Bilirubin adsorption property of sol-gel-derived titania particles for blood purification therapy

Titania (anatase) gel powders were prepared by peptizing commercially available titania sols and heating them at temperatures up to 700 degrees C, as candidates for bilirubin adsorbents for blood purification therapy. Those titania particles were in contact with a protein solution containing bilirubin and bovine serum albumin that mimics the blood of bilirubinemia patients. The amount of free or direct bilirubin in the solution insignificant. Indirect bilirubin or a bilirubin complex with albumin was adsorbed on the anatase powders, the primary particle size of which was as large as or larger than the size of an albumin molecule. The surface charge and surface charge density were only minor factors in controlling the indirect bilirubin adsorption. The present results indicated that the size of primary particles and hydrophobicity were significant for the sol-derived anatase in terms of bilirubin adsorption, and both were controllable by the heating temperature and the time period.     

Preferential adsorption of high density lipoprotein from blood plasma onto biomaterial surfaces

In the present study a two step enzyme immuno assay (EIA) was used for the investigation of the adsorption of proteins and lipoproteins from solutions and from blood plasma onto polymer surfaces. It was found that only a small adsorption of the major blood proteins occurred from plasma Evidence is presented that the reason for this adsorption behaviour is a preferential adsorption of high density lipoprotein (HDL).     

内源性高甘油三酯血症患者血浆VLDL、LDL及HDL对血凝的影响

目的：研究内源性高甘油三酯血症(HTG)患血浆极低密度脂蛋白(VLDL)、低密度脂蛋白(LDL)及高密度脂蛋白(HDL)是否发生了氧化修饰及其对血凝的影响。方法：对2l例内源性高甘油三酯血症患与2l例年龄性别相近的正常人的血脂、脂质过氧化物进行了分析。用一次性密度梯度超速离心法分离血浆VLDL、LDL及HDL，测定这三种脂蛋白的234nm光吸收、相对电泳迁移率(REM)和硫代巴比妥酸反应物质(TBARS)，分别将这三种脂蛋白加入由正常人新鲜混合血浆构成的反应系统中，按试剂盒分别测定凝血酶原时间(PT)及活化部分凝血酶原时间(APIT)。结果：内源性HTG患血浆TG含量平均升高2．73倍，HDLC下降l.7l倍，同时LPO升高1．22倍;HTG组VLDL、LDL及HDL的REM、234nm光吸收值、TBARS含量均较对照组显增加(P＜0．01)，表明内源性HTG患血浆VLDL、LDL及LDL均发生了氧化修饰生成Ox—VLDL、Ox-LDL.PT及APTT在分别加入HTG组的VLDL、LDL及HDL后均比加入相应正常组脂蛋白明显缩短(P均＜0．05)。相关分析表明，HTG组血浆VLDL及HDL相对电泳迁移率(REM)与PT呈负相关(P＜0．01)。结论：HTG患血浆VLDL、LDL及HDL发生了氧化修饰，并使PT及APTT明显缩短。     

Low density lipoprotein (LDL) heterogeneity and atherosclerosis

LDL particles constitute the predominant form of transport of cholesterol towards tissues, and a major risk factor for atheroma. The pathogenesis of atherosclerosis is, as yet, far from completely elucidated and, indeed, within the LDL particles themselves, there is a structural heterogeneity. Approximately fifteen subfractions have been isolated so far, presenting differing profiles in normal and hyperlipidemic subjects. Some of these subgroups seem to be linked with an atherogenic potential, this is the case for the smaller and the more dense of the particles. LDL particles are normally cleared from the blood stream via the BE receptor, whose synthesis is regulated by the intracellular content of cholesterol. The particules must however undergo several, in vivo and in vitro, transformations which modify their metabolism and are all the more important when their half life is prolonged. These transformations, and in particular oxydation, lead, via the macrophage pathway, to the accumulation of cholesterol, the creation of foam cells, and ultimately to the formation of the lipid streak. Antioxydants therefore open the way to new therapeutic pathways by acting in synergy with cholesterol lowering agents.     

Low density lipoprotein receptor as a candidate receptor for hepatitis C virus

Hepatitis C virus (HCV) binds to different human cell lines in vitro. However, the efficiency of adsorption is very low due mainly to a relatively small fraction of the virus being able to bind to these cells. Free low density lipoprotein (LDL > 200 microg/ml) is able to block the attachment of HCV to human fibroblasts in vitro completely. COS-7 cells being primarily not able to bind HCV were transfected with a vector containing the entire coding sequence of the human LDL-receptor (LDLR). HCV was now bound to these cells. We propose that HCV and LDL are competitive for the cellular LDLR and that LDL in sera of patients may regulate the binding of HCV to this target.     

