Haloperidol disrupts lipid rafts and impairs insulin signaling in SH-SY5Y cells

Haloperidol exerts its therapeutic effects basically by acting on dopamine receptors. We previously reported that haloperidol inhibits cholesterol biosynthesis in cultured cells. In the present work we investigated its effects on lipid-raft composition and functionality. In both neuroblastoma SH-SY5Y and promyelocytic HL-60 human cell lines, haloperidol inhibited cholesterol biosynthesis resulting in a decrease of the cell cholesterol content and the accumulation of different sterol intermediates (7-dehydrocholesterol, zymostenol and cholesta-8,14-dien-3β-ol) depending on the dose of the drug. As a consequence, the cholesterol content in lipid rafts was greatly reduced, and several pre-cholesterol sterols, particularly cholesta-8,14-dien-3β-ol, were incorporated into the cell membrane. This was accompanied by the disruption of lipid rafts, with redistribution of flotillin-1 and Fyn and the impairment of insulin-Akt signaling. Supplementing the medium with free cholesterol abrogated the effects of haloperidol on lipid-raft composition and functionality. LDL (low-density lipoprotein), a physiological vehicle of cholesterol in plasma, was much less effective in preventing the effects of haloperidol, which is attributed to the drug's inhibition of intracellular vesicular trafficking. These effects on cellular cholesterol homeostasis that ultimately result in the alteration of lipid-raft-dependent insulin signaling action may underlie some of the metabolic effects of this widely used antipsychotic.     

CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells

We investigated the membrane localization of CD95 in type I and type II cells, which differ in their ability to recruit and activate caspase-8. We found that CD95 was preferentially located in lipid rafts of type I cells, while it was present both in raft and non-raft plasma membrane sub-domains of type II cells. After stimulation, CD95 located in phospholipid-rich plasma membrane was recruited to lipid rafts in both types of cells. Similarly, CD95 cross-linking resulted in caspase-independent translocation of FADD/MORT1 and caspase-8 to the lipid rafts, which was prevented by a death domain-defective receptor. CD95 internalization was then rapid in type I and delayed in type II cells and showed a substantial correlation with the kinetics of Fas-associated death domain (FADD)and caspase-8 recruitment to lipid rafts. Finally, electron microscopy analysis showed that after CD95 stimulation lipid rafts aggregated in large clusters that were internalized in endosomal vesicles, where caspase-8 underwent massive processing. Taken together, our data demonstrate that CD95 death-inducing signaling complex formation and internalization in type I and type II cells occur in lipid rafts, which are a major site of caspase-8 activation.     

Integrin-associated proteins as potential therapeutic targets

Summary: Integrins are adhesion receptors important for hematopoiesis, leukocyte trafficking, and formation of immunological synapses; hence, they may provide targets for therapeutic intervention in leukocyte-driven pathologies. Blocking integrin–ligand binding is one strategy for inhibiting integrins; however, a complete loss of integrin function can lead to mechanism-based toxicities. Because integrin α and β subunits interact with a variety of other proteins to receive and transmit cellular signals, targeting these integrin-associated proteins may utilize alternative sites for intervention that lead to therapies with fewer side effects. This review summarizes integrin-associated proteins in leukocytes and focuses on four of these proteins with perceived therapeutic potential. Specific mutations in the α4 integrin cytoplasmic tail block or enforce binding to paxillin and thus modulate integrin signaling required for efficient cell migration. Similarly, the association of RAPL(NORE1B) with β2 integrins may participate in adhesive and migratory events in leukocytes. The β integrin cytoplasmic tail-binding protein talin is critical for increasing the affinity of integrins (activation), and blockade of talin binding can prevent leukocyte arrest on the endothelium. Finally, the membrane protein CD98 mediates β1 and β3 integrin signaling and may be involved in leukocyte functions. Identification of biologically important interactions of integrins and signaling proteins can thus pave the way to new strategies for manipulating leukocyte functions.     

Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis

Rituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic B-cell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca(2+) levels and downstream apoptotic signalling.     

