Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses.

REASONS FOR PERFORMING STUDY: Endotoxaemia currently is associated with a poor prognosis in horses. The results of recent trials in other species indicate that phospholipid emulsions reduce the deleterious effects of endotoxin (LPS). However, in a previous study in horses, a 2 h infusion of emulsion caused an unacceptable degree of haemolysis. HYPOTHESIS: Rapid administration of a lower total dose of emulsion would reduce the effects of LPS and induce less haemolysis; the emulsion would reduce inflammatory effects of LPS in vitro. METHODS: Twelve healthy horses received an i.v. infusion either of saline or a phospholipid emulsion (100 mg/kg), followed immediately by E. coli 055:B5 LPS (30 ng/kg). Clinical parameters, haematological profiles, serum tumour necrosis factor (TNF) activity, serum lipid profiles, urine analyses and severity of haemolysis were monitored before and at selected times after LPS. Monocytes were also incubated in vitro with LPS in the presence or absence of emulsion, after which TNF and tissue factor activities were determined. RESULTS: Clinical signs of endotoxaemia were reduced in horses receiving the emulsion, including clinical score, heart rate, rectal temperature, serum TNF activity, and the characteristic leucopenic response to LPS, when compared to horses not receiving the emulsion. Three horses receiving the emulsion had none, 2 had mild and one had moderate haemolysis. There were no differences in urinalysis results and creatinine concentrations, either within the groups over time or between the groups. Serum concentrations of phosphatidylcholine, bile acids and triglycerides peaked immediately after the infusion; there were no significant changes in concentrations of nonesterified fatty acids or cholesterol. Incubation of equine monocytes with emulsion prevented LPS-induced TNF and tissue factor activities. CONCLUSIONS: Rapid administration of emulsion significantly reduced inflammatory effects of LPS in vivo and caused a clinically insignificant degree of haemolysis. The results of the in vitro studies indicate that emulsion prevents not only LPS-induced synthesis of cytokines, but also expression of membrane-associated mediators (i.e. tissue factor). POTENTIAL RELEVANCE: Rapid i.v. administration of emulsions containing phospholipids that bind endotoxin may provide a clinically useful method of treating endotoxaemia in horses.

Protein-losing enteropathy in systemic lupus erythematosus: report of a severe, persistent case and review of pathophysiology.

Protein-losing enteropathy (PLE) is a rare manifestation of systemic lupus erythematosus (SLE). We report a severe and resistant case of PLE, discuss pathophysiology and possible role of cytokines in the disease process. We also present a review of the current literature.

Preoperative and intraoperative predictors of postoperative morbidity, poor graft function, and early rejection in 190 patients undergoing liver transplantation.

HYPOTHESIS: Preoperative and intraoperative variables predict in part adverse outcome after liver transplantation. DESIGN: Prospective, blinded, cohort study. SETTING: Tertiary care hospital. SUBJECTS: A total of 190 adult patients undergoing primary liver transplantation. MAIN OUTCOME MEASURE: Adverse outcome was prospectively defined as either in-hospital death or prolonged postoperative hospitalization (>14 days) associated with morbidity. Potential preoperative and intraoperative risk factors were collected. Associations were tested by univariate analysis followed by multivariate analysis in which preoperative factors were entered before intraoperative factors. RESULTS: Adverse outcome occurred in 44.7% of patients. Incidences of other complications were as follows: in-hospital mortality (8.4%), primary graft nonfunction (4.2%), poor early graft function (1.1%), and early rejection (31.2%). Univariate predictors of adverse outcome were United Network for Organ Sharing status (P =.003), Child-Turcotte-Pugh score (P =.02), POSSUM physiological score (P =.002), recipient age (P =.01), preoperative serum high-density lipoprotein cholesterol level (P =.03), preoperative serum creatinine level (P =.002), preoperative serum total IgG level (P =.004), duration in hospital preoperatively (P =.03), operative duration (P<.001), allogeneic erythrocyte transfusions (P<.001), total intraoperative fluids (P =.002), and use of inotropic agents (P =.01). In the final multivariate model, predictors of adverse outcome were United Network for Organ Sharing status (P =.03), recipient age (P =.002), and total intraoperative fluids (P =.04). Most patients who died or had a prolonged hospitalization exhibited dysfunction of more than 1 organ system, including pulmonary, renal, and infectious complications. CONCLUSIONS: Adverse outcome occurs frequently after liver transplantation, usually involves multiple organ systems, and is predicted in part by several preoperative and intraoperative factors.

