Co-administration of CSL112 (apolipoprotein A-I [human]) with atorvastatin and alirocumab is not associated with increased hepatotoxic or toxicokinetic effects in rats.

CSL112 (apolipoprotein A-I, apo AI [human]) is an investigational drug in Phase 3 development for risk reduction of early recurrent cardiovascular events following an acute myocardial infarction (AMI). Although CSL112 is known to be well tolerated with a regimen of four weekly 6 g intravenous infusions after AMI, high doses of reconstituted apo AI preparations can transiently elevate liver enzymes in rats, raising the possibility of additive liver toxicity and toxicokinetic (TK) effects upon co-administration with cholesterol-lowering drugs, i.e., HMG-CoA reductase and proprotein convertase subtilisin/kexin type 9 inhibitors. We performed a toxicity and TK study in CD rats assigned to eleven treatment groups, including two dose levels of intravenous (IV) CSL112 (140 mg/kg, low-dose; 600 mg/kg, high-dose) administered as a single dose, alone or with intravenous alirocumab 50 mg/kg/week and/or oral atorvastatin 10 mg/kg/day. In addition, control groups of atorvastatin and alirocumab alone and in combination were investigated. Results showed some liver enzyme elevations (remaining <2-fold of baseline) related to administration of CSL112 alone. There was limited evidence of an additive effect of CSL112 on liver enzymes when combined, at either dose level, with alirocumab and/or atorvastatin, and histology revealed no evidence of an increased incidence or severity of hepatocyte vacuolation compared to the control treatments. Co-administration of the study drugs had minimal effect on their respective exposure levels, and on levels of total cholesterol and high-density lipoprotein cholesterol. These data support concomitant use of CSL112 with alirocumab and/or atorvastatin with no anticipated negative impact on liver safety and TK.

The HDL from septic-ARDS patients with composition changes exacerbates pulmonary endothelial dysfunction and acute lung injury induced by cecal ligation and puncture (CLP) in mice.

BACKGROUND: Septic-acute respiratory distress syndrome (ARDS), characterized by the acute lung injury (ALI) secondary to aberrant systemic inflammatory response, has high morbidity and mortality. Despite increased understanding of ALI pathogenesis, the therapies to prevent lung dysfunction underlying systemic inflammatory disorder remain elusive. The high density lipoprotein (HDL) has critical protective effects in sepsis and its dysfunction has a manifested contribution to septic organ failure. However, the adverse changes in HDL composition and function in septic-ARDS patients are large unknown. METHODS: To investigate HDL remodeling in septic-ARDS, we analyzed the changes of HDL composition from 40 patients with septic-ARDS (A-HDL) and 40 matched normal controls (N-HDL). To determine the deleterious functional remodeling of HDL, A-HDL or N-HDL was administrated to C57BL/6 and apoA-I knock-out (KO) mice after cecal ligation and puncture (CLP) procedure. Mouse lung microvascular endothelial cells (MLECs) were further treated by these HDLs to investigate whether the adverse effects of A-HDL were associated with endothelial dysfunction. RESULTS: Septic-ARDS patients showed significant changes of HDL composition, accompanied with significantly decreased HDL-C. We further indicated that A-HDL treatment aggravated CLP induced ALI. Intriguingly, these deleterious effects of A-HDL were associated with pulmonary endothelial dysfunction, rather than the increased plasma lipopolysaccharide (LPS). Further in vitro results demonstrated the direct effects of A-HDL on MLECs, including increased endothelial permeability, enhanced expressions of adhesion proteins and pro-inflammatory cytokines via activating NF-κB signaling and decreased junction protein expression. CONCLUSIONS: Our results depicted the remodeling of HDL composition in sepsis, which predisposes lung to ARDS via inducing ECs dysfunction. These results also demonstrated the importance of circulating HDL in regulating alveolar homeostasis.

Mechanism of Trypanosoma brucei gambiense resistance to human serum.

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic ß-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.

Adaptation of Trypanosoma rhodesiense to hypohaptoglobinaemic serum requires transcription of the APOL1 resistance gene in a RNA polymerase I locus.