Effects of TREM-1 activation in human neutrophils: activation of signaling pathways, recruitment into lipid rafts and association with TLR4

Neutrophilic polymorphonuclears (PMNs) play an important role in the progression of sepsis-related inflammation and become highly activated by a wide array of ligands on the site. The triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently described receptor that has many effects on human PMN. The engagement of TREM-1 on PMN can induce phagocytosis, reactive oxygen species production and release of myeloperoxidase and IL-8. LPS has a priming effect on these functions. We show in this paper that Lyn, AKT, extracellular signal-regulated kinase 1/2 and Jak2 signaling pathways are elicited following TREM-1 engagement and activation by a monoclonal agonist antibody (anti-TREM-1) in human PMN, leading to the phosphorylation of STAT5 and RelA, a subunit of the nuclear factor-kappa B family. We also show that TREM-1 is recruited to ganglioside M1-lipid rafts in PMN upon stimulation with LPS or anti-TREM-1. Moreover, we observed that Toll-like receptor 4 and TREM-1 co-localize upon stimulation and TREM-1 engagement resulted in the phosphorylation of IL-1R-associated kinase 1, but not its stimulant-induced degradation. These data shed a new light on how various receptors implicated in the innate immune response could interact to insure an efficient inflammatory response upon pathogens-associated aggression.     

Expression of GM1, a marker of lipid rafts, defines two subsets of human monocytes with differential endocytic capacity and lipopolysaccharide responsiveness

Monocytes constitute 5-10% of total human peripheral blood leucocytes and remain in circulation for several days before replenishing the tissue macrophage populations. Monocytes display heterogeneity in size, granularity and nuclear morphology, and in the expression of cell membrane molecules, such as CD14, CD16, CD32, CD64, major histocompatibility complex class II, CCR2, CCR5, among others. This has led to the suggestion that individual monocyte/macrophage populations have specialized functions within their microenvironments. This study provides evidence for the occurrence of two peripheral blood monocyte subpopulations on the basis of their differential expression of GM1, a sphingolipid found mostly in lipid rafts, a CD14(+) GM1(low) population and a CD14(+) GM1(high) population comprising about 97.5% and 2.5% of total CD14(+) cells, respectively. GM1 expression correlates with functional differences in terms of endocytic activity, susceptibility to mycobacterial infection, and response to lipopolysaccharide (LPS) (modulation of Toll-like receptor-4 expression). CD14(+) GM1(low) cells proved to be less endocytic and more responsive to LPS, whereas CD14(+) GM1(high) cells are more endocytic and less responsive to LPS. In addition, during monocyte to macrophage differentiation in vitro, the percentage of CD14(+) GM1(high) cells increases from about 2.5% at day 1 to more than 50% at day 7 of culture. These results suggest that GM1(low) and GM1(high) monocytes in peripheral blood, represent either different stages of maturation or different subsets with specialized activities. The expression of CD16 on GM1(high) favours the first possibility and, on the other hand that up-regulation of GM1 expression and probably lipid rafts function is involved in the monocyte to macrophage differentiation process.     

The functional roles of lipid rafts in T cell activation, immune diseases and HIV infection and prevention

The first appearance of lipid rafts, or lipid rafts-like structure, was occasionally observed by cryo-electronic microscopy in 1980s as cavity, such as caveolae. However, the fully understanding of lipid raft was attributed by the studies of T cell activation, virus entry/budding, and other membrane events. During the interaction of T cell and antigen presenting cell, a highly organized structure is formed at the interface of the two cells, where cholesterol and sphingolipids are enriched, and form a liquid ordered phase that facilitates the signaling proteins on and off. Lipid rafts are also involved in virus entry and assembly. In this review, we will discuss cholesterolsphingolipid floating microdomain, the lipid raft as a unique compartment of the plasma membrane, with biological functions that ensure correct intracellular traffic of proteins and lipids, such as protein-protein interactions by concentrating certain proteins in these microdomains, while excluding others. We also discuss the disruption of lipid rafts is related to different diseases and aging, and we especially exploit the lipid rafts as pharmaceutical targets for anti-virus and anti-inflammation, particularly a new approach to control HIV infection for AIDS prevention and protection by inhibition or disruption of lipid rafts. Cellular ＆ Molecular Immunology.     

Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 (ω − 2, where ω is the GPI moiety at E160), P159 (ω − 1), and M162 (ω + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.     