Relationship of hypolipidemia to cytokine concentrations and outcomes in critically ill surgical patients.

OBJECTIVE: To determine the relationship of hypolipidemia to cytokine concentrations and clinical outcomes in critically ill surgical patients. DESIGN: Consecutive, prospective case series. SETTING: Surgical intensive care unit of an urban university hospital. PATIENTS: Subjects were 111 patients with a variety of critical illnesses, for whom serum lipid, lipoprotein, and cytokine concentrations were determined within 24 hrs of admission to a surgical intensive care unit. Controls were 32 healthy men and women for whom serum lipid, lipoprotein, and cytokine concentrations were determined. INTERVENTIONS: Blood samples were drawn on admission to the intensive care unit. Predetermined clinical outcomes including death, infection subsequent to intensive care unit admission, length of intensive care unit stay, and magnitude of organ dysfunction were monitored prospectively. MEASUREMENTS AND MAIN RESULTS: Measurements included total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, apolipoproteins A-I and B, phospholipid, triglyceride, interleukin-6, interleukin-10, soluble interleukin-2 receptor, tumor necrosis factor-alpha, and soluble tumor necrosis factor receptors p55 and p75. Mean serum lipid concentrations were extremely low: total cholesterol, 127 +/- 52 mg/dL; low-density lipoprotein cholesterol, 75 +/- 41 mg/dL; high-density lipoprotein cholesterol, 29 +/- 15 mg/dL. Total, low-density lipoprotein, and high-density lipoprotein cholesterol concentrations and apolipoprotein concentrations inversely correlated with interleukin-6, soluble interleukin-2 receptor, and interleukin-10 concentrations, whereas the triglyceride concentration correlated positively with tumor necrosis factor soluble receptors p55 and p75. Clinical outcomes were related to whether the admission cholesterol concentration was above (n = 56) or below (n = 55) the median concentration of 120 mg/dL. Each of the clinical end points occurred between 1.9- and 3.5-fold more frequently in the very low cholesterol (<120 mg/dL) group. Nine patients (8%) died during the hospitalization. Seven of the nine patients who died had total cholesterol concentrations below the median concentration of 120 mg/dL. CONCLUSIONS: Low cholesterol and lipoprotein concentrations found in critically ill surgical patients correlate with interleukin-6, soluble interleukin-2 receptor, and interleukin-10 concentrations and predict clinical outcomes.

The correlation of cytokine levels with body weight after megestrol acetate treatment in geriatric patients.

BACKGROUND: Cachexia is associated with elevated levels of cytokines in cancer and human immunodeficiency virus patients. Studies in cancer and acquired immunodeficiency syndrome patients showed that treatment with megestrol acetate (MA) is associated with improvement in appetite and weight gain. Reduction in the levels of cytokines is associated with weight gain in laboratory animals with cancer. This study evaluates the correlation between changes in cytokine (or their receptor) levels and weight following MA treatment in geriatric weight-loss patients. METHODS: Veterans Administration Medical Center nursing home patients (N = 69) with a weight loss of > or =5% of usual body weight over the past 3 months or body weight 20% below their ideal body weight participated in a 12-week, randomized, double-blind, placebo-controlled trial, with an additional 13-week follow-up period. Patients were randomly assigned to receive a placebo or MA oral suspension of 800 mg/d for 12 weeks. Levels of the following cytokines (or their receptors) were measured at baseline and after 12 weeks of treatment: tumor necrosis factor soluble receptor (TNFR) subunits. TNFR-p55 and TNFR-p75: interleukin 6 (IL-6); and the soluble interleukin-2 receptor (sIL-2R). The subjects' weight and body composition were measured at the start of the study. Weight and mortality were followed up for another 13 weeks after discontinuing the MA study drug. RESULTS: Elevated levels of IL-6 in almost all geriatric cachexic patients, compared with normal volunteers (mean, <4.6 pg/ml). were noted at baseline. At 12 weeks after the study drug treatment, there was a decrease in cytokine levels (or their receptors) in the MA group (mean change in IL-6, 3.63+/-6.62 pg/ml; TNFR-p55, -0.06+/-0.11 ng/ml; TNFR-p75. -0.01+/-0.29 ng/ml; and sIL-2R, 0.08+/-0.07 ng/ml) and the placebo group (mean change in IL-6, -2.08+/-3.92 pg/ml; TNFR-p55, -0.02+/-0.08 ng/ml; TNFR-p75, -0.20+/-0.18 ng/ml; and sIL-2R, 0.02+/-0.03 ng/ml). Although the change in cytokine levels was not statistically significant between the two groups, significant negative correlation (p < .05) was found. For example, increased weight correlated with decreased sIL-2R levels (r = .36) and TNFR-p75 (r = -.31; fat-free mass (FFM) gain and reduction of sIL-2R (r = -.39), TNFR-p75 (r = -.30). There was a significant correlation between weight gain and reduction of TNFR-p75 (r = .54), TNFR-p55 (r- = .47), and sIL-2R (r = -.53); FFM gain and reduction of sIL-2R (r = -.59), TNFR-p75 (r = -.41), TNFR-p55 (r = -.42); and fat gain and reduction of TNFR-p75 (r = -.41) in the MA group (p < .05), but not in the placebo group. CONCLUSIONS: Although there was no significant change in cytokine levels between the two groups, the reduction in cytokine levels after MA treatment correlated with improvement in weight, fat mass, and FFM at 12 weeks.