HB-H-8树脂吸附低密度脂蛋白的体外实验研究

目的 目前,用于降低血液中低密度脂蛋白(LDL)的吸附分离材料尚存在需要改进的地方.HB-H-8树脂在吸附LDL方面尚无应用,本研究对HB-H-8树脂的吸附性能进行了初步评价.方法 首先实验室制备HB-H-8树脂,经过处理后用其对LDL高于正常范围的患者血清标本进行体外动态和静态吸附实验.结果 体外动态吸附结果表明,HB-H-8树脂对LDL的吸附饱和时间为2h,最适吸附温度为恒温37℃,体外静态吸附结果显示HB-H-8树脂对LDL平均吸附率为63.2％,而对高密度脂蛋白(HDL)的平均吸附率为1.9％.结论 HB-H-8树脂对LDL具有良好的特异性吸附性能,有望开发为一种低密度脂蛋白的血液净化医用吸附材料.     

Low density lipoprotein receptors in cultured skin fibroblasts from psoriasis patients

Low density lipoprotein (LDL) receptor activity, measured as 125I-LDL association and degradation at 37 degrees C, was determined in cultured fibroblasts from involved as well as uninvolved skin obtained from 20 psoriasis patients. The same analyses were conducted in fibroblasts from two reference groups consisting of 19 heterozygotes for familial hypercholesterolemia and 16 normal subjects, respectively. Psoriasis patients had significantly lower LDL receptor activity than normals, and it was comparable to that of the heterozygotes for familial hypercholesterolemia. The reduced LDL receptor activity was not accompanied by an increase in total serum cholesterol. The psoriasis patients had a significant reduction in apo-B concentration, but did not differ from the normals in the other serum lipid or lipoprotein parameters. There was no difference in LDL receptor activity between involved and uninvolved skin from psoriasis patients. These results suggest that there is an abnormal cell membrane in dermal fibroblasts from psoriasis patients. Since their total serum cholesterol is normal, their low LDL receptor activity may be confined to dermal cells, leaving the hepatic lipid metabolism normal. The pathogenetic significance of this finding is unknown.     

Low density lipoprotein receptor expression and function in human polymorphonuclear leucocytes

Low density lipoprotein receptors (LDLR), capable of internalizing LDL, are expressed in polymorphonuclear neutrophils (PMN). The expression was assessed using anti-LDLR antibody by flow cytometry. The internalization of LDL was assessed by: (i) quantification of the uptake of labelled LDL with 1,1′-dioctadecyl-3,3,3′,3′ tetramethyl-indocarboxycyanine perchlorate (DiI) by flow cytometry; and (ii) the binding of LDL-¹²⁵I. In fresh purified cells, Lineweaver–Burk analysis of LDL binding (LDL-DiI) revealed that the calculated Kd (internalized LDL) for PMN (15.0 × 10⁻⁹ m) is lower than the Kd for monocytes (1.1 × 10⁻⁷ m) and the Kd for lymphocytes (3.2 × 10⁻⁷ m). Scatchard analysis (LDL-¹²⁵I) revealed 25 000 binding sites and a Kd of 9.6 × 10⁻⁹ m for PMN. The interaction of LDL with its receptor caused a two-fold fast (peak at 1 min) and transient increase in the oxidative burst, measured by the formation of 2′,7′ dicholoflurescein (DCF) by flow cytometry. This effect was not observed in monocytes or lymphocytes, and it was blocked by anti-LDLR antibody. The stimulation of LDL was optimal at 10 μg of protein/ml. LDL was able to suppress DCF formation induced by phorbol myristate acetate (PMA) and PMA was unable to further stimulate LDL-treated cells, suggesting protein kinase-C (PKC) involvement in LDL effects. Using a PKC assay, LDL was shown to induce a two-fold increase in PKC translocation to the membrane. Thus, LDL increases PMN oxidative burst through a PKC-dependent pathway.     

