[Coronary heart disease in childhood in familial hypercholesteremia. Maximum therapy with LDL apheresis]. / Koronare Herzkrankheit im Kindesalter bei familiärer Hypercholesterinämie. Maximaltherapie unter Einsatz der LDL-Apherese.

In children with familial hypercholesterolemia, coronary heart disease requires both medical theraphy and LDL apheresis. We report a case of verified occlusion of the anterior descending branch of the left coronary artery in a 10-year-old patient. The pathological findings revealed by ergometry established the diagnosis. The goal was to achieve the greatest possible reduction of lipid parameters and fibrinogen by lowering plasma viscosity employing LDL apheresis. It is astonishing that this treatment is also well tolerated by children. The basic vascular approaches suffice and shunt operations are not absolutely necessary. The efficacy of this method became vividly apparent by the changes in the skin lesions. Additional angiographic follow-up and further clinical course wil provide information on the usefulness of this treatment strategy with maximum lipid theraphy and the expected improvement in prognosis.

Binding kinetics of triclosan (Irgasan) to alloplastic vascular grafts: an in vitro study.

The aim of this study was to investigate the binding kinetics of triclosan (Irgasan) to alloplastic vascular grafts and to examine its antimicrobial activity against various microbial pathogens in vitro. Vascular grafts made by Intergard (Intervascular), Fluoropassiv (Vascutek), and Gore-tex (Gore) were examined. Grafts were incubated in 10 g/L triclosan (Irgasan), dried, sterilized, and incubated in RPMI medium. One-centimeter segments of the grafts were resected under sterile conditions at intervals of minutes, then hours, followed by days and up to 4 weeks. Samples were stored frozen at -20 degrees C for the measurement of triclosan bound to the vascular graft by high-performance liquid chromatography (HPLC). The binding kinetics under perfusion conditions were determined for Intergard grafts, which were perfused with 50 mL of nutrient medium for 24 hr. Samples were taken at various time intervals for the measurement of triclosan. The antimicrobial activity of triclosan against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans as well as Enterococcus faecium was determined. Triclosan effectively binds to vascular graft without the use of intermediate binding substances. It stayed on the graft for the duration of 4 weeks. Under both static and perfusion conditions, the binding kinetics are similar. Triclosan binds most effectively to Intergard grafts, less so to Fluoropassiv grafts, and not at all to Gore-tex material. Antimicrobial activity of triclosan is very effective against S. aureus and E. faecium but not against P. aeruginosa.

Association of serum tumor necrosis factor levels with decrease of cholesterol during septic shock.

Recent studies suggest that release of cytokines during inflammatory states such as septic shock leads to hypocholesterolemia. To examine whether tumor necrosis factor alpha (TNF), which is the major cytokine in inflammatory disease, causes hypocholesterolemia, we measured serum levels of total (bioactive and receptor-bound) TNF, cholesterol, Apo B, and Apo A1 in seven patients with septic shock over a period of 8 days. Since elevated serum TNF levels are accompanied by the release of soluble TNF receptors, levels of TNF receptors p55 and p75 were also measured. Patients with septic shock had significantly higher serum TNF and TNF receptor levels compared with healthy controls. Increased cytokine levels were accompanied by a significant decline in total serum cholesterol apolipoprotein A1 and B. In vitro studies with cultured human skin fibroblasts, human umbilical vein endothelial cells, and HepG2 hepatoma cells showed that TNF increased the degradation of 125I-labeled low-density lipoprotein in all the cell lines tested. Recombinant soluble TNF receptors inhibited the TNF-induced stimulation of low-density lipoprotein receptor in a concentration-dependent manner. However, the calculated ratio of TNF receptors to total TNF measured in serum of these patients was not able to counteract the stimulatory effect of TNF, possibly due to the higher molar excess of TNF receptors required to achieve this effect in vitro. Our data strengthen the hypothesis that serum values of total TNF determine the extent of hypocholesterolemia during sepsis and septic shock despite the presence of a high concentration of TNF receptors. Studies with recombinant TNF also confirm the role of TNF in hypocholesterolemia in inflammation.