Quality of life and stimulation of weight gain after treatment with megestrol acetate: correlation between cytokine levels and nutritional status, appetite in geriatric patients with wasting syndrome.

BACKGROUND: The geriatric wasting syndrome (GWS) has been associated with proinflammatory cytokines, depression and progressive decline in quality of life (QOL). The objective of this study was to evaluate the correlation between the changes in cytokine levels and appetite, nutritional markers, and QOL in geriatric patients with GWS following a randomized clinical trial of megestrol acetate (MA) versus placebo. METHODS: This was a prospective, double-blind, placebo-controlled trial. We evaluated 69 predominantly male (3 females) nursing home residents with weight loss of > or =5% of their usual body weight over the past three months or body weight 20% below their ideal body weight. Patients were randomly assigned to receive either placebo or megestrol acetate (MA) oral suspension (O.S.) 800 mg/day for 12 weeks and were then followed for 13 weeks off treatment. Data on appetite, weight, nutritional status, QOL and cytokine levels were collected at baseline and week 12. The correlation between appetite, weight, nutritional status, sense of well being and cytokine level changes in response to MA treatment was examined at week 12. RESULTS: Appetite, sense of well being, and QOL assessed by an "enjoyment list" significantly improved in the MA arm. Rising prealbumin showed a negative correlation with decreasing IL-6 (r = -0.51), TNFR-p 55 (r = -0.49) and sIL-2R (r = -0.38). There was also an improvement in prealbumin and a decrease in IL-6 and TNFR-p55 in the MA-arm (p < 0.01). A correlation between a decrease in the IL-6 levels and improvement in depression (r = 0.50) was seen in the MA arm as well. Improvement in appetite positively correlated with increased enjoyment of life (r = -0.41), less depression (r = -0.34), improved sense of well being (r = 0.36), prealbumin gain (r = 0.30), and weight gain (r = 0.38) by 12 weeks. Also, improvement in appetite positively correlated with improvement in nutritional parameters such as prealbumin, albumin, fat free mass and weight in the MA arm. CONCLUSIONS: In a geriatric nursing home population with weight loss, reduction in cytokine levels after MA treatment correlates with improvement in appetite, prealbumin, albumin, and improvement in quality of life.

Interleukin-6 predicts hypoalbuminemia, hypocholesterolemia, and mortality in hemodialysis patients.

