eschar tissue obtained through debridement of burn wounds. We determined the cellular profile and the cell numbers immediately after isolation and after 2 and 14 days of fibroblast culture using flow cytometry and cell counting with a cytometer. In addition, parts of the isolated cell suspensions were seeded directly into a porous collagen dermal substitute to investigate contraction over time. Various cell types were isolated from the three different tissues, but after 14 days of culturing predominantly fibroblasts (>90%) were detected. Keratinocytes, granulocytes and macrophages, if present, disappeared within 14 days. In the cell populations derived from dermal tissue, the percentage of myofibroblasts had decreased significantly by day 14 (from 8% to 3%, P=0.028). In contrast, this percentage had increased in the cell populations derived from fat and eschar (from 23% to 40% and from 20% to 38%, respectively). The fibroblast yield from dermal tissue after 2 weeks of culturing (50 x 10(6) cells/g of tissue) was significantly higher than the yield from fat and eschar tissue (2 x 10(6) cells/g of each tissue, P=0.029). Immunohistochemistry of collagen matrices seeded and cultured with fat- and eschar-derived cells revealed a high prevalence of myofibroblasts, whereas hardly any myofibroblasts were detected in the matrices seeded with dermal cells. The contraction of the eschar matrices was highest (74+/-6% remaining area), whereas dermal matrices contracted significantly less (92+/-7% remaining area, P=0.029) with intermediate contraction for fat matrices. We conclude that fibroblast cultures can be established from dermal tissue, fat tissue and eschar tissue. Dermis is the best fibroblast source for use in skin substitutes as it yields the highest numbers of fibroblasts with minimal numbers of myofibroblasts.

Quality of secondary cardiovascular prevention in specialised care in the Netherlands: the SOLID study.

OBJECTIVE: To determine the extent to which Dutch patients with a history of cardiovascular disease and high cholesterol levels, treated in specialised care, are achieving low-cholesterol targets as defined by national guidelines. DESIGN: Hospital-based cohort study. SETTING: Practices of 41 hospital-based cardiologists and internists in the Netherlands. SUBJECTS: 7377 patients. RESULTS: Forty-one percent of the patients with an indication for secondary cardiovascular prevention by lipid-lowering drug treatment were receiving medication and were achieving cholesterol targets, 42% were receiving lipid-lowering medication but had cholesterol levels above target, 11% were not receiving treatment, and 5% had no recent lipid measurements. CONCLUSION: Compared with previous studies, the SOLID study shows that a relatively large percentage of the Dutch patients under specialised care with a history of cardiovascular disease and an indication for cholesterol-reducing therapy are currently being treated. A considerable proportion of the patients, however, are still not receiving optimal treatment and more than 10% are not being treated at all.

Uptake and processing of liposomal phospholipids by Kupffer cells in vitro.

We investigated the intracellular metabolic fate of [Me-14C]choline-labeled phosphatidylcholines and sphingomyelin taken up by rat Kupffer cells in maintenance culture during interaction with large unilamellar liposomes composed of cholesterol, labeled choline-phospholipid and phosphatidylserine (molar ration 5:4:1). With both labeled compounds only small proportions of water-soluble radioactivity were found to accumulate in the cells and in the culture medium, suggesting limited phospholipid degradation. However, after a lag period of 30 min progressively increasing proportions of cell-associated liposomal phospholipid were found to be converted to cellular phospholipid, nearly all of which was phosphatidylcholine. This conversion as well as the limited release of water-soluble label from the cells was inhibited by the lysosomotropic agents ammonium chloride and chloroquine. With [Me-14C]choline-labeled lysophosphatidylcholine, label was found to become cell-associated far in excess of an encapsulated liposomal label, [3H]inulin. Without a lag period virtually all of this was rapidly converted to phosphatidylcholine, a process which was not inhibited by the lysosomotropic agents. It is concluded that Kupffer cells, after endocytosis of liposomes, degrade the liposomal phospholipids effectively but reutilize the choline moiety for de novo synthesis of cellular phosphatidylcholine.

Preferential amplification and phenotypic selection in a population of deleted and wild-type mitochondrial DNA in cultured cells.

In order to study the still poorly understood dynamics of mitochondrial gene segregation, we attempted to alter the percentage of deleted mtDNA (del-mtDNA) over wild-type mtDNA in cell-culture by manipulating respiratory chain capacity. For this purpose, we used a cell-line harbouring a 6-kb mtDNA-deletion which normally was present in 70% of the molecules. The results show that in the presence of low concentrations of doxycycline (DC), an inhibitor of mitochondrial protein synthesis, the average percentage of del-mtDNA in culture steadily declined. After short-term DC treatment most cells still contained del-mtDNA and removal of DC led to a rapid increase in the proportion of del-mtDNA. In contrast, long-term DC treatment rendered del-mtDNA undetectable by Southern analysis, reflecting the complete absence of del-mtDNA in most cells. In this case, del-mtDNA in culture remained at a constant low level after removal of the drug. The results clearly show the importance of phenotypic selection in the segregation of a deleterious mtDNA mutation.

