Paediatric high-grade osteosarcoma and its prognostic factors: a 10-year retrospective study.

INTRODUCTION: This retrospective study was conducted to identify the characteristics of paediatric high-grade osteosarcoma and define its prognostic factors. METHODS: We identified paediatric patients (aged <19 years at diagnosis) diagnosed with high-grade osteosarcoma from 1 January 2009 to 31 December 2018 in two hospitals in Hong Kong, then retrospectively evaluated their medical records to identify prognostic factors. RESULTS: In total, 52 patients were included in this study (22 girls, 42.3%). Femoral tumour was the most common form of osteosarcoma. Most patients (78.8%) had localised disease at diagnosis. The lung was the most common site of metastasis. Almost half (n=23, 46.9%) of the patients showed a good response to chemotherapy (ie, chemonecrosis >90%). Most patients (n=40, 80%) underwent limb-salvage surgery. The event-free survival and overall survival rates were 55.8% and 71.2%, respectively. Prognostic factors independently associated with poor event-free survival and poor overall survival were the presence of metastasis at diagnosis, poor tumour chemonecrosis, and the need for amputation. CONCLUSION: This multicentre review of paediatric high-grade osteosarcoma showed that the baseline patient demographics, event-free survival, and overall survival in Hong Kong were similar to previous findings in other countries. Patients with metastatic disease at diagnosis and poor chemonecrosis had worse survival outcomes. Molecular analyses of genetic abnormalities may help to identify targeted therapies in future studies.

Characterization of high density lipoprotein binding to human adipocyte plasma membranes.

Freshly isolated human adipocytes showed specific uptake of 125I-labeled human high density lipoprotein (HDL2 and HDL3), a portion of which could be released by subsequent incubation with excess unlabeled ligand. To study the mechanism of HDL binding, sucrose gradient-purified adipocyte plasma membranes were incubated with radioiodinated lipoprotein particles under equilibrium conditions in the absence (total binding) or presence (nonspecific binding) of 100-fold excess unlabeled ligand. Specific binding of HDL2 and HDL3, calculated by subtracting nonspecific from total binding, was Ca++ independent, unaffected by EDTA, and not abolished by pronase treatment of the membranes. Modification of HDL3 by reductive methylation or cyclohexanedione treatment also failed to affect its binding to adipocyte plasma membranes. High salt concentration (200 mM NaCl) inhibited specific binding of HDL2 and HDL3 but had no effect on LDL binding. A significant portion of 125I-HDL2 or 125I-HDL3 binding was consistently inhibited by adding excess unlabeled LDL, but this inhibition was incomplete as compared with a similar molar excess of unlabeled HDL2 or HDL3. The role of apoproteins (apo) in HDL binding to adipocyte membranes was examined by comparing binding of HDL2 and HDL3 isolated from normal, abetalipoproteinemic (abeta) and apo E-deficient (apo E0) plasma. Specific binding was observed with all normal and mutant HDL particles. Furthermore, a significant portion (61-78%) of abeta-HDL2, apo E0-HDL2, and apo E0-HDL3 binding was inhibited by adding 100-fold excess of unlabeled low density lipoproteins (LDL). The cross-competition of LDL and HDL binding was confirmed by the ability of normal, abeta, and apo E0-HDL2 to completely inhibit 125I-LDL binding. These data suggest that HDL binding is independent of apo E and that the responsible apoprotein(s) of HDL complete with LDL-apo B for binding to the same or closely related site in the adipocyte plasma membrane. Normal and apo E0-HDL3 binding was also completely inhibited by normal HDL2, which suggested that HDL2 and HDL3 probably bind to the same site. Scatchard analysis of normal HDL2, normal HDL3, and apo E0-HDL3 binding data best fitted a one-component binding profile with similar equilibrium dissociation constants (40-96 nM). HDL3 binding was found to be effectively inhibited by anti-human apo AI or anti-human apo AII, but not by anti-human apo B antisera. This binding was also unaffected by monoclonal anti-human apo B or E antibodies known to inhibit binding of apo B or apo E containing lipoprotein to the LDL receptor of cultured fibroblasts. These findings, taken together, suggest that human fat cells possess HDL binding sites with apo AI and /or apo AII specificity. The significant but partial inhibition of HDL2 and HDL3 binding by LDL along with the complete inhibition of LDL binding by HDL2 and HDL3 tends to exclude a single binding site that interacts both lipoproteins and favors the interpretation that LDL and HDL particles bind to multiple recognition sites or to different conformation of the same lipoprotein binding domain on the human fat cell.