Low serum albumin and low serum cholesterol levels are among the most consistent predictors of mortality in patients with end-stage renal disease (ESRD) undergoing hemodialysis. Hypoalbuminemia is often interpreted as a marker of poor nutrition, but serum albumin and cholesterol levels can also be low as part of a cytokine-mediated acute-phase reaction to acute or chronic inflammation. Here we report the results from a 900-day prospective study designed to determine whether tumor necrosis factor-alfa (TNF-alpha) and interleukin-6 (IL-6) predict serum albumin and cholesterol levels and mortality in a group of 90 ambulatory, adult hemodialysis patients with no acute infection, hospitalization or surgery, and no known acquired immunodeficiency syndrome (AIDS), malignancy, or liver disease. Measurable levels of TNF-alpha and/or IL-6 were found in 89 of 90 patients. Significant relationships were found between TNF-alpha and IL-6 and the degree of hypoalbuminemia and dyslipoproteinemia. IL-6 was the strongest predictor of mortality in univariate and multivariate analysis, followed by age, albumin level, and body mass index (BMI). Although the cause of hypercytokinemia was not addressed in this study, the data support the view that hypoalbuminemia and hypocholesterolemia are negative acute-phase responses to inflammatory stimuli. These results suggest that efforts to identify the nature of the stimuli for cytokine production and to lower cytokine levels in hemodialysis patients might be effective in improving the survival of patients undergoing hemodialysis.

Increasing hepatic cholesterol 7alpha-hydroxylase reduces plasma cholesterol concentrations in normocholesterolemic and hypercholesterolemic rabbits.

The effect of bile acid depletion and replacement with glycodeoxycholic acid on plasma cholesterol concentrations, hepatic low-density lipoprotein (LDL) receptor binding and messenger RNA (mRNA) levels, and hepatic activities and mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol 7alpha-hydroxylase was investigated in 19 New Zealand white (NZW) and 15 Watanabe heritable hyperlipidemic (WHHL) rabbits. Bile acid depletion was produced by external bile drainage for 5 days, which maximized cholic acid synthesis. Replacement was achieved by infusing glycodeoxycholic acid intraduodenally for 24 hours so that the hepatic bile acid flux reached prefistula levels. Plasma and liver cholesterol concentrations were 13 times and 50% greater, respectively, hepatic LDL receptor-mediated binding was 26% less, and cholesterol 7alpha-hydroxylase activity and mRNA levels were 62% and 86% less in WHHL than NZW rabbits. After bile drainage, plasma cholesterol concentrations decreased 29% in NZW rabbits and 40% in WHHL rabbits and were associated with a 2.1-fold increase in hepatic LDL receptor-mediated binding in the NZW rabbits, but there was no change in the WHHL rabbits. Cholesterol 7alpha-hydroxylase activity and mRNA levels increased three and four times in NZW and WHHL rabbits, respectively, although liver cholesterol levels remained unchanged. Replacement with exogenous glycodeoxycholic acid increased plasma cholesterol concentrations 1.7 times in NZW rabbits and decreased enhanced cholesterol 7alpha-hydroxylase activity 54%, mRNA levels 86%, cholic acid synthesis 38%, and hepatic LDL receptor-mediated binding 57% in NZW rabbits. Bile acid depletion stimulated cholic acid synthesis by up-regulating cholesterol 7alpha-hydroxylase to use cholesterol and reduce plasma concentrations substantially in both NZW and WHHL rabbits, although LDL receptors did not function in WHHL rabbits. Glycodeoxycholic acid replacement inhibited elevated cholesterol 7alpha-hydroxylase, cholic acid synthesis, and hepatic LDL receptor binding to reestablish baseline plasma cholesterol levels in NZW rabbits. Hypercholesterolemia in WHHL rabbits was related to the combination of dysfunctional LDL receptors and inhibited cholesterol 7alpha-hydroxylase. Plasma cholesterol concentrations were reduced significantly when cholesterol 7alpha-hydroxylase was stimulated even in the absence of LDL receptor function.

Low lipid concentrations in critical illness: implications for preventing and treating endotoxemia.