Sol-gel derived aluminosilicate coatings on alumina as substrate for osteoblasts

Rat bone marrow stromal cell differentiation on aluminosilicate 3Al₂O₃–2SiO₂ coatings was investigated. Thin ceramic coatings were prepared on -alumina substrates by the sol–gel process and calcined in order to establish an amorphous aluminosilicate ceramic phase with and without nanosized transitional mullite crystals. In addition, coatings of thermally sprayed aluminosilicate and diphasic γ-alumina–silica nanosized colloids were prepared. Cell culture testing by rat osteoblasts showed good biocompatibility for aluminosilicates with sustained normal osteoblast functions. Despite mutual disparities in physical and chemical nanostructures, the culture findings suggested fairly similar osteoblast response to all tested coatings. The results suggest that topographical frequency parameters and chemical uniformity are important parameters in determining the best conditions for osteoblasts on sol–gel derived aluminosilicate materials.     

CRP促进THP-1来源的巨噬细胞血凝素样氧化型低密度脂蛋白受体-1的表达

目的探讨C反应蛋白(CRP)对单核细胞株(THP-1)来源的巨噬细胞血凝素样氧化型低密度脂蛋白受体-1(LOX-1)蛋白和mRNA的表达及相关信号转导通路的影响。方法用佛波醇脂诱导THP-1分化为巨噬细胞。用不同浓度CRP在体外干预,分别采用RT-PCR和细胞酶联免疫测定干预后巨噬细胞LOX-1抗原蛋白和mRNA的表达。同时观察当NF-κB、AP-1和MARK信号通路抑制剂存在时,CRP对LOX-1抗原和mRNA表达的影响。结果CRP促进巨噬细胞LOX-1蛋白和mRNA表达增加。NF-κB的抑制剂BAY11-7085能抑制CRP对LOX-1蛋白和mRNA表达的诱导作用。结论CRP能在转录及转录后水平诱导THP-1来源的巨噬细胞表达LOX-1,这种调节作用可能是通过NF-κB信号传导通路实现的。     

Plasma filtration adsorption dialysis (PFAD): a new technology for blood purification

Severe sepsis is one of the most significant challenges in critical care. Despite all the developments achieved in infectious diseases and critical care, along with numerous attempts to develop treatments, the mortality rate of severe sepsis and septic shock remains unacceptably high. The pathophysiology of severe sepsis and septic shock is only partially understood. Circulating pro- inflammatory and anti-inflammatory mediators appear to participate in the complex cascade of events which leads to deranged microcirculatory function, as we know from the peak concentration hypothesis. Therapeutic trials targeting single pro-inflammatory and anti-inflammatory mediators failed to demonstrate any benefit, suggesting that the unselective removal of different mediators may be a more appropriate approach. In severe sepsis several blood purification techniques, such as continuous hemofiltration (CVVH), high volume hemofiltration (HVHF), pulse high volume hemofiltration (HVHF), plasma filtration, plasma dsorption, coupled plasma filtration adsorption (CPFA), have been proposed but such techniques appear to have both theorical as well as practical limitations. Plasma Filtration Adsorption Dialysis (PFAD) is a new extracorporeal treatment which combines different principles of blood purification in a single device. The core of this technique is a new dialyzer composed by three suitable compartments that provide specific functions. The association of multiple principles permits specific removal of molecules implicated in the pathophysiology of patient's disease and re-establishment of hydro-electrolyte, acid-base equilibrium, if renal dysfunction-failure is present. The final target of PFAD is to obtain complete purification by combining principles of physics and chemistry to remove hydrophilic and hydrophobic molecules with a very wide range of weights.     

Low density lipoprotein receptor activity in human leukemic cells--relation to chromosome aberrations

Chromosome analysis and low density lipoprotein (LDL) receptor activity of leukemic cells from 38 patients with acute non-lymphocytic leukemia were correlated. Clonal chromosome aberrations were found in 22 patients, and an extra chromosome 8 was found in 7 of them. LDL receptor activity was significantly higher in patients with an extra chromosome 8 than in patients with other abnormalities or a normal karyotype. Chromosome 8 may harbor genes of importance for the expression of the LDL receptor.     