Transfer of free and esterified cholesterol from low-density lipoproteins and high-density lipoproteins to human adipocytes.

Cholesterol stored in human adipose tissue is derived from circulating lipoproteins. To delineate the cholesterol transport function of LDL and HDL, the movement of radiolabelled esterified cholesterol and free cholesterol from labelled LDL and HDL to human adipocytes was examined in the present study. LDL and HDL were enriched and labelled in esterified cholesterol with [14C]cholesterol by the action of plasma lipid transfer proteins and lecithin-cholesterol acyltransferase. Doubly labelled (3H,14C) LDL and HDL were prepared by exchanging free [3H]cholesterol into the 14C-labelled lipoproteins. 14C-labelled lipoprotein and 3H-labelled lipoprotein were also prepared separately and mixed to yield a mixed doubly labelled lipoprotein. Relative to the total amount added, proportionally more free than esterified cholesterol was transferred to the adipocytes upon incubation with any doubly labelled LDL and HDL. The calculated mass of free and esterified cholesterol transferred, however, varied with different labelled lipoproteins. 3H- and 14C-labelled LDL or HDL transferred 2-3-fold more esterified than free cholesterol while the reverse occurred with the mixed doubly labelled LDL or HDL. Thus, free cholesterol-depleted particles preferentially transferred cholesterol ester to the fat cells. In the presence of the homologous unlabelled native lipoprotein, the transfers of free and esterified cholesterol from labelled LDL or HDL were specifically inhibited. Selective transfer of esterified cholesterol relative to apoprotein was also observed when esterified cholesterol uptake from both LDL and HDL was assayed along with the binding of 125I-labelled lipoprotein. The cellular accumulation of cholesterol ether-labelled HDL (a non-hydrolyzable analogue of cholesterol ester) exceeded that of cholesterol ester consistent with significant hydrolysis of the latter physiological substrate. These results demonstrate preferential transfer of free cholesterol and esterified cholesterol over apoprotein for both LDL and HDL in human adipocytes. Furthermore, the data suggest that the cholesterol ester transport function of LDL and HDL can be enhanced by free cholesterol depletion and cholesterol ester enrichment of the particles, and affirms a role for adipose tissue in the metabolism of lipid-modified lipoproteins.

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus.

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the identity of the fatty acyl chains can uniquely specify or determine which side of the membrane individual phosphatidylethanolamine molecules come to occupy. Although fatty acyl chain asymmetry and phosphatidylethanolamine asymmetry are correlated in VSV, no simple rules can be discerned which uniquely relate the two paramaters.

The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes.

Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL3). This binding was independent of apolipoprotein E as HDL3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL3. To examine the importance of apolipoprotein A-I, the major HDL3 apolipoprotein, in the specific binding of HDL3 to human adipocytes, HDL3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL3, a 92 +/- 3.1% reduction (mean +/- S.E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [3H]cholesteryl ether labeled HDL3, was also significantly reduced (about 35% reduction, P less than 0.005) by substituting apolipoprotein A-II for A-I in HDL3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I.

Hypothyroidism reduces HDL binding to rat liver cells.

The plasma clearance rate of high density lipoprotein is reduced in the hypothyroid rat. Because the liver is an important site of HDL-cholesterol catabolism, the present study was undertaken to investigate whether thyroid hormone deficiency affects binding of HDL to liver cells. Male Wistar rats were made hypothyroid by feeding propylthiouracil (0.1% w/w). Liver cells were isolated by in situ perfusion of the liver with a buffered collagenase solution. 125I-labelled rat HDL binding to isolated liver cells was carried out at low temperature on ice. For both control and hypothyroid rat liver cells, 125I-HDL binding was significantly inhibited by excess unlabelled rat HDL and also by human HDL3 and human LDL but was unaffected by the addition of 10 mM EDTA. From Scatchard analysis of dose-response studies, hypothyroid cells displayed a lower HDL binding capacity (P less than 0.01) and a higher binding affinity (P less than 0.025) compared to control cells. These results suggest that thyroid hormone affects the expression of the HDL binding site in liver cells which may contribute to the reduced HDL clearance in the hypothyroid animal.

Weight loss in massive obesity: reciprocal changes in plasma HDL cholesterol and HDL binding to human adipocyte plasma membranes.

