Knockout of CLTC gene reduces but not completely block SFTSV infection.

Clathrin is a key protein for viruses to enter host cells. Previous studies often use clathrin inhibitors or gene knockdown technology to partially inhibit the function of clathrin, but whether SFTSV can infect host cells without clathrin expression remains unclear. In this research, a clathrin heavy chains (CLTC) knockout A549 cell line was established by CRISPR/Cas9 technology, and the knockout of CLTC was verified by PCR, Western blot, immunofluorescence and T7E1 analysis. The off-target effect was evaluated by PCR combined with Sanger sequencing. Furthermore, this research verified that SFTSV infection was significantly inhibited, but not completely blocked, due to the deletion of CLTC protein. Our research also found that lipid raft inhibitor Filipin, other than macropinocytosis inhibitor EIPA, could significantly reduce SFTSV infection, and the inhibition was more obviously observed when Filipin was used in CLTC knockout cells. These result indicated that clathrin-dependent and lipid raft mediated endocytosis are the major two mode used by SFTSV entry. In conclusion, this study constructed a CLTC knockout cell line, which, for the first time, established a cell model for the study of the function of CLTC protein, and provided direct evidence that SFTSV pendent could still infect cells without clathrin. Additionally, we confirmed that lipid raft mediated endocytosis, as a clathrin-independent pathway, could be another key mode for SFTSV entry.

Functional analysis of two SLC9A6 frameshift variants in lymphoblastoid cells from patients with Christianson syndrome.

BACKGROUND: Christianson syndrome (CS) is caused by mutations in SLC9A6 and is characterized by global developmental delay, epilepsy, hyperkinesis, ataxia, microcephaly, and behavioral disorder. However, the molecular mechanism by which these SLC9A6 mutations cause CS in humans is not entirely understood, and there is no objective method to determine the pathogenicity of single SLC9A6 variants. METHODS: Trio-based whole exome sequencing (WES) was carried out on two individuals with suspicion of CS. qRT-PCR, western blot analysis, filipin staining, lysosomal enzymatic assays, and electron microscopy examination, using EBV-LCLs established from the two patients, were performed. RESULTS: Trio-based WES identified a hemizygous SLC9A6 c.1560dupT, p.T521Yfs*23 variant in proband 1 and a hemizygous SLC9A6 c.608delA, p.H203Lfs*10 variant in proband 2. Both children exhibited typical phenotypes associated with CS. Expression analysis in EBV-LCLs derived from the two patients showed a significant decrease in mRNA levels and no detectable normal NHE6 protein. EBV-LCLs showed a statistically significant increase in unesterified cholesterol in patient 1, but only non-significant increase in patient 2 when stained with filipin. Activities of lysosomal enzymes (ß-hexosaminidase A, ß-hexosaminidase A + B, ß-galactosidase, galactocerebrosidase, arylsulfatase A) of EBV-LCLs did not significantly differ between the two patients and six controls. Importantly, by electron microscopy we detected an accumulation of lamellated membrane structures, deformed mitochondria, and lipid droplets in the patients' EBV-LCLs. CONCLUSIONS: The SLC9A6 p.T521Yfs*23 and p.H203Lfs*10 variants in our patients result in loss of NHE6. Alterations of mitochondria and lipid metabolism may play a role in the pathogenesis of CS. Moreover, the combination of filipin staining with electron microscopy examination of patient lymphoblastoid cells can serve as a useful complementary diagnostic method for CS.

Erythrocyte Plasma Membrane Lipid Composition Mirrors That of Neurons and Glial Cells in Murine Experimental In Vitro and In Vivo Inflammation.

Lipid membrane turnover and myelin repair play a central role in diseases and lesions of the central nervous system (CNS). The aim of the present study was to analyze lipid composition changes due to inflammatory conditions. We measured the fatty acid (FA) composition in erythrocytes (RBCs) and spinal cord tissue (gas chromatography) derived from mice affected by experimental allergic encephalomyelitis (EAE) in acute and remission phases; cholesterol membrane content (Filipin) and GM1 membrane assembly (CT-B) in EAE mouse RBCs, and in cultured neurons, oligodendroglial cells and macrophages exposed to inflammatory challenges. During the EAE acute phase, the RBC membrane showed a reduction in polyunsaturated FAs (PUFAs) and an increase in saturated FAs (SFAs) and the omega-6/omega-3 ratios, followed by a restoration to control levels in the remission phase in parallel with an increase in monounsaturated fatty acid residues. A decrease in PUFAs was also shown in the spinal cord. CT-B staining decreased and Filipin staining increased in RBCs during acute EAE, as well as in cultured macrophages, neurons and oligodendrocyte precursor cells exposed to inflammatory challenges. This regulation in lipid content suggests an increased cell membrane rigidity during the inflammatory phase of EAE and supports the investigation of peripheral cell membrane lipids as possible biomarkers for CNS lipid membrane concentration and assembly.