Validation of an automated enzyme immunoassay for Interleukin-6 for routine clinical use.

Serum levels of Interleukin-6 (IL-6), a proinflammatory cytokine, are increased in early stages of inflammatory diseases such as infection and sepsis. Assay systems which permit its measurement within a few hours and as a single measurement have not been reported so far. We therefore evaluated a now commercially available automated method for IL-6 measurement on the Cobas Core immunological analyzer (Roche Diagnostic Systems) which enables single IL-6 measurement within about 1 hour. The automated assay correlates well with an established, manual microtiter plate assay (Biosource GmbH) which uses the same antibodies and reagents (r=0.98). Accuracy of the automated method was established by adding known amounts of IL-6 international reference preparation. Recovery of the international standard was in the range of 92104%. The automated assay had a precision of singletons below 6% and was linear up to 2800 pg/ml. This automated assay provides a suitable, convenient and time saving method for measurement of IL-6 serum levels in the routine clinical laboratory.

Dysregulation of monocytic nuclear factor-kappa B by oxidized low-density lipoprotein.

Nuclear factor-kappa B (NF-kappa B)/Rel transcription factors may be involved in atherosclerosis, as is suggested by the presence of activated NF-kappa B in human atherosclerotic lesions. The aim of the present study was to investigate the effects of oxidized LDL (oxLDL) on the NF-kappa B system in human THP-1 monocytic cells as well as adherent monocytes. Our results demonstrate that short-term incubation of these cells with oxLDL activated p50/p65 containing NF-kappa B dimers and induced the expression of the target gene IL-8. This activation of NF-kappa B was inhibited by the antioxidant and H2O2 scavenger pyrrolidine dithiocarbamate and the proteasome inhibitor PSI. The oxLDL-induced NF-kappa B activation was accompanied by an initial depletion of I kappa B-alpha followed by a slight transient increase in the level of this inhibitor protein. In contrast, long-term treatment with oxLDL prevented the lipopolysaccharide-induced depletion of I kappa B-alpha, accompanied by an inhibition of both NF-kappa B activation and the expression of tumor necrosis factor-alpha and interleukin-1 beta genes. These observations provide additional evidence that oxLDL is a potent modulator of gene expression and suggest that (dys)regulation of NF-kappa B/Rel is likely to play an important role in atherogenesis.

Determination of LDL- and scavenger-receptor activity in adherent and non-adherent cultured cells with a new single-step fluorometric assay.

Lipoproteins labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) are widely used to visualize LDL-and scavenger-receptor activity in cultured cells. The purpose of this study was to evaluate a new single-step fluorometric assay with high sensitivity for the quantitative determination of the LDL- or scavenger-receptor activity in adherent and non-adherent cells. We used an aqueous solution of 1 g/l SDS dissolved in 0.1 M NaOH to lyse the cells after incubation with DiI-LDL or DiI-acetylated LDL. This allows for the first time the determination of fluorescence intensity and cell protein in the same sample without prior lipid extraction of the fluorochrome. Fluorescence of the cell lysates was determined in microtiter plates with excitation-emission set at 520 and 580 nm, respectively. This rapid method demonstrates high specificity for determining the LDL- and scavenger-receptor activity in cultured cells (e.g., human skin fibroblasts from patients with and without familial hypercholesterolemia; human U-937 monocyte and murine P388 D1 macrophage cell lines). The validity of our fluorescence assay is demonstrated by comparison of cellular uptake and metabolism of lipoproteins labeled with both, DiI and 125iodine. The rapidity and accuracy of this assay allows its routine application for studying receptor-mediated lipoprotein uptake in various cell types.

Early prediction of outcome in score-identified, postcardiac surgical patients at high risk for sepsis, using soluble tumor necrosis factor receptor-p55 concentrations.