Serum lipid profile as a predictor of dengue severity: A systematic review and meta‐analysis

Dengue virus is known to modulate host cell lipid metabolism in order to promote efficient viral replication. Recent studies have focused on circulating lipids as potential biomarkers of dengue severity; however, the results obtained so far lack the consistency to establish a definite relationship between the two. Therefore, in the present study, we investigated serum lipids as potential biomarkers of dengue severity by conducting a meta‐analysis of the currently available clinical data. Nine studies that evaluated 1,953 patients were included in the review, many of which were cross‐sectional (44.4%) and conducted in Asian countries (55.5%). These studies observed the presence of lipids in serum samples (77%) of patients in the acute phase of the disease (88.8%). Circulating total‐cholesterol (P = .001) and LDL (P = .001) levels, but not HDL (P = .07), VLDL (P = .9) and triglyceride (P = .57) levels, were inversely and significantly correlated with dengue severity. Total cholesterol (P ≤ .001) and LDL (P = .001) were also useful in determining the risk of hypovolemic shock in patients with severe dengue. Subgroup analysis showed that factors, such as design (cross‐sectional vs cohort), racial‐ethnic differences (Asian vs Latin Americans), and age range (children vs adult) influenced the correlation and also contributed to the high level of heterogeneity in the studies. Our meta‐analysis demonstrates that total‐cholesterol and LDL‐cholesterol levels should be explored as routine laboratory markers for dengue severity, as they will help in employing an appropriate patient therapy, and thus optimize the use of available resources.     

Siglecs as potential targets of therapy in human mast cell- and/or eosinophil-associated diseases

Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of vertebrate glycan-binding cell-surface proteins. The majority mediate cellular inhibitory activity once engaged by specific ligands or ligand-mimicking molecules. As a result, Siglec engagement is now of interest as a strategy to therapeutically dampen unwanted cellular responses. When considering allergic inflammation, human eosinophils and mast cells express overlapping but distinct patterns of Siglecs. For example, Siglec-6 is selectively and prominently expressed on mast cells while Siglec-8 is highly specific for both eosinophils and mast cells. This review will focus on a subset of Siglecs and their various endogenous or synthetic sialoside ligands that regulate eosinophil and mast cell function and survival. It will also summarize how certain Siglecs have become the focus of novel therapies for allergic and other eosinophil- and mast cell-related diseases.     

The role of tyrosine kinases in systemic lupus erythematosus and their potential as therapeutic targets

The autoimmune disease systemic lupus erythematosus is characterized by loss of tolerance to nuclear antigens. Breakdown of tolerance is associated with alterations in T-cell and B-cell receptor signal transduction, including increased protein phosphorylation that may underlie pathogenesis and explain the characteristic hyperactivity of T and B cells and other immune cells in active disease. Tyrosine kinases play a central role in signaling processes in cells known to be important in the pathogenesis of autoimmune diseases. Considerable progress has been made in understanding the function of tyrosine kinases in immune cell signaling pathways. In this review, we will summarize the function of tyrosine kinases and their novel inhibitors from studies made in animal lupus models and systemic lupus erythematosus patients.     

Effects of beta-bungarotoxin and tityustoxin on accumulation of putative amino acid neurotransmitters by rat cortex synaptosomes

¹⁴C-labelled glutamate and γ-aminobutyrate, preloaded into purified synaptosomes from rat cortex, were released by β-bungarotoxin and depolarization with 55 mM K⁺ or tityustoxin; in all cases the induced release was higher in the presence of Ca²⁺. Thus, these neurotoxins exert similar effects in vitro on central synapses, which release neuroactive amino acids, to those previously observed in the peripheral nervous system. Accumulation of these two putative transmitters by synaptosomes was de creased by either toxin, probably due to their disruption of the membrane potential. β-Bungarotoxin, a homogeneous protein which exhibits phospholipase activity in the presence of deoxycholate and Ca²⁺, but not Sr²⁺, augmented the respiration rate of synaptic mitochondria. Although K⁺-stimulated release was observed in the absence of Ca^{2 +} and the presence of Sr²⁺, the action of the toxin on the release of amino acids and lactate dehydrogenase was diminished.These results, therefore, support the view that phospholipase activity of β-bungarotoxin may play an important part in increasing the release of transmitters observed both in vivo and in vitro. The effects of tityustoxin on both the accumulation and release of the putative transmitters were abolished by tetrodo toxin; K⁺-induced depolarisation greatly decreased its potentiating action on release, possibly suggesting that the binding of tityustoxin, like other similar scorpion toxins, is dependent on membrane potential.     