OBJECTIVES: To determine the prevalence and clinical significance of hypolipidemia found in critically ill patients, and whether the addition of a reconstituted lipoprotein preparation could inhibit the generation of tumor necrosis factor-alpha (TNF-alpha) in acute-phase blood taken from these patients. SETTING: Surgical intensive care unit (ICU) of a large urban university hospital. DESIGN: Prospective case series. PATIENTS: A total of 32 patients with a variety of critical illnesses had lipid and lipoprotein concentrations determined. Six patients and six age- and gender-matched control subjects had whole blood in vitro studies of the effect of lipoprotein on lipopolysaccharide mediated TNF-alpha production. INTERVENTIONS: Blood samples were drawn on admission to the ICU and over a subsequent 8-day period. MEASUREMENTS AND MAIN RESULTS: Mean serum lipid and lipoprotein values obtained from patients within 24 hrs of transfer to the surgical ICU were extremely low: mean total cholesterol was 117 mg/dL (3.03 mmol/L), low-density lipoprotein cholesterol 71 mg/dL (1.84 mmol/L), and high-density lipoprotein cholesterol 25 mg/dL (0.65 mmol/L). Only the mean triglyceride concentration of 105 mg/dL (1.19 mmol/L), and the mean lipoprotein(a) concentration of 25 mg/dL (0.25 g/L) were within the normal range. During the first 8 days following surgical ICU admission, there were trends toward increasing lipid and lipoprotein concentrations that were significant for triglycerides and apolipoprotein B. Survival did not correlate with the lipid or lipoprotein concentrations, but patients with infections had significantly lower (p = .008) high-density lipoprotein cholesterol concentrations compared with noninfected patients. Lipopolysaccharide-stimulated production of TNF-alpha in patient and control blood samples was completely suppressed by the addition of 2 mg/mL of a reconstituted high-density lipoprotein preparation. CONCLUSIONS: Patients who are critically ill from a variety of causes have extremely low cholesterol and lipoprotein concentrations. Correction of the hypolipidemia by a reconstituted high-density lipoprotein preparation offers a new strategy for the prevention and treatment of endotoxemia.

Visualization of hepatitis C virions and putative defective interfering particles isolated from low-density lipoproteins.

Hepatitis C virus (HCV) in highly infectious sera has been shown to be predominantly associated with low-density lipoproteins. To determine whether the association is specific to low-density lipoproteins (LDL) or very low-density lipoproteins (VLDL), we fractionated HCV-containing plasma by a column chromatographic procedure known to separate these classes. Hepatitis C virus RNA detected by polymerase chain reaction (PCR) was associated primarily with the very low-density (VLDL) fraction. However, it could not be ruled out that virus-associated LDL may have eluted with this fraction. Hepatitis C virus virions isolated from sera having sufficient titre for visualization by electron microscopy are generally coated with antiviral antibodies, therefore we utilized the lipid association to isolate antibody-free virions. Very low-density lipoproteins were isolated by ultracentrifugal flotation and then treated with deoxycholate to release the virions. These were then isolated in a highly purified form by centrifugation in a sucrose gradient. The 1.10-1.11 g ml-1 region of the gradients contained 60-70 nm particles. Particles with similar surface structure but having a diameter of only 30-40 nm constituted about 30% of the total. The latter may represent defective interfering particles. The identity of both small and large particles with HCV virions and associated particles was confirmed by their trapping on grids by an anti-HCV E2 monoclonal antibody, and by their aggregation by rabbit antiserum to an amino-terminal peptide of E1. Thus, both E1 and E2 epitopes are displayed on the surface of intact HCV virions.

Unexpected inhibition of cholesterol 7 alpha-hydroxylase by cholesterol in New Zealand white and Watanabe heritable hyperlipidemic rabbits.

We investigated the effect of cholesterol feeding on plasma cholesterol concentrations, hepatic activities and mRNA levels of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase and hepatic LDL receptor function and mRNA levels in 23 New Zealand White (NZW) and 17 Watanabe heritable hyperlipidemic (WHHL) rabbits. Plasma cholesterol concentrations were 9.9 times greater in WHHL than NZW rabbits and rose significantly in both groups when cholesterol was fed. Baseline liver cholesterol levels were 50% higher but rose only 26% in WHHL as compared with 3.6-fold increase with the cholesterol diet in NZW rabbits. In both rabbit groups, hepatic total HMG-CoA reductase activity was similar and declined > 60% without changing enzyme mRNA levels after cholesterol was fed. In NZW rabbits, cholesterol feeding inhibited LDL receptor function but not mRNA levels. As expected, receptor-mediated LDL binding was reduced in WHHL rabbits. Hepatic cholesterol 7 alpha-hydroxylase activity and mRNA levels were 2.8 and 10.4 times greater in NZW than WHHL rabbits. Unexpectedly, cholesterol 7 alpha-hydroxylase activity was reduced 53% and mRNA levels were reduced 79% in NZW rabbits with 2% cholesterol feeding. These results demonstrate that WHHL as compared with NZW rabbits have markedly elevated plasma and higher liver cholesterol concentrations, less hepatic LDL receptor function, and very low hepatic cholesterol 7 alpha-hydroxylase activity and mRNA levels. Feeding cholesterol to NZW rabbits increased plasma and hepatic concentrations greatly, inhibited LDL receptor-mediated binding, and unexpectedly suppressed cholesterol 7 alpha-hydroxylase activity and mRNA to minimum levels similar to WHHL rabbits. Dietary cholesterol accumulates in the plasma of NZW rabbits, and WHHL rabbits are hypercholesterolemic because reduced LDL receptor function is combined with decreased catabolism of cholesterol to bile acids.