OBJECTIVE: To investigate the prognostic value of increased serum concentrations of soluble tumor necrosis factor (TNF) receptors in patients at high risk for sepsis. DESIGN: Prospective study. SETTING: Cardiac surgical intensive care unit in a University Hospital. PATIENTS: Those 27 of 870 consecutive postcardiac surgical patients who met a previously validated high-risk criterion for imminent sepsis (Acute Physiology and Chronic Health Evaluation II [APACHE II] score of > or = 24 on the first postoperative day [day 1]). In this population, systemic inflammatory response syndrome was present in 96% of the patients and the in-hospital mortality rate was 30%. In addition, ten postcardiac surgical patients with an uncomplicated course (mortality rate 0%) were studied for comparison. INTERVENTIONS: Blood sampling for measurements of serum concentrations of TNF and soluble TNF receptors 55 kilodalton (TNF receptor-p55) and 75 kilodalton (TNF receptor-p75) on days 1, 2, 3, and 5. MEASUREMENTS AND MAIN RESULTS: Compared with the ten patients with an uncomplicated course (group A), the high-risk patients had significantly higher baseline (day 1) serum concentrations of soluble TNF receptor-p55 (9.2 vs. 4.2 ng/mL) and soluble TNF receptor-p75 (9.2 vs. 5.5 ng/mL). These high-risk patients could be further differentiated into two subgroups: one (B) with a prompt decrease in APACHE II score and a good prognosis (mortality rate 0%) and another (C) with a persisting high risk of sepsis and mortality rate (40%, p < .05). Although baseline APACHE II score was similar in both high-risk subgroups, soluble TNF receptor-p55 concentrations were significantly higher in subgroup C compared with subgroup B already at baseline (10.7 vs. 4.7 ng/mL). The receiver operating characteristic curve for subgroup classification by soluble TNF receptor-p55 was in a discriminating position with an area (0.773 +/- 0.096), confirming soluble TNF receptor-p55 as a predictor of mortality. TNF and soluble TNF receptor-p75 concentrations were less predictive at baseline. CONCLUSIONS: This study suggest that increased soluble TNF receptor-p55 concentrations in the serum of postcardiac surgical patients allow earlier prognostication of subsequent hospital course than APACHE II scores alone. This study further suggests that the combination of physiologic scores and cytokine receptor measurements could improve the predictive power of early postoperative risk stratification.

Study of causes underlying the low atherosclerotic response to dietary hypercholesterolemia in a selected strain of rabbits.

We have recently characterized a strain of rabbits that shows a low atherosclerotic response (LAR) to dietary hypercholesterolemia in contrast to the usual high atherosclerotic response (HAR) of rabbits [1]. Presently, we have focused on three well established and important stages of atherogenesis, i.e., monocyte adhesion to endothelium, cell mediated peroxidative modification of lipoproteins and induction of a receptor that recognizes modified low density lipoprotein (LDL). The results obtained show that (1) beta-very low density lipoprotein (beta-VLDL) from LAR and HAR rabbits enhanced monocyte adhesion to endothelial cells to the same extent; (2) Cell mediated peroxidation of LDL and beta-VLDL, tested by loss of alpha-tocopherol and formation of thiobarbituric acid reacting substances (TBARS), was compared using macrophages, fibroblasts and smooth muscle cells (SMC) of LAR and HAR rabbits and no significant differences were found; (3) Induction of scavenger receptor by phorbol ester (phorbol 12-myristate 13-acetate (PMA)) and platelet-derived growth factor-BB (PDGF-BB) was determined in SMC or fibroblasts from LAR and HAR rabbits using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated LDL (DiL-acLDL). We found a significantly higher uptake of DiI-acLDL in SMC and fibroblasts derived from HAR rabbits as compared with cells from LAR rabbits. Similar results were also obtained with [125I]-acLDL in fibroblasts from LAR and HAR rabbits with respect to cellular lipoprotein degradation after PMA pretreatment. Even though the attenuated atherosclerotic response to hypercholesterolemia of LAR rabbits may have multiple underlying causes, the most prominent so far is an apparent difference in inducibility of scavenger receptor in SMC and fibroblasts.