Human obesity is frequently associated with elevated plasma triglyceride and cholesterol concentrations and reduced high density lipoprotein (HDL) cholesterol, abnormalities that commonly revert to normal levels with weight loss. This study was undertaken to examine possible mechanism(s) associated with the changes in plasma HDL cholesterol concentrations in massively obese patients after weight loss. Ten massively obese patients (two men and eight women, age = 37.8 +/- 2.4 years) were studied before, during, and after 1 year of weight loss and weight maintenance following gastric stapling. Total cholesterol and low density lipoprotein cholesterol were within the normal range for sex and age before weight loss and did not change significantly during or after weight reduction. In the females, HDL cholesterol concentrations increased from 0.96 +/- 0.06 mmol/L to 1.23 +/- 0.3 mmol/L (mean +/- SEM, n = 8, P less than .05) with weight reduction. In the two men, plasma HDL cholesterol concentrations were, respectively, 1.22 and 0.65 mmol/L before and 1.23 and 0.98 mmol/L after weight loss. Specific binding of 125I-HDL2 and 125I-HDL3 to purified plasma membranes was determined using abdominal and omental fat depot before and after weight loss in six of the ten obese patients. An average reduction of 30% to 40% in 125I-HDL2 and 125I-HDL3 binding capacity to these membranes occurred after weight loss. Furthermore, a positive correlation (r = .65, n = 10, P less than .05) was observed between plasma HDL cholesterol and triglyceride concentrations before weight loss but not after weight loss (r = .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Use of [125I]deoxycytidine to detect herpes simplex virus-specific thymidine kinase in tissues of latently infected guinea pigs.

The footpad skin and the lumbosacral dorsal root ganglia were removed from inbred guinea pigs at different times after subcutaneous infection with herpes simplex virus type 2 (HSV-2) in both hind footpads. These tissues, shown by our previous study to harbor latent HSV, were dispersed into single cells. The presence of virus-specific thymidine kinase (TK) in these cells was assayed by the uptake and phosphorylation of [125I]deoxycytidine in culture. [125I]deoxycytidine was shown to be a specific substrate for the HSV-coded TK. The method could detect herpes TK activity in a culture of 10(6) cells with less than 0.1% of the cells being virally infected. The enzyme was readily detected in footpad cells of acutely (24 h) but not of latently (14 days to 1 year) infected guinea pigs. No herpes TK was found either in the sensory ganglionic cells of guinea pigs during the early and late phases of latent infection. It is concluded that HSV-2, while residing in the footpads and the lumbosacral ganglia of the guinea pig during latent infection, does not express any viral TK function.

Enhanced binding of phospholipase-A2-modified low density lipoprotein by human adipocytes.

Recognition of low density lipoprotein (LDL) by human adipocytes is not dependent on the classical LDL (apoprotein B-E) receptor. To assess whether LDL phospholipids have a role in adipocyte-LDL interactions, binding studies were carried out with human LDL modified with cobra venom phospholipase A2 (PLA2) and freshly isolated adipocytes and purified adipocyte plasma membranes prepared from surgical biopsies. LDL incubated with PLA2 showed increased monoacylphospholipid content, decreased diacylphospholipid content, and increased anodic migration on agarose gel electrophoresis. LDL cholesterol, triglyceride, and protein content remained unchanged. Typically, modification of 16 and 47% of LDL phospholipids enhanced specific binding of 125I-labelled LDL to plasma membranes progressively from 3.1 micrograms LDL bound/mg membrane protein (control) to 5.8 and 28.2 micrograms LDL bound/mg membrane protein, respectively. Nonspecific binding was not altered significantly. Excess unlabelled native LDL and high density lipoprotein (HDL3) effectively inhibited binding of PLA2-modified LDL. Freshly isolated adipocytes also showed enhanced binding and uptake of PLA2-modified LDL (0.1 vs. 0.9 micrograms LDL/10(6) cells x 2 h), control vs. modified). The results demonstrate that alterations of LDL phospholipids significantly enhance LDL binding and suggest a regulatory role for phospholipids in lipoprotein-cell interaction. Furthermore, the results support the view that human adipose tissue may be involved in the metabolism of modified lipoproteins, in vivo.

Enhanced binding of low and high density lipoproteins to human adipocyte plasma membranes: effects of temperature and proteases.

The specific binding of 125I-labelled low density lipoprotein ([125I]LDL to human adipocyte plasma membranes was higher at 37 than at 0 degree C. Prior treatment of membranes with pronase had no effect on LDL binding measured at 0 degree C but consistently stimulated binding at 37 degrees C. Plasmin was similar to pronase in enhancing LDL-specific binding, but thrombin was not as effective. 125I-labelled high density lipoprotein ([125I]HDL2) specific binding to human adipocyte plasma membranes was similarly sensitive to temperature and pronase treatment. Addition of the protease inhibitor aprotinin in the adipocyte membrane binding assay significantly reduced [125I]LDL binding at 37 degrees C (p less than 0.05), suggesting the involvement of a protease activity intrinsic to the lipoproteins and (or) membranes. These data demonstrate that both LDL and HDL binding in human adipocyte plasma membranes can be "up-regulated" by specific proteolytic perturbations in a temperature-dependent manner.

