Stefan Barlage

The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.

Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism. It is characterized by absence of plasma high-density lipoprotein (HDL) and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes. Although low HDL cholesterol is associated with an increased risk for coronary artery disease, this condition is not consistently found in TD pedigrees. Metabolic studies in TD patients have revealed a rapid catabolism of HDL and its precursors. In contrast to normal mononuclear phagocytes (MNP), MNP from TD individuals degrade internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism. HDL-mediated cholesterol efflux and intracellular lipid trafficking and turnover are abnormal in TD fibroblasts, which have a reduced in vitro growth rate. The TD locus has been mapped to chromosome 9q31 (ref. 9). Here we present evidence that TD is caused by mutations in ABC1, encoding a member of the ATP-binding cassette (ABC) transporter family, located on chromosome 9q22–31 (ref. 10). We have analysed five kindreds with TD and identified seven different mutations, including three that are expected to impair the function of the gene product. The identification of ABC1 as the TD locus has implications for the understanding of cellular HDL metabolism and reverse cholesterol transport, and its association with premature cardiovascular disease.

Changes in HDL-associated apolipoproteins relate to mortality in human sepsis and correlate to monocyte and platelet activation.

ObjectiveLipoproteins modulate vascular cell function in inflammation. In this study, we analyzed whether plasma concentrations of lipoproteins and apolipoproteins in human sepsis are related to patient survival and the activation of blood monocytes and platelets.DesignObservational study.SettingMedical and surgical intensive care units (ICU) of a university hospital.Patients151 consecutive patients after sepsis criteria had been met for the first time.InterventionsNone.MeasurementsPlasma lipoproteins, apolipoproteins, platelet CD62P-expression, monocyte HLA-DR-expression, SAPS II-scores (Simplified Acute Physiology Score) and 30-day-mortality were recorded.ResultsTotal cholesterol, high-density-lipoprotein (HDL) and low-density-lipoprotein (LDL) cholesterol, apolipoprotein (apo)-AI and apo-B were all found to be significantly lower in non-survivors than in survivors. In contrast to other (apo)lipoproteins, apo-AI and HDL cholesterol further decreased in non-survivors during the ICU stay. Logistic regression analysis revealed apo-AI to be an independent predictor of 30-day-mortality. A significant inverse correlation was found for apo-AI/HDL-cholesterol and platelet activation. Later in the course of the disease, HLA-DR expression on monocytes correlated positively to apo-AI and apo-CI concentrations and inversely to the apo-E concentration.ConclusionLow apo-AI is independently related to 30-day mortality in human sepsis and the decrease in apo-AI/HDL cholesterol correlates to increased platelet activation. Moreover, changes in apolipoproteins supposed to modulate lipopolysaccharide effects, such as apo-CI and apo-E, correlate to monocyte activation.

E-LDL and Ox-LDL differentially regulate ceramide and cholesterol raft microdomains in human Macrophages†

Atherosclerosis is characterized by the generation of lipid‐loaded macrophage‐derived foam cells. To study the effect of different types of atherogenic lipoproteins, human macrophages were loaded with enzymatically degraded low density lipoprotein (E‐LDL) or oxidized LDL (Ox‐LDL). Cellular cholesterol content was increased by E‐LDL, whereas Ox‐LDL increased the ceramide content. Cell surface expression analysis by flow cytometry and confocal microscopy revealed that Ox‐LDL increased ceramide and lactosylceramide expression compared to E‐LDL loading and induced ceramide rafts, whereas loading with E‐LDL induced cholesterol‐rich microdomains. Formation of different rafts may have consequences for raft associated signaling in cholesterol homeostasis and apoptosis in human macrophages.

The role of plasma phospholipid transfer protein (PLTP) in HDL remodeling in acute-phase patients

During reverse cholesterol transport plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and pre-beta-HDL. The acute-phase response is accompanied with dramatic changes in lipid metabolism including alterations in HDL concentration, composition, and thereby its function as a substrate for HDL remodeling proteins in circulation. To evaluate how acute-phase HDL (AP-HDL) functions in PLTP-mediated HDL conversion, we collected plasma samples from patients with severe acute-phase response (n=17), and from healthy controls (n=30). Subsequently, total HDL (1.063<d<1.21 g/ml) was isolated from patients and controls and incubated in the absence and presence of purified PLTP. The results show that HDL isolated from the acute-phase patients is converted by PLTP in vitro in a corresponding manner as normal HDL. In the combined population, C-reactive protein correlated significantly with lecithin-cholesterol acyltransferase (LCAT) activity (r=-0.53), cholesterol ester transfer protein activity (r=-0.80), PLTP activity (r=0.44), and PLTP mass (r=-0.66). When compared to the controls, the patients had 31% higher PLTP activity, but 52% lower PLTP mass leading to a 165% higher PLTP specific activity in the patients. The present data indicate that during the acute-phase response, plasma PLTP activity and mass are strongly affected by the lipoprotein distribution as well as lipid composition. We suggest that the decrease of HDL during the acute phase is caused by reduced LCAT and increased PLTP activities both increasing the plasma levels of lipid-poor apoA-I particles.