A basic model for the association of ligands with membrane cholesterol: application to cytolysin binding.

Almost all the cholesterol in cellular membranes is associated with phospholipids in simple stoichiometric complexes. This limits the binding of sterol ligands such as filipin and perfringolysin O (PFO) to a small fraction of the total. We offer a simple mathematical model that characterizes this complexity. It posits that the cholesterol accessible to ligands has two forms: active cholesterol, which is that not complexed with phospholipids; and extractable cholesterol, that which ligands can capture competitively from the phospholipid complexes. Simulations based on the model match published data for the association of PFO oligomers with liposomes, plasma membranes, and the isolated endoplasmic reticulum. The model shows how the binding of a probe greatly underestimates cholesterol abundance when its affinity for the sterol is so weak that it competes poorly with the membrane phospholipids. Two examples are the understaining of plasma membranes by filipin and the failure of domain D4 of PFO to label their cytoplasmic leaflets. Conversely, the exaggerated staining of endolysosomes suggests that their cholesterol, being uncomplexed, is readily available. The model is also applicable to the association of cholesterol with intrinsic membrane proteins. For example, it supports the hypothesis that the sharp threshold in the regulation of homeostatic endoplasmic reticulum proteins by cholesterol derives from the cooperativity of their binding to the sterol weakly held by the phospholipids. Thus, the model explicates the complexity inherent in the binding of ligands like PFO and filipin to the small accessible fraction of membrane cholesterol.

First Discovery of Cholesterol-Lowering Activity of Parthenolide as NPC1L1 Inhibitor.

Elevated cholesterol significantly increases the risk of developing atherosclerosis and coronary heart disease. The key to treating hypercholesterolemia is lowering plasma cholesterol levels. There have been no studies on the cholesterol-lowering potential of parthenolide (PTL), a naturally occurring small molecule from Tanacetum parthenium. Here, we first put forth PTL's cholesterol-lowering ability to inhibit cellular uptake of cholesterol in a dose-dependent manner. Its performance was on par with the positive control drug, ezetimibe. Niemann-Pick C1 Like-1 (NPC1L1) has been identified as a potential therapeutic target for hypercholesterolemia. The interaction of PTL with NPC1L1 could be explained by the results of molecular docking and filipin staining further reinforces this hypothesis. Furthermore, PTL reduced the expression of NPC1L1 in HepG2 cells in a concentration-dependent manner, which suggests that PTL functions as a potential NPC1L1 inhibitor with therapeutic potential for hypercholesterolemia.

Molecular and functional study of pediatric patients with Niemann-Pick C in Argentina.

Niemann-Pick type C (NP-C) is a rare, autosomal recessive disorder. At least 95% of all the cases with this disease are due to mutations in the NPC1 gene. The clinical signs and symptoms of NP-C are classified into visceral, neurological and psychiatric. Our aim is to report the clinical findings, molecular results and filipin staining of 4 patients. The age of onset, expressed as median and range, was 0.2 (0.08-4.0) years and the age of diagnosis was 4.0 (2.5-8.9) years. Neurological and/or visceral manifestations were presented in our patients. Foamy cells in bone marrow biopsy were found in two patients. Through a molecular analysis of NPC1 gene, one non-reported (novel) and 4 previously described mutations were found. The filipin staining showed a positive pattern in all the patients. The diagnostic confirmation of these pediatric patients means a contribution to the casuistry of this disease in Argentina.