Asymmetric distribution of phosphatidylethanolamine in the membrane of vesicular stomatitis virus.

The membrane-impermeable reagent trinitrobenzenesulfonate has been shown to react only with the surface components of vesicular stomatitis virus (VSV) membranes. When the amount of phosphatidylethanolamine (PE) available to modification by trinitrobenzenesulfonate in intact virions was determined, it was found that 36% of the total membrane PE was converted to the trinitrophenyl derivative. The same proportion of the total membrane PE was reactive after removal of the surface glycoprotein by trypsin digestion, but disruption of the virus membrane by sonication rendered all of the PE reactive. These results indicate that PE is asymmetrically distributed in the VSV membrane; 36% is present in the outer lipid leaflet, whereas 64% is found on the inner layer.

Characterization of low density lipoprotein binding to human adipocytes and adipocyte membranes.

125I-labeled low density lipoprotein (LDL) binding to purified plasma membranes prepared from freshly isolated human adipocytes was saturable, specific, and displaceable by unlabeled ligand. The maximum specific binding capacity measured at saturating concentrations of 125I-LDL was 1.95 +/- 1.17 micrograms of LDL bound/mg of membrane protein (mean +/- S.D., n = 16). In contrast to cultured fibroblasts, specific binding of LDL to adipocyte membranes was calcium-independent, was not affected by EDTA or NaCl, and was not destroyed by pronase. Plasma membranes purified directly from homogenized adipose tissue also showed calcium-independent LDL specific binding (0.58 +/- 0.33 micrograms of LDL bound/mg of membrane protein, mean +/- S.D. n = 11). Specific binding, internalization, and degradation of 125I-methylated LDL was demonstrated in isolated adipocytes and competition experiments showed that native and methylated LDL interacted with adipocytes through some common recognition mechanism(s). Compared to native LDL, specific binding of methylated LDL to adipocyte membranes was significantly reduced (43%), indicating that interaction of LDL with adipocyte was dependent in part on the lysine residues of apolipoprotein B. LDL binding to adipocyte plasma membranes was also competitively inhibited by human high density lipoprotein subfractions HDL2 and HDL3. Thus, LDL metabolism in mature adipocytes appears to be regulated by mechanisms distinctly different from a variety of cultured mesenchymal cells. In addition, the ability of adipocytes to bind, internalize, and degrade significant amounts of methylated LDL supports the view that adipose tissue is involved in the metabolism of modified lipoproteins in vivo.

Selective uptake of HDL cholesterol ester by human fat cells.

In humans, high-density lipoprotein (HDL)-cholesterol ester turnover exceeds that of HDL apoproteins by severalfold or more, suggesting an independent catabolic fate of these constituents. The present study investigated the cellular uptake and dissociation of HDL labeled in its apoproteins with 125I and in its cholesterol ester with [3H]cholesteryl palmityl ether, a nonhydrolyzable cholesterol ester analogue. Approximately 50% of cell-associated 125I-HDL2 and 125I-HDL3 was released from prelabeled adipose cells by incubating the latter in the presence or absence of unlabeled lipoproteins for 2 h. The uptake of HDL-cholesterol ester by human fat cells as reflected by [3H]cholesteryl palmityl ether was 5-18 times greater than that predicted from the uptake of 125I-HDL2 and 125I-HDL3 and was irreversible. Analysis of dissociated 125I-HDL3 demonstrated changes to both higher and lower density fractions compared with the starting material. There was a high correlation between the cellular uptake of HDL3-cholesterol ester and HDL3-apoprotein uptakes (r = 0.90, P less than 0.01), suggesting that HDL-cholesterol ester uptake requires a specific apoprotein interaction or binding step. The selective uptake and retention of HDL-cholesterol ester by isolated adipocytes implies that human fat tissue may play a role in regulating the lipid composition of plasma HDL.

Interactions of high density lipoprotein subclasses (HDL2 and HDLc) with dog adipocytes: selective effects of cholesterol and saturated fat feeding.