The role of lipids in nephrosclerosis and glomerulosclerosis.

Hyperlipidemia and lipoprotein abnormalities are often encountered in patients with nephrotic syndrome or chronic renal disease and also in those undergoing haemodialysis and with renal transplant. Even though the significance of lipid deposition in renal tissue and the role of lipoproteins in the pathogenesis of renal disease in man is unclear, experimental and clinical data indicate a possible damaging effect of a disturbed lipid metabolism on the kidney. In humans, glomerular lipid deposition is observed in genetic diseases such as Fabry's disease, lecithin:cholesterol acyltransferase activity (LCAT) deficiency and arteriohepatic dysplasia, and in diseases with acquired disturbance of lipid metabolism such as nephrotic syndrome and cholestatic liver disease. Studies on animals with lupus nephritis, aminonucleoside nephrosis, reduced renal mass, diabetes mellitus or systemic hypertension have shown that cholesterol can increase the incidence of glomerulosclerosis. As most of these studies have been performed in the rat, which has a different lipoprotein profile to that of man, these results should be carefully interpreted with regard to their relevance for humans. In vitro cell culture studies on human glomerular cells have given some preliminary insights into the cellular mechanisms of lipid induced glomerular damage. Apo E-containing lipoproteins, which are pathologically elevated in many renal diseases, are avidly taken up by human mesangial cells. These cells seem to play a central role in the initiation of glomerulosclerosis by inducing proliferation and production of excess extracellular matrix. Lipoproteins are able to stimulate DNA synthesis in these cells, and increase the synthesis of mitogens and extracellular matrix protein. The pathogenic role of oxidized lipoproteins has not yet been defined. Human mesangial cells do not seem to take up these modified lipoproteins. However, macrophages infiltrate glomeruli and may constitute the stimulus for the generation of minimally modified lipoproteins and their cellular uptake. The data from animal experiments suggest that treatment that corrects hyperlipidemia may have an ameliorative effect on renal function. Thus, there are strong indications that lipoproteins may play a critical role in mediating the development of glomerulosclerosis.

Arterial hypertension and hyperlipidemia as determinants of glomerulosclerosis.

Arterial hypertension is a dominant pathogenetic factor for glomerulosclerosis. Nevertheless metabolic factors such as hyper- or dyslipoproteinemia may significantly modify and accelerate the process of glomerular scarring. Hyperlipidemia and lipoprotein abnormalities are often encountered in patients with nephrotic syndrome and chronic renal disease. Although the significance of lipid deposition in renal tissue and the role of lipoproteins in the pathogenesis of renal disease in man has not yet been clearly defined, experimental and clinical data indicate a damaging effect of disturbed lipid metabolism on the kidney. In humans glomerular lipid deposition is observed in several genetic diseases, including lecithin-cholesterol acyltransferase activity deficiency. Studies on animals with reduced renal mass, diabetes mellitus or arterial hypertension have shown that hypercholesterolemia increases the incidence of glomerulosclerosis. Especially the interaction of arterial hypertension and dyslipoproteinemia leads to a rapid and pronounced development of glomerulosclerosis. As most of these studies have been performed in the rat, which has a different lipoprotein profile than man, these results should be carefully interpreted with regard to their relevance for humans. In vitro cell culture studies on human glomerular cells have provided insight into the possible cellular mechanisms of lipid-induced glomerular damage. Apoprotein E containing lipoproteins that are pathologically elevated in many renal diseases are avidly taken up by human glomerular cells. Mesangial cells seem to play a central role in the initiation of glomerulosclerosis by proliferation and production of excess extracellular matrix. Lipoproteins are able to stimulate DNA synthesis in these cells and increase the synthesis of mitogens and matrix proteins. The pathogenetic role of modified, oxidized lipoproteins has not yet been elucidated.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of long-term treatment with simvastatin on plasma lipids and lipoproteins in patients with primary hypercholesterolemia.