Niemann-Pick tipo C (NP-C) es una enfermedad poco frecuente, con un patrón de herencia autosómico recesivo. Al menos el 95% de los casos se producen por mutaciones en el gen NPC1. Los signos y síntomas clínicos de NP-C se clasifican en viscerales, neurológicos y psiquiátricos. En este trabajo presentamos los hallazgos clínicos, los resultados moleculares y la tinción con filipina de 4 pacientes con NP-C. La edad de presentación de los primeros síntomas, expresada como mediana y rango, fue de 0.2 años (0.08-4.0) años y la edad del diagnóstico fue 4.0 (2.5-8.9) años. Los pacientes presentaron manifestaciones neurológicas y / o viscerales. Se encontraron células espumosas en la biopsia de médula ósea en 2 pacientes. El análisis molecular del gen NPC1 encontró 1 variante nueva y 4 previamente publicadas. La tinción de filipina mostró un patrón positivo en todos los pacientes. La confirmación diagnóstica de este grupo de pacientes pediátricos significa un aporte a la casuística de esta enfermedad en Argentina.

Genome-Guided Discovery of Antifungal Filipins from a Deep-Sea-Derived Streptomyces antibioticus.

Nine new (1-3, 5-8, 11, and 12; named filipins VI-XIV) and three known (4, 9, and 10) filipin-type polyene macrolides were isolated from the deep-sea-derived Streptomyces antibioticus OUCT16-23 using a genome-guided strategy coupled with bioassay. Their structures were elucidated based on the extensive MS and NMR spectroscopic analyses together with ECD calculations. In an antifungal assay, compounds 4, 5, and 7-10 showed different degrees of growth inhibition against Candida albicans with minimum inhibitory concentrations (MICs) of 1.56-12.5 µg/mL, by which the alkyl side-chain substitution affecting the activity was preliminarily studied. A biosynthetic pathway to 1-12 in S. antibioticus OUCT16-23 is also proposed.

Evaluation of Cardiotonic Steroid Modulation of Cellular Cholesterol and Phospholipid.

We have previously shown that 21-benzylidene digoxin (21-BD) increases the total cholesterol and phospholipid content on the membrane of HeLa cells. Lipid modulation caused by cardiotonic steroids (CTS) is still unexplored. Therefore, the aim of the present study was to evaluate the cholesterol and phospholipid modulation of the cell membrane caused by ouabain and 21-BD and the possible involvement of the caveolae on this modulation. For this, one cell line containing caveolae (HeLa) and other not containing (Caco-2) were used. The modulation of the lipid profile was evaluated by total cholesterol and phospholipids measurements, and identification of membrane phospholipids by HPTLC. The cholesterol distribution was evaluated by filipin staining. The caveolin-1 expression was evaluated by Western Blotting. Ouabain had no effect on the total membrane lipid content in both cell lines. However, 21-BD increased total membrane phospholipid content and had no effect on the membrane cholesterol content in Caco-2 cells. CTS were not able to alter the specific phospholipids content. In the filipin experiments, 21-BD provoked a remarkable redistribution of cholesterol to the perinuclear region of HeLa cells. In Caco-2 cells, it was observed only a slight increase in cholesterol, especially as intracellular vesicles. The caveolin-1 expression was not altered by any of the compounds. Our data mainly show different effects of two cardiotonic steroids. Ouabain had no effect on the lipid profile of cells, whereas 21-BD causes important changes in cholesterol and phospholipid content. Therefore, the modulation of cholesterol content in the plasma membrane of HeLa cells is not correlated with the expression of caveolin-1.

Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells.

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

Lactoferrin perturbs lipid rafts and requires integrity of Pma1p-lipid rafts association to exert its antifungal activity against Saccharomyces cerevisiae.

Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs ergosterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma1-10 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi.

Isolation of Lipid Rafts (Detergent-Resistant Microdomains) and Comparison to Extracellular Vesicles (Exosomes).

Lipid rafts (LRs) represent cellular microdomains enriched in sphingolipids and cholesterol which may fuse to form platforms in which signaling molecules can be organized and regulated (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1) 62-75, 2003). In a proposed Model 1 (Cheng et al., J Exp Med 190:1549-1550, 1999) the LR has a well-ordered central core composed mainly of cholesterol and sphingolipids that is surrounded by a zone of decreasing lipid order. Detergents such as Triton X-100 can solubilize the core (and a significant amount of phosphoglyceride), but the LRs will be insoluble at 4 °C and be enriched in a well-characterized set of biomarkers. Model 2 proposes that the LRs are homogeneous, but there is selectivity in the lipids (and proteins) extracted by the 1% Triton X-100. Model 3 proposes LRs with distinct lipid compositions are highly structured and can be destroyed by binding molecules such as beta-methylcyclodextrin or filipin. These may be Caveolin in some cell types but not in brain. Since it is unlikely that two LR preparations will be exactly the same this review will concentrate on LRs defined as "small (50 nm) membranous particles which are insoluble in 1% Triton X-100 at 4 °C and have a low buoyant density (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1):62-75, 2003; Testai et al., J Neurochem 89:636-644, 2004). We will present a generic method for isolating LRs for both lipidomic, proteomic, and cellular signaling analysis [1-6].