Human apolipoprotein L1 (APOL1) kills African trypanosomes except Trypanosoma rhodesiense and Trypanosoma gambiense, the parasites causing sleeping sickness. APOL1 uptake into trypanosomes is favoured by its association with the haptoglobin-related protein-haemoglobin complex, which binds to the parasite surface receptor for haptoglobin-haemoglobin. As haptoglobin-haemoglobin can saturate the receptor, APOL1 uptake is increased in haptoglobin-poor (hypohaptoglobinaemic) serum (HyHS). While T. rhodesiense resists APOL1 by RNA polymerase I (pol-I)-mediated expression of the serum resistance-associated (SRA) protein, T. gambiense resists by pol-II-mediated expression of the T. gambiense-specific glycoprotein (TgsGP). Moreover, in T. gambiense resistance to HyHS is linked to haptoglobin-haemoglobin receptor inactivation by mutation. We report that unlike T. gambiense, T. rhodesiense possesses a functional haptoglobin-haemoglobin receptor, and that like T. gambiense experimentally provided with active receptor, this parasite is killed in HyHS because of receptor-mediated APOL1 uptake. However, T. rhodesiense could adapt to low haptoglobin by increasing transcription of SRA. When assayed in Trypanosoma brucei, resistance to HyHS occurred with pol-I-, but not with pol-II-mediated SRA expression. Similarly, T. gambiense provided with active receptor acquired resistance to HyHS only when TgsGP was moved to a pol-I locus. Thus, transcription by pol-I favours adaptive gene regulation, explaining the presence of SRA in a pol-I locus.

BH3 domain-independent apolipoprotein L1 toxicity rescued by BCL2 prosurvival proteins.

The potent trypanolytic properties of human apolipoprotein L1 (APOL1) can be neutralized by the trypanosome variant surface antigen gene product known as serum resistance-associated protein. However, two common APOL1 haplotypes present uniquely in individuals of West African ancestry each encode APOL1 variants resistant to serum resistance-associated protein, and each confers substantial resistance to human African sleeping sickness. In contrast to the dominantly inherited anti-trypanosomal activity of APOL1, recessive inheritance of these two trypanoprotective APOL1 alleles predisposes to kidney disease. Proposed mechanisms of APOL1 toxicity have included BH3 domain-dependent autophagy and/or ion channel activity. We probed these potential mechanisms by expressing APOL1 in Xenopus laevis oocytes. APOL1 expression in oocytes increased ion permeability and caused profound morphological deterioration (toxicity). Coexpression of BCL2 family members rescued APOL1-associated oocyte toxicity in the order MCL1 ∼ BCLW > BCLXL ∼ BCL2A1 ≫ BCL2. Deletion of nine nominal core BH3 domain residues abolished APOL1-associated toxicity, but missense substitution of the same residues abolished neither oocyte toxicity nor its rescue by coexpressed MCL1. The APOL1 BH3 domain was similarly dispensable for the ability of APOL1 to rescue intact mice from lethal trypanosome challenge. Replacement of most extracellular Na(+) by K(+) also reduced APOL1-associated oocyte toxicity, allowing demonstration of APOL1-associated increases in Ca(2+) and Cl(-) fluxes and oocyte ion currents, which were similarly reduced by MCL1 coexpression. Thus APOL1 toxicity in Xenopus oocytes is BH3-independent, but can nonetheless be rescued by some BCL2 family proteins.

Oxidized HDL induces cytotoxic effects: implications for atherogenic mechanism.

Atherosclerosis can be considered as an inflammatory disease and oxidized low-density lipoprotein (oxLDL) is a critical factor in atherogenesis. Although high-density lipoprotein (HDL) is generally an antiatherogenic lipoprotein, this property can be compromised by functional impairment mainly due to oxidative modification. As such, understanding the proatherogenic properties exerted by oxidized-HDL (oxHDL) becomes more important. This study was focused on examining the role of oxHDL as a proatherogenic agent, using oxLDL as a positive control. The comparative toxicity of oxHDL and oxLDL having same range of malondialdehyde, to monocytes was evaluated. After treatment, markers for oxidative stress, inflammation, and cytotoxicity were quantitated. The results showed that like oxLDL, oxHDL induced significant oxidative stress, cytotoxicity, and release of TNF -alpha and MMP-9 in monocytes/macrophages, but was less potent than oxLDL in promoting these proatherogenic effects. Further, the effects of oxHDL for the enhanced formation of MMP-9 were found to be mediated by NADPH oxidase/ROS-JNK/ERK pathway, as one mechanism.

Recombinant high density lipoprotein nanoparticles for target-specific delivery of siRNA.