Apolipoproteins B and E, and angiotensin I-converting enzyme (ACE) genetic polymorphisms in Italian women with coronary artery disease (CAD) and their relationships with plasma lipid and apolipoprotein levels

The Xba I, Eco RI and the signal peptide insertion/deletion ( I/D ) polymorphic sites of APOB gene, the Cfo I polymorphic site of apolipoprotein E gene (APOE), and the insertion/deletion polymorphism of angiotensin I-converting enzyme (ACE) gene were studied using polymerase chain reaction (PCR) in 55 postmenopausal women with coronary artery disease (CAD) and in 119 control women of equivalent age. Patients and controls were recruited from the population of Rome, considered representative of Central and Southern Italy. There were no significant differences in allele frequencies between the two groups, though APOB X-, R- and I, APOE*3 , and ACE D alleles were slightly more frequent in the cases than in the controls. The patients did not differ from the controls for plasma total cholesterol (TC), HDL-cholesterol, LDL-cholesterol, and apoAI values, while they presented significantly higher levels of triglycerides and apoB, and lower apoE levels. TC, apoE, and apoB quantitative values, adjusted for age, varied significantly among APOB Xba I and APOE genotypes. APOB X-X-genotype was associated in patients with a significantly lower mean TC concentration than the other two genotypes pooled together. APOE 3–2 genotype in the controls had significantly lower TC levels with respect to the other two pooled genotypic classes and higher apoE levels compared to 3–3 and 4–3 genotypes. In the patients, 3–2 genotype had significantly lower apoB levels than the pooled 3–3 and 4–3 class. We conclude that in the Italian women the DNA polymorphisms studied in this work do not seem to be important risk factors for CAD occurrence; that apoE quantitation could be another useful parameter to identify subjects at risk of CAD; and that APOB X -and APOE*2 are the alleles that most influence the interindividual plasma lipid variation among CAD female patients.     

T-cell surface molecules involved in the induction and expression of lymphokine-activated killing of autologous and allogeneic tumor targets

The cellular interactions and the surface molecules involved in the generation and the expression of lymphokine-activated killer-cell (LAK) activitiesin vitro in blood mononuclear cells (BMN) from cancer patients and healthy individuals against autologous and allogeneic tumors were studied. The depletion of a plastic-adherent population(s) from BMN at the initiation ofin vitro cultures in recombinant interleukin-2 (rIL-2) markedly interfered with the generation of LAK activities. Readdition of the same number of irradiated autologous plastic-adherent cells to the nonadherent population restored the generation of LAK. The requirement of the plasticadherent population(s) inin vitro induction of LAK activities was observed only in autologous situations. Furthermore, selective modulations of CD3 and CD2 receptors on BMN with the appropriate monoclonal antibodies (MAb) during the induction phases of LAK responses profoundly inhibited the generation of LAK. Thus, unhindered expressions of CD2 molecules and CD3 molecules were necessary for the maximum cytotoxic activation of non-antigen-driven effector cells in short-term cultures in rIL-2.     

Isolation of two CD50 (ICAM-3)-negative Jurkat T-cell clones and their application for analysis of CD50 function

Abstract: The leukocyte differentiation antigen CD50 (intercellular adhesion molecule-3, ICAM-3), mediates cell-cell adhesion through its ligand LFA-1 and is a transducting receptor molecule during T-cell activation. Since CD50 homologues in other species have not yet been identified, the role of this molecule can only be analyzed in human cell models. Thus, to better study CD50 function in T cells, we have obtained two CD50-negative T-cell clones, named CAMY.l and CAMY.2. These clones were derived from the Jurkat T-cell variant PPL.l. Data from analysis of protein expression, specific mRNA content and calcium mobilization assays have confirmed the absence of functional CD50 molecules on these two clones. Thus, CAMY.l and CAMY.2 show no CD50 expression by phenotypical and immunoprecipit-ation analysis. CD50-sperific mRNA content is undetectable by Northern blot analysis in these clones and, only, when RT-PCR was performed could specific mRNA be detected. Additionally, CD50 cross-linking on theses clones shows no increase in intracellular calcium. Transfection of CD50 cDNA on CAMY cells restores not only CD50 surface expression, but its functional ability to induce calcium mobilization, CD69 upregulation and cell morphological changes. The CAMY.l and CAMY.2 clones provide useful model systems to analyze CD50 function in T cells.     