Cellular glycosaminoglycans and low density lipoprotein receptor are involved in hepatitis C virus adsorption

The initial binding of Hepatitis C virus (HCV) to the cell membrane is a critical determinant of pathogenesis. Two putative HCV receptors have been identified, CD81 and low-density lipoprotein receptor (LDLr). CD81 interacts in vitro with the HCV E2 envelope glycoprotein, and LDLr interacts with HCV present in human plasma. In order to characterize these potential receptors for HCV, virus from plasma, able to replicate in cell culture, was inoculated on Vero cells or human hepatocarcinoma cells. HCV adsorption was assessed by quantitating cell-associated viral RNA by a real-time RT-PCR method. Anti-LDLr antibody, low and very low density lipoproteins inhibited significantly HCV adsorption, confirming the role of LDLr as HCV receptor. Only one out of the two anti-CD81 antibodies used in this study led to a partial inhibition of HCV binding. This study also highlights a role for glycosaminoglycans (GAGs) in HCV adsorption: treatment of virus with heparin led to 70% inhibition of attachment, as did desulfation of cellular GAGs. Treatment of Vero cells with heparin-lyase significantly inhibited virus attachment but by only 30%. These results demonstrate the complexity of the HCV binding step in which LDLr interacts strongly with HCV, whereas the interaction of HCV with GAGs and particularly with CD81 seem to be more moderate.     

Low density lipoprotein inactivates Vibrio vulnificus cytolysin through the oligomerization of toxin monomer

Blood lipoprotein has been shown to be an important defense factor against the bacterial infection. Lipoprotein is scavenger of bacterial endotoxin (lipopolysaccharide, LPS). However, there is little evidence showing its protective action against the bacterial exotoxin. We have previously demonstrated that cholesterol inactivates Vibrio vulnificus cytolysin (VVC) through oligomerization of the toxin monomer. The aim of the present study was to investigate the relationship between VVC and low-density lipoprotein cholesterol (LDL-C). LDL induced the oligomerization of VVC in a dose-dependent manner. The oligomerization of VVC monomer with LDL was demonstrated by immunoblotting, which exhibited 200-kDa bands corresponding to a tetramer of the native VVC of relative molecular weight of 51,000. Moreover, LDL inactivated hemolytic activity of VVC in a dose-dependent manner, and this response was not changed by the modifications of LDL (heat denaturation of proteins and peroxidation of phospholipids), suggesting that LDL-C is associated with the defense action against VVC. These results suggest that LDL-C inactivates VVC through the induction of toxin oligomerization.The first two authors contributed equally to this work     

Low density lipoprotein promotes human naive T cell differentiation to Th1 cells

Oxidized LDL (oxLDL) in the arterial wall and its incorporation into foam cells leads to inflammation and nucleation of atherosclerotic plaque; this is opposed by HDL. OxLDL and HDL regulate activation of macrophages and endothelial cells, and study of T cell participation has been limited to mature, differentiated cells such as Th1 cells, which perpetuate atherogenesis by promoting cell-mediated responses and inflammation. Immature naïve T cells, just emerged from the thymus, have remained largely unstudied. We hypothesized that LDL and HDL provide selective modulation of immature naïve T cell differentiation and participation in plaque development. In our in vitro model, naïve cells become activated and differentiate to mature effector T cells that are Th1, Th2 or Treg cells. Addition of oxLDL favored differentiation to Th1 cells, reduced Th2 cell activity and prolonged cell survival. In contrast, HDL inhibited T cell proliferation and reduced cell survival. The data suggest a novel mechanism where oxLDL enhances differentiation of human naïve CD4+ T cells to Th1 cells capable of promoting inflammation and plaque progression, and this is opposed by HDL.     

Low density lipoprotein receptor activity in cultured skin fibroblasts from octa- and nonagenarians

Low density lipoprotein (LDL) receptor activity determined as association or degradation of 125I-LDL at 37 degrees C, was measured in cultured skin fibroblasts from 48 elderly, independent subjects (84-95 years old). Data concerning these subjects were compared with those from a reference group of 27 younger, healthy subjects (5-64 years old) and 23 heterozygotes for familial hypercholesterolemia (9-67 years old). The median (10th-90th percentile) association and degradation value for the older subjects were 294 (172-535) ng 125I-LDL/mg protein/6h and 191 (99-376) ng 125I-LDL/mg protein/6h, respectively. These values did not differ significantly from the corresponding values in the normal subjects (296 (60-491) ng 125I-LDL/mg protein/6h and 171 (99-275) ng 125I-LDL/mg protein/6h, respectively). Thus, maximal LDL receptor activity in fibroblasts from the older subjects seems to be comparable to that of younger, healthy controls. However, the elderly subjects had markedly higher values for total serum cholesterol and LDL cholesterol than the normal controls. There was a significant increase in LDL receptor activity with age in the normal controls. Such an increase was not found in heterozygotes for familial hypercholesterolemia.     