We investigated long-term hypolipidemic effects and clinical safety of simvastatin, a new competitive inhibitor of 3-hydroxy-methylglutaryl coenzyme A reductase in 24 patients with familial and non-familial hypercholesterolemia. Patients received up to 40 mg simvastatin for a period of 30 months. Significant decreases were noted in plasma cholesterol (30%), plasma triglycerides (25%), very low density lipoprotein-cholesterol (26%), and low density lipoprotein-cholesterol (40%), whereas an increase in plasma high density lipoprotein-cholesterol (11%) was observed. Furthermore, the percentage decrease in plasma low density lipoprotein cholesterol was independent of individual baseline concentrations. Simvastatin did not alter the composition of low density lipoproteins or high density lipoproteins. The percentage decrease in total plasma and low density lipoprotein-cholesterol was independent of apoprotein E isoforms and low density lipoprotein-receptor activity as assayed in cultured fibroblasts. The drug therapy was well tolerated and clinical examinations revealed no adverse effects. Clinical chemistry indices and hematological, as well as endocrinological parameters remained within normal limits and ranges.

Low-density lipoprotein plasmaphaeresis with and without lovastatin in the treatment of the homozygous form of familial hypercholesterolaemia.

A 7-year-old girl with homozygous familial hypercholesterolaemia and plasma low-density lipoprotein(LDL)-cholesterol levels of 820 mg/dl (21.2 mmol/l) and progressive xanthomata was treated with heparin extracorporeal low-density lipoprotein precipitation (HELP) to lower her plasma LDL. On weekly HELP treatment she maintained her pre-HELP treatment LDL-cholesterol levels at 409 mg/dl (10.6 mmol/l). The long-term HELP treatment was well tolerated and led to regression of her xanthomata. Subsequently, lovastatin [Mevacor; Merck Sharp & Dohme, Westpoint, Pa., USA (20 mg/day)] was added to the regimen, causing a further 20% decrease in her pre-HELP treatment plasma LDL-cholesterol levels. Lovastatin alone did not sufficiently lower her plasma LDL and could not replace the weekly HELP therapy. Our data show that lovastatin is an effective adjunctive therapy for lowering plasma LDL-cholesterol in a homozygous patient, once plasma LDL levels have already been lowered by regular HELP treatment.

Receptor mediated uptake of apo B and apo E rich lipoproteins by human glomerular epithelial cells.

Various pathological disorders are accompanied by the deposition of lipids into glomerular cells. To gain insight into these disorders, it is essential to know if glomerular cells possess lipoprotein receptors. We therefore characterized the activity of lipoprotein receptors in cultured epithelial cells of the human glomerulus. Podocytes were chosen as they are directly exposed to lipoproteins in pathological states like in glomerular proteinuria (such as, nephrotic syndrome). Isolated human glomeruli (purity greater than 95%) were incubated in buffered RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum at 37 degrees C and 5% CO2. Outgrowing cells were vimentin and keratin positive. Monolayer cultures of human glomerular epithelial cells upon incubation in lipoprotein deficient serum for 48 hours expressed a receptor-dependent uptake of lipoproteins. These cells showed about 10% of the maximal capacity for LDL uptake as compared to fibroblasts; however, the Km values for binding, internalization and degradation were similar in the cultures of glomerular epithelial cells and fibroblasts. The Km values for degradation of LDL, chylomicron remnants, beta-VLDL from cholesterol-fed rabbits and VLDL from familial LCAT-deficiency patients were 14.2, 4.9, 2.9, 4.5 micrograms protein/ml medium, respectively, for glomerular epithelial cells. The avid uptake of 125I-labeled apo E-containing lipoproteins was further substantiated by their poor displacement by a 25-fold excess of unlabeled LDL and their ability to down regulate the apo B,E receptor activity. LDL as well as beta-VLDL were able to suppress the incorporation of 14C acetate into sterols and to stimulate 3H-cholesterylester formation. These experiments show that cultured glomerular epithelial cells express lipoprotein receptor activity. Plasma concentrations of apo E-containing lipoproteins are increased in certain renal diseases (such as, nephrotic syndrome); these lipoproteins could be rapidly removed by glomerular epithelial cells and lead to lipid deposition in glomeruli.