Role of invariant natural killer T cells in immune regulation and as potential therapeutic targets in autoimmune disease

Autoimmune diseases are caused by pathogenic antibody and/or T-cell responses that are left unchecked by regulatory immune mechanisms. Recent studies in immunology have focused on subsets of regulatory T cells (T_regs) that can suppress autoimmune responses. Invariant natural killer T (iNKT) cells are a subset of T_regs that recognize glycolipid antigens in the context of CD1d proteins. iNKT cells play a suppressive role in several autoimmune diseases and, therefore, are attractive targets for development of immunotherapies for these diseases. While preclinical studies with reagents, such as the sea sponge-derived iNKT-cell antigen α-galactosylceramide, have been promising, there are substantial concerns about treating humans with autoimmunity, or at risk of developing autoimmunity, with these reagents.     

Prevalence of hepatitis B virus MHR mutations and their correlation with genotypes and antiviral therapy in chronically infected patients in Serbia

Understanding the prevalence and diversity of HBsAg variants in a population is fundamental to assay design and planning vaccination programs. It has been shown that mutations within the S gene, caused by selection or natural variation, can lead to false‐negative results in assays for HBsAg, or have clinical implications, such as evading anti‐HBV immunoglobulin therapy or vaccine‐induced immunity. The region of HBsAg where most of these mutations occur is known as the major hydrophilic region (MHR). The aim of this study was to determine the prevalence and mutational patterns of MHR mutations in patients with chronic hepatitis B, and their correlation with patient characteristics, viral factors and antiviral therapy. The study comprised 164 plasma samples from patients with chronic hepatitis B, of which, 34.8% were on long‐term lamivudine monotherapy. Direct sequencing of part of the S/pol gene was used for identification of HBsAg mutations, HBV genotypes, subgenotypes and HBsAg subtypes. The overall frequency of MHR mutations was 22.6%, but it varied significantly between untreated and treated patients (16.8% vs. 33.3%). The most frequent substitution was at position 120 (9.1%) whereas the most common vaccine‐escape position, 145, was affected in 1.8% of isolates. The presence of MHR mutations was correlated with genotype D, subgenotype D3, and ayw2/ayw3 HBsAg subtypes and to older age (>40 years). It is concluded that natural viral variability present in a geographical region, duration of infection, and antiviral therapy are among the major factors associated with the occurrence of MHR mutations. J. Med. Virol. 82: 1160–1167, 2010. © 2010 Wiley‐Liss, Inc.     

p47phox基因第10外显子多态性与脑卒中的关联及其对血脂的影响

目的探讨p47phox基因第10外显子C923T(Ala308Val)多态性与湖南地区汉族人群脑卒中的关联及其对血脂的影响。方法采用PCR-单链构象多态技术和DNA直接测序法检测湖南地区汉族220例脑卒中患者、20个脑卒中家系成员和100名健康对照者的p47phox基因第10外显子C923T(Ala308Val)多态性;酶法测定总胆固醇、甘油三酯、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇;免疫比浊法测定apoA-Ⅰ及apoB100;ELISA法测定血清脂蛋白α水平。结果湖南省汉族人p47phox基因第10外显子C923T(Ala308Val)多态存在CC、CT、TT3种基因型,脑卒中组及其各亚型组C923T(Ala308Val)多态基因频率分布与对照组比较,差异无统计学意义(P>0.05);分析不同基因型对血脂、脂蛋白水平的影响,发现脑梗死组和对照组CT基因型血清甘油三酯水平均明显高于CC基因型(P<0.05)。结论湖南省汉族人群p47phox基因C923T(Ala308Val)多态性可能与脑卒中的易患性无关联,可能与脑梗死患者血脂代谢相关。