Therapeutic trial of reconstituted human high-density lipoprotein in a canine model of gram-negative septic shock.

In a controlled, randomized trial, the authors investigated the effects of reconstituted human high-density lipoprotein (R-HDL) on survival, endotoxemia, cytokine production and pathophysiologic and metabolic events in an animal model of gram-negative septic shock. At 0.5, 8 and 16 hr after implantation of a clot infected with Escherichia coli, canines received intravenous R-HDL (n = 13), control lipid (n = 7) or human serum albumin (HSA, n = 7) divided into three doses (0.3, 0.1 and 0.1 g/kg, respectively) at an hourly rate of 0.1 g/kg. All animals were treated with antibiotics and fluids. Animals treated with R-HDL had lower levels of circulating endotoxin and tumor necrosis factor and a smaller decrease in white blood cell counts than did animals treated with lipids and HSA (all P < .05). The survival times of lipid- and HSA-treated animals were similar (P = .3) and were significantly greater than those of R-HDL-treated animals (P = .02). During the first 6 hr after clot implantation, R-HDL-treated animals had significantly greater abnormalities in liver function test findings compared with lipid- and HSA-treated animals (all P < .05). For the first 24 hr, R-HDL-treated animals had significant increases in HDL levels; however, there were no significant relationships between these levels and the constituents of HDL (apolipoprotein AI and phosphatidylcholine) or liver function abnormalities and survival times (all r < .2, P > .3). In normal animals, administration of R-HDL (in similar doses) caused transient elevation of liver enzymes; in animals given sterile clot i.p., R-HDL caused seizures.(ABSTRACT TRUNCATED AT 250 WORDS)

Reconstituted high-density lipoprotein neutralizes gram-negative bacterial lipopolysaccharides in human whole blood.

We have tested hypotheses relating lipoprotein structure to function as measured by the relative ability to neutralize endotoxin by comparing natural human lipoproteins, a chemically defined form of reconstituted high-density lipoprotein (R-HDL), and a lipid emulsion (Intralipid). The human whole-blood system was used as an in vitro model of lipopolysaccharide (LPS) binding protein and CD14-dependent activation of cytokine production. When lipoproteins were compared on the basis of protein content, R-HDL was most effective in reducing tumor necrosis factor alpha (TNF-alpha) production followed in order by very low density lipoprotein, low-density lipoprotein, Intralipid, and natural HDL. However, when these particles were compared by protein, phospholipid, cholesterol, or triglyceride content by stepwise linear regression analysis, only phospholipid was correlated to effectiveness (r2 = 0.873; P < 0.0001). Anti-CD14 monoclonal antibodies MY4 and 3C10 inhibited LPS binding protein and CD14-dependent activation of TNF-alpha production by LPS at LPS concentrations up to approximately 1.0 ng/ml. R-HDL (2 mg of protein per ml) blocked TNF-alpha production by LPS from both smooth- and rough-type gram-negative bacteria at concentrations up to 100 ng of LPS per ml but had little effect on heat-killed gram-positive Staphylococcus aureus and no effect on other LPS-independent stimuli tested. These results support our hypothesis that LPS is neutralized by binding to phospholipid on the surface of R-HDL and demonstrate that R-HDL is a potent inhibitor of the induction of TNF-alpha by LPS from both rough- and smooth-form gram-negative bacteria in whole human blood.

Dextran-sulfate cellulose adsorption for lowering Lp[a].