Heterogeneity of human lipoprotein Lp[a]: cytochemical and biochemical studies on the interaction of two Lp[a] species with the LDL receptor.

Human Lp[a] can be fractionated into two species with different affinities for lysine-Sepharose. Forty to 81% of the total Lp[a] in the density fraction 1.055-1.15 g/ml from five individuals was retained by this affinity column. The remaining unretained Lp[a] species with no apparently functional lysine binding site was similar to the retained species in its electrophoretic mobility, lipid, protein, and apolipoprotein composition, and the heterogeneity was not related to apo[a] size polymorphism. Interaction of the two species with the low density lipoprotein (LDL) receptor was studied in human fibroblasts. Using gold-labeled lipoproteins and an immunochemical procedure, both Lp[a] species could be located in clusters on the cell surface, but the extent of labeling was far lower than that seen with LDL. Both Lp[a] variants were less effective than LDL in 1) down-regulation of LDL-receptor activity; 2) suppression of cellular sterol synthesis; and 3) stimulation of cholesteryl ester formation in human fibroblasts. Although degradation of both species of Lp[a] by the perfused rat liver was stimulated after estrogen induction of hepatic LDL-receptor activity, the stimulation amounted to only a quarter of that seen with LDL. The heterogeneity of Lp[a] with respect to the ability to bind epsilon-aminocarboxylic acid will need to be considered in studying the physiological role of this lipoprotein. Both Lp[a] species exhibited a similar interaction with the LDL-receptor in vitro, and confirmed previous investigations that Lp[a] is only a poor ligand for the LDL-receptor.

Influence of smoking on cellular lipid metabolism.

Smoking and hyper-beta-lipoproteinemia, are well-established risk factors for the early development of atherosclerotic disease. The presence of both factors augments the potential risk. Since the influence of smoking on the plasma lipoprotein profile in humans seems to be either absent or minor, we were interested in whether cigarette smoking develops a direct influence on lipid metabolism at the cellular level. Our findings showed that smoking has no effect on either the high affinity LDL receptor activity or on the activity of HMG-CoA reductase, acid lipase, or cholesterol ester hydrolase.

Differential induction of rat liver microsomal UDP-glucuronosyltransferase activites by various inducing agents.

The selectivity of various inducers of UDP-glucuronosyltransferase was investigated in rat liver microsomes and compared with their effect on monooxygenase reactions. (1) Similar to 3-methyl-cholanthrene beta-naphthoflavone selectively stimulated the glucuronidation of 1-naphthol and 4-methylumbelliferone (GT1 substrates). (2) In contrast, DDT preferentially enhanced the glucuronidation of morphine, 4-hydroxybiphenyl (GT2 substrates) and bilirubin, similar to phenobarbital. (3) Colfibric acid and bezafibrate selectively enhanced bilirubin glucuronidation without affecting GT1 and GT2 reactions. (4) Similar to ethoxyquin and Aroclor 1254, trans-stilbene oxide enhanced both GT1 and GT2 activities but not bilirubin glucuronidation. (5) In contrast to 3-methylcholanthrene-type inducers which induce both cytochrome P-450MC and GT1, probably through a common receptor protein, ethoxyquin and trans-stilbene oxide markedly induced GT1 reactions without affecting benzo[a]pyrene monooxygenase.