Direct cytosolic siRNA delivery by reconstituted high density lipoprotein for target-specific therapy of tumor angiogenesis

We described here the mechanisms by which small interfering RNA (siRNA) molecules incorporated in reconstituted high density lipoprotein (rHDL) were efficiently transferred into the cytoplasm of cells to perform target-specific therapy of tumor angiogenesis. Using fluorescent-tagged apolipoprotein A-I (apoA-I) and cholesterol-conjugated siRNA (Chol-siRNA), it was confirmed with FACS and confocal microscopic measurements that Chol-siRNA-loaded rHDL nanoparticles (rHDL/Chol-siRNA complexes) were successfully established and apoA-I certainly was attached to the surface of Chol-siRNA-loaded lipoplexes (Lipos/Chol-siRNA complexes). Stably assembled rHDL/Chol-siRNA complexes demonstrated proper nanosize, quasi-spherical shape and improved nuclease protection over naked Chol-siRNA. It was also interesting to note that rHDL provided a highly effective approach to transfer Chol-siRNA across the membrane directly into the cytoplasm via the scavenger receptor BI (SR-BI)-mediated non-endocytotic mechanism, thereby bypassing endo-lysosomal trapping. We also showed clear evidence that the in vitro implementation of rHDL for Chol-siRNA-VEGF (Chol-siRNA targeting vascular endothelial growth factor gene) delivery markedly promoted RNA interference (RNAi)-mediated degradation of VEGF mRNA, resulting in down-regulation of secreted VEGF protein. In vivo fluorescence imaging indicated that near-infrared (NIR) dye Cy5 labeled Chol-siRNA-loaded rHDL nanoparticles (rHDL/Cy5-Chol-siRNA complexes) displayed long circulation time, SR-BI positive tumor-selective targeting, and efficient cytosolic delivery capabilities. Furthermore, intravenous administration of Chol-siRNA-VEGF-loaded rHDL nanoparticles (rHDL/Chol-siRNA-VEGF complexes) significantly enhanced anti-tumor efficacy against breast cancer, decreased VEGF expression level, and inhibited formation of intratumoral microvessels at the tumor tissue. It was concluded that rHDL possessed therapeutic potential and versatility in mediating Chol-siRNA-VEGF direct cytosolic delivery for target-specific anti-angiogenic therapy in breast cancer.     

High density lipoprotein uptake by freshly isolated human peripheral blood T lymphocytes

A high density lipoprotein (HDL) receptor/binding site has been identified on peripheral blood lymphocytes, and some of its properties were compared with those of HDL receptors on other cell types. Binding studies were performed using fluorescent (dioctadecylindocarbocyanine)-labelled HDL (DiI-HDL) and analyzed by fluorescence microscopy and quantitative flow cytometry. Uptake of low levels of DiI-HDL during a 2 h incubation at 37 degrees C was a property of all lymphocytes, i.e. not of one particular subset only. Visual inspection of these cells in the fluorescence microscope revealed both membrane and cytoplasmic fluorescence, indicating that DiI-HDL become internalized during the 2 h incubation; internalization appeared to be a receptor-mediated process. In competitive binding studies, apo E-free HDL competed effectively for DiI-HDL binding, whereas LDL competed very weakly. Two features of DiI-HDL uptake are demonstrated which are unique to lymphocytes: 1) it was enhanced 3-6-fold by inclusion of EDTA in the incubation medium or by incubating in Ca2+/Mg2+ free medium, and 2) it was saturable at 37 degrees C.     

A novel approach for blood purification: mixed-matrix membranes combining diffusion and adsorption in one step

Hemodialysis is a commonly used blood purification technique in patients requiring kidney replacement therapy. Sorbents could increase uremic retention solute removal efficiency but, because of poor biocompatibility, their use is often limited to the treatment of patients with acute poisoning. This paper proposes a novel membrane concept for combining diffusion and adsorption of uremic retention solutes in one step: the so-called mixed-matrix membrane (MMM). In this concept, adsorptive particles are incorporated in a macro-porous membrane layer whereas an extra particle-free membrane layer is introduced on the blood-contacting side of the membrane to improve hemocompatibility and prevent particle release. These dual-layer mixed-matrix membranes have high clean-water permeance and high creatinine adsorption from creatinine model solutions. In human plasma, the removal of creatinine and of the protein-bound solute para-aminohippuric acid (PAH) by single and dual-layer membranes is in agreement with the removal achieved by the activated carbon particles alone, showing that under these experimental conditions the accessibility of the particles in the MMM is excellent. This study proves that the combination of diffusion and adsorption in a single step is possible and paves the way for the development of more efficient blood purification devices, excellently combining the advantages of both techniques.