The Streptomyces filipinensis Gamma-Butyrolactone System Reveals Novel Clues for Understanding the Control of Secondary Metabolism.

Streptomyces γ-butyrolactones (GBLs) are quorum sensing communication signals triggering antibiotic production. The GBL system of Streptomyces filipinensis, the producer of the antifungal agent filipin, has been investigated. Inactivation of sfbR (for S. filipinensis γ-butyrolactone receptor), a GBL receptor, resulted in a strong decrease in production of filipin, and deletion of sfbR2, a pseudo-receptor, boosted it, in agreement with lower and higher levels of transcription of filipin biosynthetic genes, respectively. It is noteworthy that none of the mutations affected growth or morphological development. While no ARE (autoregulatory element)-like sequences were found in the promoters of filipin genes, suggesting indirect control of production, five ARE sequences were found in five genes of the GBL cluster, whose transcription has been shown to be controlled by both S. filipinensis SfbR and SfbR2. In vitro binding of recombinant SfbR and SfbR2 to such sequences indicated that such control is direct. Transcription start points were identified by 5' rapid amplification of cDNA ends, and precise binding regions were investigated by the use of DNase I protection studies. Binding of both regulators took place in the promoter of target genes and at the same sites. Information content analysis of protected sequences in target promoters yielded an 18-nucleotide consensus ARE sequence. Quantitative transcriptional analyses revealed that both regulators are self-regulated and that each represses the transcription of the other as well as that of the remaining target genes. Unlike other GBL receptor homologues, SfbR activates its own transcription whereas SfbR2 has a canonical autorepression profile. Additionally, SfbR2 was found here to bind the antifungal antimycin A as a way to modulate its DNA-binding activity.IMPORTANCEStreptomyces GBLs are important signaling molecules that trigger antibiotic production in a quorum sensing-dependent manner. We have characterized the GBL system from S. filipinensis, finding that two key players of this system, the GBL receptor and the pseudo-receptor, each counteracts the transcription of the other for the modulation of filipin production and that such control over antifungal production involves an indirect effect on the transcription of filipin biosynthetic genes. Additionally, the two regulators bind the same sites, are self-regulated, and repress the transcription of three other genes of the GBL cluster, including that encoding the GBL synthase. In contrast to all the GBL receptors known, SfbR activates its own synthesis. Moreover, the pseudo-receptor was identified as the receptor of antimycin A, thus extending the range of examples supporting the idea of signaling effects of antibiotics in Streptomyces The intricate regulatory network depicted here should provide important clues for understanding the regulatory mechanism governing secondary metabolism.

In vitro cultured human endometrial cells release extracellular vesicles that can be uptaken by spermatozoa.

Extracellular vesicles (EVs) derived from different parts of the male reproductive tract can be internalized by human spermatozoa affecting their maturation and regulating their functions. Here we demonstrate that EVs derived from the female tract can be uptaken by sperm and affect their competence. Primary endometrial cells release EVs with a diameter between 50 and 350 nm and bear the standard vesicle and exosome marker proteins CD63, CD9, TSG101 and ALIX. The uptake of dye-labelled endometrial cell-derived EVs by spermatozoa, quantified as fluorescence intensity, was significantly higher when EVs were derived from cells in the proliferative phase. Vital, motile fluorescent sperm could be appreciated after a 48-hour co-incubation with endometrial cells previously labelled with the Vybrant™ DiO dye. EV internalization by sperm was blocked at 4 °C and by incubation with filipin, suggesting an energy-dependent process probably attributable to the lipid-raft domain mediated-endocytosis. Sperm ability to undergo capacitation and acrosome reaction was stimulated by endometrial cell-derived EVs as manifested by the increased protein tyrosine phosphorylation and evident reactivity when stimulated with a calcium ionophore. Based on these findings, EVs exchange may be suggested as an emerging way through which female reproductive tract cells can interact with the passing spermatozoa.