Adipose tissue is a cholesterol storage organ and derives its cholesterol primarily from circulating lipoproteins. The present study shows that adipocytes isolated from canine omental fat tissue interact specifically with high density lipoprotein subfractions lacking or enriched in apolipoprotein E, namely canine high density lipoprotein-2 (HDL2) and HDLc, respectively. While 125I-labeled HDL2 binding was inhibited similarly by both excess unlabeled HDLc and HDL2, 125I-labeled HDLc interaction was inhibited by its homologous ligand only. Paired studies showed that the amount of HDLc associated with adipocytes was significantly higher compared to HDL2. The effect of a short-term cholesterol and saturated fat feeding on adipocyte-HDL interaction was examined using fat cells obtained from dogs before and again 3 weeks after a diet supplemented with cholesterol (1% w/w) and saturated fat (30% lard, w/w). Significant increases in body weight and omental fat cell weight occurred after fat feeding. The amount of 125I-labeled HDL2 that could be bound to adipocytes increased after the diet, whether expressed on a per cell basis (P less than 0.005) or per unit cell surface (P less than 0.025). The amount of cell-associated 125I-labeled HDLc, however, was not significantly affected by the cholesterol-rich diet. The characteristics of HDLc and HDL2 dissociation were assessed by examining the release of labeled lipoproteins from adipocytes preincubated with 125I-labeled HDLc and 125I-labeled HDL2. HDL2 dissociation from adipocytes was significantly decreased (P less than 0.05) following the diet and may explain in part the apparent increase in cell-associated 125I-labeled HDL2.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional variation in high-density lipoprotein binding to human adipocyte plasma membranes of massively obese subjects.

Obesity is associated with significant changes in cholesterol and lipoprotein metabolism. High density lipoprotein (HDL) cholesterol is often reduced and adipose tissue cholesterol stores are increased in obese individuals. This prompted a study on the binding of HLD fractions (HDL2 and HDL3) to adipocyte plasma membranes obtained from massively obese subjects (BMI greater than 37 kg m-2) undergoing gastroplasty. Regional variation in HDL binding to these adipocyte plasma membranes was demonstrated. Membranes derived from the abdominal subcutaneous depot exhibited similar binding affinity (Kd) but higher binding capacity (Bmax) for HDL2 and HDL3 than that from the omental depot. There was significant inter-individual variation in Bmax but the amount of HDL2 or HDL3 bound to the two depots of the same individual was positively correlated (HDL2, r = 0.66, P less than 0.05; HDL3, r = 0.88, P less than 0.01). While HDL2 binding showed a higher affinity (lower Kd) than HDL3, a significant positive correlation existed between HDL2 and HDL3 binding to the same adipocyte membranes (r = 0.89, P less than 0.01). A significant inverse correlation (P less than 0.05) was also observed between HDL2 and HDL3 binding to adipocyte membranes and plasma HDL-cholesterol concentration. These results suggest that adipose tissue is an important site of HDL metabolism and the subcutaneous fat depot may play a proportionally more significant role due to its higher HDL binding capacity. It is further suggested that increased HDL binding and metabolism by the expanded adipose tissue mass may contribute to reduced plasma HDL-cholesterol levels frequently associated with obesity.

Regional variation in HDL metabolism in human fat cells: effect of cell size.

Abdominal obesity is related to reduced plasma high-density lipoprotein (HDL) cholesterol, and both are associated with cardiovascular disease risk. We have observed that plasma membranes from abdominal subcutaneous adipocytes have a greater HDL binding capacity than omental fat cell plasma membranes. The present study examined whether these binding characteristics could be due to differences in fat cell size or cholesterol concentration between the two adipose depots. Abdominal subcutaneous and deep omental fat were obtained from massively obese patients at surgery. Subcutaneous abdominal fat cells were significantly larger and their cellular cholesterol content greater than omental adipocytes. The uptake of HDL by collagenase-isolated fat cells was studied by incubating the cells for 2 h at 37 degrees C with 10 micrograms/ml 125I-HDL2 or 125I-HDL3. In both depots, the cellular uptake of 125I-HDL2 and 125I-HDL3 was specifically inhibited by addition of 25-fold excess unlabeled HDL and a close correlation was observed between the cellular uptake of 125I-HDL2 and 125I-HDL3. In obese patients, the uptake of 125I-HDL was higher in subcutaneous cells than in omental cells [5.85 +/- 0.53 vs. 2.74 +/- 0.30 pmol X 2 h-1. (10(6) cells)-1]. The cellular 125I-HDL uptake was significantly correlated with adipocyte size and fat cell cholesterol content but not with adipocyte cholesterol concentration. These results suggest that the higher HDL uptake observed in subcutaneous cells compared with omental cells in obesity is the result of differences in adipocyte size rather than differences in the cholesterol concentration (cholesterol-to-triglyceride ratio).(ABSTRACT TRUNCATED AT 250 WORDS)