Dextran sulfate selectively adsorbs lipoproteins that contain apolipoprotein-B100, including those associated with apolipoprotein-a, from human plasma with high affinity. Dextran sulfate immobilized on cellulose is incorporated into the Liposorber LA-15 system (Kaneka Corporation, Osaka, Japan). This system was evaluated in a nine-center controlled study of patients with familial hypercholesterolemia (FH) who had not responded adequately to diet and drug therapy. There were 54 patients with heterozygous FH: 45 randomized to treatment, 9 controls) and 10 patients with homozygous FH, all of whom received LDL apheresis. Removal of both LDL and Lp[a] was specific and highly efficient, > 90% of theory. Plasma LDL and Lp[a] concentrations were effectively lowered by repetitive LDL apheresis during the study and returned to baseline levels 3 to 4 weeks after the last apheresis treatment. The kinetics of the LDL rebound fit a simple one-pool kinetic model with a constant synthetic rate and constant fractional catabolic rate. The kinetics of the Lp[a] rebound are more complex and may require a model that includes a large extravascular Lp[a] pool in slow equilibrium with the plasma pool.

In vivo protection against endotoxin by plasma high density lipoprotein.

Overwhelming bacterial infection is accompanied by fever, hypotension, disseminated intravascular coagulation, and multiple organ failure leading to death in 30-80% of cases. These classical symptoms of septic shock are caused by potent cytokines that are produced in response to endotoxin released from Gram-negative bacteria. Treatments with antibodies and receptor antagonists to block endotoxin or cytokine mediators have given mixed results in clinical trials. High density lipoprotein (HDL) is a natural component of plasma that is known to neutralize endotoxin in vitro. We report here that raising the plasma HDL concentration protects mice against endotoxin in vivo. Transgenic mice with 2-fold-elevated plasma HDL levels had more endotoxin bound to HDL, lower plasma cytokine levels, and improved survival rates compared with low-HDL mice. Intravenous infusion of HDL also protected mice, but only when given as reconstituted HDL prepared from phospholipid and either HDL apoprotein or an 18-amino acid peptide synthesized to mimic the structure of apolipoprotein A-I of HDL. Intact plasma HDL was mildly toxic, and HDL apoprotein was ineffective. The effectiveness of the reconstituted peptide renders very unlikely any significant contribution to protection by trace proteins in apo-HDL. These data suggest a simple leaflet insertion model for binding and neutralization of lipopolysaccharide by phospholipid on the surface of HDL. Plasma HDL may normally act to protect against endotoxin; this protection may be augmented by administration of reconstituted HDL or reconstituted peptides.

Immunochemical studies of extracellular glycoproteins (X-GPs) of goldfish brain.

Exoglycoproteins (X-GPs) are a family of soluble glycoproteins which are the most prominent constituent of the extracellular compartment of goldfish brain. On conventional two-dimensional polyacrylamide gels they typically display two primary molecular weight forms, averaging about 33 and 38 kDa, each appearing as a row of five to seven individual spots. When X-GP antibodies were applied by Western blotting, gels of goldfish brain extract prepared without a reducing agent showed, in addition to the primary molecular weight groups, at least one row of spots of slightly lower molecular weight and a major array of spots in the range of 45-60 kDa. The latter presumably represent dimers of the primary X-GP forms since they gave rise to the primary forms upon treatment with a reducing agent. However, on gradient gels prepared without detergents or reducing agents, X-GPs identified by immunostaining appeared only at 200 kDa and above, indicating that these proteins naturally occur in the form of large particles. Deglycosylation of the brain extract by N-glycosidase F reduced the molecular weight of each primary X-GP form by about 5 kDa, but did not abolish the microheterogeneity, which is at least partly due to minor differences in primary structure among the proteins in individual spots. Both rows of spots in the deglycosylated sample showed a coordinated shift toward the basic side of the gel, and a prominent new spot appeared on the basic end of the lower molecular weight group, which probably represents the fully deglycosylated form of the most abundant X-GP isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of lipoproteins on human glioblastoma growth in vitro.

Experiments were performed using an established human glioblastoma cell line to determine the effect of lipoproteins on regulating their growth. It was found that synthetic and natural human high density lipoproteins (HDL) were effective in inhibiting tumor cell growth in a nontoxic, dose-dependent manner, and that the LD50 was 10-fold lower than that for normal rat astrocytes grown under identical conditions. In the presence of the antioxidant, glutathione, essentially all of the growth-inhibiting properties of HDL could be reversed suggesting that oxidized lipids from the HDL interacting with the plasma membranes of the glioblastoma cells were responsible for the growth-inhibiting effect observed. The markedly lower concentration of HDL required to inhibit glioblastoma cells in culture compared to normal astrocytes suggested that the mechanism of HDL-induced inhibition may be important for tumor growth in vivo. One possible mechanism under investigation is the possibility of HDL modulation of a membrane-associated, tumor-specific phosphatase.