Disease control efficacy of 32,33-didehydroroflamycoin produced by Streptomyces rectiviolaceus strain DY46 against gray mold of tomato fruit.

Despite the efficacy of synthetic fungicides in controlling postharvest diseases, public concerns regarding chemical residues in food and an increase in drug-resistant strains of pathogens have led to a need for new agents to control postharvest diseases. The current study was performed to find control agents of microbial origin that are effective on gray mold of tomato fruits. We recently isolated Streptomyces rectiviolaceus DY46, which has antagonistic activity against various plant pathogenic fungi. The incidence of gray mold of tomato fruits was markedly reduced by 80.0% in tomatoes treated with the cell extract of Streptomyces rectiviolaceus DY46 compared with the control tomatoes. The active ingredient was purified from the cell extract of DY46 and identified to be 32,33-didehydroroflamycoin (DDHR). DDHR displayed MICs (minimal inhibitory concentrations) against the mycelial growth of various plant pathogenic fungi at concentrations of 8-64 mg L-1. The incidence of gray mold in tomato fruits inoculated with conidial suspension (104 conidia mL-1) of Botrytis cinerea was markedly reduced by 88.9% in tomatoes treated with DDHR (100 mg L-1) compared with the control. The DDHR residue in tomato fruit was significantly diminished 2 d after treatment. These results show that DDHR would be relatively safe for use as a postharvest fungicide.

Macropinocytosis dependent entry of Chikungunya virus into human muscle cells.

Chikungunya virus (CHIKV) is a re-emerging arbovirus known to cause chronic myalgia and arthralgia with high morbidity. CHIKV is now considered endemic in many countries across Asia and Africa. In this study, the susceptibility of various human, mammalian and mosquito cell lines to CHIKV infection was evaluated. CHIKV infection was found to be cell-type dependent and virus strain-specific. Furthermore, SJCRH30 (human rhabdomyosarcoma cell line) was showed to be highly permissive to CHIKV infection, with maximum production of infectious virions observed at 12 h.p.i. Pre-infection treatment of SJCRH30 with various inhibitors of endocytosis, including monodansylcadaverine (receptor-mediated endocytic inhibitor), dynasore (clathrin-mediated endocytic inhibitor), as well as filipin (caveolin-mediated endocytosis inhibitor), resulted in minimal inhibition of CHIKV infection. In contrast, dose-dependent inhibition of CHIKV infection was observed with the treatment of macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Furthermore, siRNA-mediated knockdown of sortin nexin 9 (SNX9) a protein involved in macropinosome formation, also resulted in a significant dose-dependent reduction in viral titre. By performing a virus entry assay, CHIKV particles were also observed to colocalize with FITC-dextran, a macropinosome marker. This study shows for the first time, that the infectious entry of CHIKV into human muscle cells is mediated by macropinocytosis. Together, the data from this study may pave the way for the development of specific inhibitors that target the entry process of CHIKV into cells.

BODIPY-cholesterol can be reliably used to monitor cholesterol efflux from capacitating mammalian spermatozoa.

Capacitation is the final maturation step spermatozoa undergo prior to fertilisation. The efflux of cholesterol from the sperm membrane to the extracellular environment is a crucial step during capacitation but current methods to quantify this process are suboptimal. In this study, we validate the use of a BODIPY-cholesterol assay to quantify cholesterol efflux from spermatozoa during in vitro capacitation, using the boar as a model species. The novel flow cytometric BODIPY-cholesterol assay was validated with endogenous cholesterol loss as measured by mass spectrometry and compared to filipin labelling. Following exposure to a range of conditions, the BODIPY-cholesterol assay was able to detect and quantify cholesterol efflux akin to that measured with mass spectrometry. The ability to counterstain for viability is a unique feature of this assay that allowed us to highlight the importance of isolating viable cells only for a reliable measure of cholesterol efflux. Finally, the BODIPY-cholesterol assay proved to be the superior method to quantify cholesterol efflux relative to filipin labelling, though filipin remains useful for assessing cholesterol redistribution. Taken together, the BODIPY-cholesterol assay is a simple, inexpensive and reliable flow cytometric method for the measurement of cholesterol efflux from spermatozoa during in vitro capacitation.

Drugs That Modify Cholesterol Metabolism Alter the p38/JNK-Mediated Targeted and Nontargeted Response to Alpha and Auger Radioimmunotherapy.