Predictors of blood loss after coronary artery bypass grafting.

OBJECTIVE To assess the predictive value of variables possibly associated with blood loss after coronary artery bypass grafting (CABG). DESIGN A prospective study. SETTING A university hospital. PARTICIPANTS Eighty-nine patients scheduled for elective CABG. INTERVENTIONS Blood samples were drawn before and after surgery. Chest tube drainage was measured hourly until removal of drains. MEASUREMENTS AND MAIN RESULTS Activation of coagulation and fibrinolysis, routine clotting tests, and expression of platelet surface antigens were analyzed using flow cytometry. A significant correlation was found among blood loss and activated partial thromboplastin time, fibrinogen, prothrombin fragment 1 + 2, D-dimers, platelet count, GPIb and P-selectin expression on platelets, use of internal thoracic artery, cross-clamp time, and thrombin-antithrombin III complex. In a multiple regression model, glycoprotein (GP) Ib expression on platelets, platelet count, use of internal thoracic artery, and D-dimers were significantly associated with blood loss. Logistic regression analysis showed that GPIb and D-dimers predicted an increased blood loss with a positive predictive value of 73% and a negative predictive value of 91%. CONCLUSIONS Postoperative D-dimers and GPIb expression may be useful to exclude nonsurgical causes in bleeding patients after CABG.

High density lipoprotein modulates platelet function.

Platelet activation by atherogenic lipoproteins can be antagonized by high density lipoprotein (HDL), probably via interaction with the ATP‐binding cassette transporter A1 (ABCA1).

Regulation of surface CD163 expression and cellular effects of receptor mediated hemoglobin-haptoglobin uptake on human monocytes and macrophages.

Local dysregulation of iron metabolism is suggested to contribute to atherosclerotic lesion development through hemoglobin scavenging pathways. We evaluated the effects of CD163‐mediated uptake of hemoglobin‐haptoglobin (HbHp) complexes on surface CD163 and intracellular heme oxygenase‐1 expression and the secretion of pro‐ and antiinflammatory cytokines by macrophages. We found that increased availability of HbHp complexes triggers the upregulation of surface CD163, and also results in a dose‐dependent secretion of IL‐6 and IL‐10.

Effects of Extracorporeal Circulation and Heparin on the Phenotype of Platelet Surface Antigens following Heart Surgery

In this prospective study, the time-dependent effects of extracorporeal circulation and heparin-mediated effects on platelet surface antigens in vitro were investigated using whole blood flow cytometry. Blood samples were drawn prior to and following extracorporeal circulation in 89 patients. The response of surface antigen expression (glycoprotein IIb/IIIa, P-selectin, and glycoprotein Ib) with and without in vitro stimulation was measured. A significant correlation of the duration of extracorporeal circulation with the postoperative response of glycoprotein IIb/IIIa, glycoprotein Ib, and P-selectin to in vitro activation was found. Postoperative P-selectin and glycoprotein Ib expression stimulated with ADP correlated to blood loss. Heparin in vitro significantly reduced glycoprotein Ib expression. Heparin, as well as the duration of extracorporeal circulation, independently correlated to phenotypic changes of platelets following extracorporeal circulation. The significant correlation of these variables to postoperative blood loss demonstrates their relevance to platelet function in vivo.

Optimization of three‐ and four‐color multiparameter DNA analysis in lymphoma specimens

Simultaneous analysis of DNA and immunophenotype of lymphoma cells by flow cytometry allows the calculation of the proliferative activity and aneuploidy in even a small lymphoma population. Unfavorable DNA binding characteristics or spectral features of DNA dyes impair the accuracy of multiparameter DNA analysis and limit their clinical application. We describe here a reliable and reproducible application of both three‐ and four‐color multiparameter DNA analysis.

MK-383 (tirofiban) induces a GPIIb/IIIa receptor conformation which differs from the resting and activated receptor.

The platelet integrin αIIb β3 (GPIIb/IIIa) acts as a receptor for fibrinogen, playing a critical role in platelet aggregation. GPIIb/IIIa antagonists, which block the receptor-ligand interaction, have been accused of causing occasional thrombocytopenia, probably via drug-induced platelet activation or immunogenic neoepitopes. We, therefore, analyzed the effects of the GPIIb/IIIa antagonist MK-383 (tirofiban) on platelet activation and GpIIb/IIIa conformation. At a concentration of 10-7 mol/l, MK-383 completely inhibited fibrinogen binding to in vitro stimulated platelets. Simultaneously, the GPIIb/IIIa expression density increased, similar to that on activated platelets, but no effect on P-selectin expression or the formation of platelet-leukocyte aggregates could be observed, indicating that MK-383 binding did not induce general platelet activation. The GPIIb/IIIa receptor conformation was further analyzed by fluorescence resonance energy transfer analysis between fluorochrome-labeled antibodies against different GpIIb/IIIa epitopes. As a result, MK-383 induced a receptor conformation that differed from the resting as well as the activated receptor as induced by ADP or TRAP-6. This conformational modulation of GPIIb/IIIa presents an interesting mechanism which may be linked to receptor recruitment without inducing general platelet activation.