High-frequency analysis of the signal-averaged ECG. Correlation with left ventricular mass in rabbits.

Standard electrocardiographic (ECG) criteria have exhibited poor correlation with left ventricular mass and poor sensitivity for left ventricular hypertrophy at acceptable levels of specificity. To assess the ability of the high-frequency filtered signal-averaged ECG to improve ECG correlation with left ventricular mass, signal-averaged orthogonal lead recordings in 29 normal rabbits and seven rabbits with left ventricular hypertrophy due to chronic aortic regurgitation were compared with left ventricular mass corrected for body weight. Voltage of the vector QRS complex was integrated over the total duration of the QRS after separate filtering with standard frequency (0-100 Hz) low-pass and high-frequency (44 Hz) high-pass filters. Measurement of individual X, Y, and Z lead R and S wave voltage was performed on averaged, standard frequency filtered complexes, and the maximal spatial vector magnitude was determined from the standard frequency filtered vectors. Voltage of the 44 Hz high-pass filtered vector QRS complex integrated over the total duration of the QRS (high-frequency vector integral) correlated closely with indexed left ventricular mass (r = 0.84, p less than 0.0001), significantly better than the correlation of standard frequency vector integral or maximal spatial vector magnitude voltages (r = 0.35 and r = 0.61, each p less than 0.01 vs high-frequency vector integral) and the correlation of orthogonal lead X R wave or lead Y S wave voltages (r = 0.55 and r = 0.37, respectively, each p less than 0.01 vs high-frequency vector integral).(ABSTRACT TRUNCATED AT 250 WORDS)

Control of variance in experimental studies of hyperlipidemia using the WHHL rabbit.

Between-animal variability has frustrated many experimental studies in outbred animal models of human disease. Variability that arises from genetic heterozygosity can be minimized by use of experimental designs that match littermates (polyzygotic twins) across control and treatment groups. Poor breeding vigor has prevented use of this experimental design in the WHHL rabbit model of hyperlipidemia and atherosclerosis. A comparison of reproduction in WHHL and normal rabbits demonstrated that litter size is limited by functional deficits at ovulation, implantation, and gestation in WHHL females. Superovulation of females reliably produced expanded litters of WHHL rabbits. Plasma lipids were measured in expanded litters of Japanese White WHHL (JW-WW) and English Half-lop WHHL (EHL-WW) rabbits. The variance of plasma cholesterol within sibships was two- to three-fold less than that between-litters. Intraclass correlation of total cholesterol within litters of EHL-WW was 0.72 and within litters of JW-WW was 0.67. These data provide evidence of genetic modulation of hypercholesterolemia in WHHL rabbits and demonstrate that experimental designs in which littermates are paired across groups can decrease the number of animals needed or increase the sensitivity of hypothesis tests by two- to threefold.

Humoral immune response following extracorporeal immunoadsorption therapy of patients with hypercholesterolemia.

Low-density lipoprotein apheresis (LDL-apheresis) is an extracorporeal procedure that preferentially removes LDL cholesterol from the blood. One of the primary techniques for performing this procedure uses immunoadsorption columns containing monospecific polyclonal sheep antibodies to human LDL covalently coupled to a gel filtration medium. LDL-apheresis has generally been well-tolerated, with chills, fever, or flushing occurring rarely. The possibility of an immune reaction was investigated as a basis for these reactions observed in 12 of the 1312 procedures performed. Antibodies to sheep IgG developed in 12 of the 15 patients treated with LDL-apheresis as a result of the shedding of small quantities of the sheep immunoglobulin from the columns. A column acid-washing procedure minimized the quantity of shed antibody but did not prevent immunization of the patient. The clinical reactions were probably unrelated to shedding and immunization, as the reactions occurred even in patients who were not immunized to the sheep IgG. Immunization to ethylene oxide was not the cause, as determined by a radioallergosorbent test. The reactions were more likely related to the activation of complement, as indicated by the generation of C3a des Arg by the columns and an increase in C3a des Arg levels systemically.