PURPOSE: For the development of new anticancer therapeutic radiopharmaceuticals, including alpha particle emitters, it is important to determine the contribution of targeted effects in irradiated cells, and also of nontargeted effects in nonirradiated neighboring cells, because they may affect the therapeutic efficacy and contribute to side effects. EXPERIMENTAL DESIGN: Here, we investigated the contribution of nontargeted cytotoxic and genotoxic effects in vitro and in vivo (in xenografted mice) during alpha (212Pb/212Bi, 213Bi) and Auger (125I) radioimmunotherapy (RIT). RESULTS: Between 67% and 94% (alpha RIT) and 8% and 15% (Auger RIT) of cancer cells were killed by targeted effects, whereas 7% to 36% (alpha RIT) and 27% to 29% (Auger RIT) of cells were killed by nontargeted effects. We then demonstrated that the nontargeted cell response to alpha and Auger RIT was partly driven by lipid raft-mediated activation of p38 kinase and JNK. Reactive oxygen species also played a significant role in these nontargeted effects, as demonstrated by NF-κB activation and the inhibitory effects of antioxidant enzymes and radical scavengers. Compared with RIT alone, the use of RIT with ASMase inhibitor (imipramine) or with a lipid raft disruptor (e.g., methyl-beta-cyclodextrin or filipin) led to an increase in clonogenic cell survival in vitro and to larger tumors and less tissue DNA damage in vivo. These results were supported by an inhibitory effect of pravastatin on Auger RIT. CONCLUSIONS: Cell membrane-mediated nontargeted effects play a significant role during Auger and alpha RIT, and drugs that modulate cholesterol level, such as statins, could interfere with RIT efficacy.

Analyzing T-Cell Plasma Membrane Lipids by Flow Cytometry.

Plasma membrane lipid rafts are highly ordered membrane microdomains enriched for glycosphingolipids and cholesterol, which play an important role during T-cell antigen receptor (TCR) signaling. Our previous work has demonstrated that plasma membrane lipid composition is an important determinant of human CD4+ T-cell function and that defects in lipid raft expression contribute to CD4+ dysfunction in patients with autoimmunity. In this chapter we share three flow cytometry-based methods to quantitatively analyze plasma membrane lipid composition in primary human CD4+ T cells. We describe the quantification of glycosphingolipid expression using cholera toxin subunit B, cholesterol expression using filipin staining, and membrane "lipid order" using di-4-ANEPPDHQ. These methods can easily be adapted to analyze different cell types.

Plasma lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment ex vivo.

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20-200 mg/dl FC; coefficient of variation <20%) and sensitively (lower limit of quantitation <1 mg/ml FC), enabling LP-X detection in FLD, cholestatic, and even fish-eye disease patients. rhLCAT incubation with FLD plasma ex vivo reduced LP-X dose dependently, generated HDL, and decreased lipoprotein FC content. Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.

Intracellular and Plasma Membrane Cholesterol Labeling and Quantification Using Filipin and GFP-D4.

Cholesterol, a major component of biological membranes, is rapidly trafficked and unevenly distributed between organelles. Anomalies of intracellular cholesterol distribution are the hallmark of a number of lysosomal lipid storage disorders. A major methodological obstacle for studying cholesterol trafficking is tracing this molecule in situ. The use of fluorescent probes that specifically bind cholesterol allows the visualization and imaging of cellular cholesterol. Here, we describe a series of assays optimized for quantifying free cholesterol in cell populations and at the single cell level, both at the plasma membrane and inside cells. These methods use two fluorescent probes: the D4 fragment of perfringolysin O fused to GFP (GFP-D4) and the polyene macrolide filipin. First, we report a robust method for quantifying plasma membrane cholesterol by flow cytometry using the GFP-D4 probe. Second, to optically distinguish and quantify intracellular cholesterol accumulation, we have adapted the classical filipin cholesterol staining protocol. Indeed, we observed that treatment of living cells with methyl-ß-cyclodextrin, a chemical known to extract cholesterol from the plasma membrane, improves the visualization of the intracellular cholesterol pool with filipin. To complement these staining procedures, we developed an image analysis protocol based on image segmentation to quantify, in a robust manner, intracellular cholesterol stained with filipin. Thus, this chapter is a guideline for cellular cholesterol staining and signal quantification.