Spontaneous release of Fc receptor-like material from human lymphoblastoid cell lines.

In the culture medium of some human lymphoblastoid cell lines material is released with the following properties: (a) hemagglutination reaction of IgG-sensitized erythrocytes; (b) enhancement of precipitation of DNA-anti-DNA complexes; (c) inhibition of binding of C1q to immune complexes; (d) inhibition of immune complex binding to lymphocytes; (e) inhibition of antibody-dependent lymphocytotoxicity. The material is not identical with C1q or rheumatoid factor, it is heat resistant (30 min at 56 degrees C); the molecular weight is about 100 000 daltons and it is capable of inhibiting antibody production in vitro. It is suggested that this material consists of Fc receptors spontaneously shed from lymphocyte membranes.

The mitochondrial DNA mutation ND6*14,484C associated with leber hereditary optic neuropathy, leads to deficiency of complex I of the respiratory chain.

The electron transfer activity of Complex I of the respiratory chain and Complex I-linked ATP synthesis were investigated in leukocytes of four males affected by Leber hereditary optic neuropathy and a mutation in the ND6 gene at nucleotide position 14,484 of mtDNA. The electron transfer activity in leukocytes of the patients was about 35% of that in control leukocytes, whereas the Complex I-linked ATP synthesis showed a decrease of only about 20%. This demonstrates that all three mtDNA mutations that are clearly associated with Leber hereditary optic neuropathy result in deficiency of Complex I. However, the relationship between these mtDNA mutations, the function of Complex I and the phenotypic profile remains elusive.

Sensory and hedonic judgments of common foods by lean consumers and consumers with obesity.

OBJECTIVE: To establish differences between lean subjects and subjects with obesity in subjective reports of predominant taste and texture attributes of common foods, and the relationships of these qualities and foods with overall liking. RESEARCH METHODS AND PROCEDURES: Twenty lean (body mass index: 20 to 25 kg/m2) nondieting healthy adults and 23 (body mass index: > or = 30 kg/m2) nondieting healthy adults with obesity assigned ratings for pleasantness and predominant sensory attributes to 50 common foods in 14 food groups using a questionnaire. They completed the Dutch Eating Behavior Questionnaire. Body composition was assessed by bioelectrical impedance. RESULTS: There were no significant group differences between pleasantness scores overall; however, lean subjects assigned higher scores to fruits and vegetables, to foods self-classified as sweet and as salty/savory, and to foods grouped in the highest and lowest quintiles of percentage food energy from fat. There were no group differences in free-choice texture descriptors applied to foods, but correspondence analysis revealed that subjects with obesity associated coarse, slippery, tough, and fatty textures with "dislike extremely," whereas lean subjects associated grainy, moist, doughy, and fibrous texture with "like extremely." Analyses reduced texture terms to 39 key descriptors for use in subsequent work. Groups did not significantly differ in reported external or restrained eating behaviors, but subjects with obesity scored significantly higher for reported emotional eating. DISCUSSION: Differences in preferences for listed common foods exist between weight status groups according to taste, food group, and texture. Consumers can make textural descriptions of foods that could be used to assess sensory and hedonic qualities of actual food choices.

Familial mitochondrial DNA depletion in liver: haplotype analysis of candidate genes.

Two sons and one daughter of healthy consanguineous parents presented with fatal hepatic failure in association with severe depletion of mitochondrial (mt)DNA in liver; a third son is healthy. Other published cases of mtDNA depletion concern single members of a family, which excludes the use of haplotype analysis. In the family presented here, the inheritance of the genes for mitochondrial transcription factor A (mtTFA), nuclear respiratory factor 1 (NRF-1), mitochondrial single-stranded DNA-binding protein (mtSSBP), and endonuclease G (EndoG) was studied using microsatellite markers linked to these genes. The inheritance of the gene for mtDNA polymerase (pol gamma) was studied using a polymorphic CAG repeat present within the coding region of the gene. EndoG and mtSSBP were excluded, but mtTFA remains a candidate. Pol gamma or NRF-1 involvement would be compatible only with autosomal dominant inheritance. Coding sequence analysis of NRF-1 and mtTFA revealed no novel mutations in affected individuals.