PURPOSE: Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for treatments of numerous diseases. However, the progress towards broad application of siRNA requires the development of safe and effective vectors that target to specific cells. In this study, we developed a novel recombinant high density lipoprotein (rHDL) vector with high siRNA encapsulation efficiency. METHODS: They were prepared by condensing siRNA with various commercial cationic polymers and coating the polyplex with a layer of lipids and apolipoprotein AI (apo AI). The rHDL nanoparticles were used to transfect SMMC-7721 hepatoma cells with stable luciferase expression. The uptake and intracellular trafficing of siRNA were also investigated. RESULTS: Characterization studies revealed these rHDL nanoparticles had similar physical properties as natural HDLs. The various rHDL formulations had high silencing efficiency (more than 70% knockdown) in hepatocytes with minimum cytotoxicity. Moreover, the uptake of rHDL by SMMC-7721 was confirmed to be mediated through the natural HDL uptake pathway. CONCLUSIONS: The work described here demonstrated the optimized rHDL nanoparticles may offer a promising tool for siRNA delivery to the liver.

Preparation and characterization of a lovastatin-loaded protein-free nanostructured lipid carrier resembling high-density lipoprotein and evaluation of its targeting to foam cells.

This study was designed to investigate whether a non-protein nanostructured lipid carrier (NLC) resembling high-density lipoprotein (HDL) could deliver a hydrophobic anti-atherogenic drug, lovastatin, to foam cells. Lovastatin-loaded NLC (LT-NLC) was prepared by a nanoprecipitation/solvent diffusion method. The LT-NLC-apoprotein (LT-NLC-apo) was prepared by incubating LT-NLC with native HDL. The physicochemical parameters of LT-NLC were characterized in terms of particle size, zeta potential, morphology, entrapment efficiency, and crystallization behavior. Targeting behavior and mechanism were demonstrated by the incubation of LT-NLC-apo with a RAW 264.7 macrophage-derived foam cell model in the presence or absence of very-low-density lipoprotein (VLDL) and lipase. The results showed that LT-NLC was solid spherical or oval in shape with an average diameter of 13.8 ± 2.2 nm, zeta potential of -29.3 ± 0.2 mV and entrapment efficiency of 96.2 ± 1.3%. Phagocytosis studies showed that uptake of LT-NLC-apo by macrophages was significantly lower than LT-NLC (p < 0.01), suggesting that LT-NLC-apo could possibly escape recognition from macrophages in vivo. The uptake was increased twofold when LT-NLC-apo was incubated with transfected foam cells containing VLDL and lipase. These results indicated that non-protein NLC resembling HDL could be a useful tool to deliver lipophilic anti-atherogenic drugs to foam cells, and that uptake could be enhanced by the VLDL receptor pathway.

D4F alleviates the C/EBP homologous protein-mediated apoptosis in glycated high-density lipoprotein-treated macrophages by facilitating autophagy.

The present study aimed to investigate the role of D4F, an apolipoprotein A-I mimetic peptide, in macrophage apoptosis induced by the glycated high-density lipoprotein (gly-HDL)-induced endoplasmic reticulum (ER) stress C/EBP homologous protein (CHOP) pathway, and unravel the regulatory role of autophagy in this process. Our results revealed that except for suppressing the accumulation of lipids within RAW264.7 macrophages caused by gly-HDL, D4F inhibited gly-HDL-induced decrease in the cell viability and increase in lactate dehydrogenase leakage and cell apoptosis, which were similar to 4-phenylbutyric acid (PBA, an ER stress inhibitor). Besides, similar to PBA, D4F inhibited gly-HDL-induced ER stress response activation evaluated through the decreased PERK and eIF2α phosphorylation, together with reduced ATF6 nuclear translocation as well as the downregulation of GRP78 and CHOP. Interestingly, D4F facilitated gly-HDL-triggered activation of autophagy, measured as elevated levels of beclin-1, LC3-II, and ATG5 expressions in macrophages. Furthermore, the inhibition effect of D4F on gly-HDL-induced ER stress-CHOP-induced apoptosis of macrophages was restrained after beclin-1 siRNA and 3-methyladenine (3-MA, an inhibitor of autophagy) treatments, while this effect was further reinforced after rapamycin (Rapa, an inducer of autophagy) treatment. Furthermore, administering D4F or Rapa to T2DM mice upregulated LC3-II and attenuated CHOP expression, cell apoptosis, and atherosclerotic lesions. However, the opposite results were obtained when 3-MA was administered to these mice. These results support that D4F effectively protects macrophages against gly-HDL-induced ER stress-CHOP-mediated apoptosis by promoting autophagy.

Triglyceride-rich lipoproteins prime aortic endothelium for an enhanced inflammatory response to tumor necrosis factor-alpha.

High levels of triglyceride-rich lipoproteins (TGRLs) in blood are linked to development of atherosclerosis, yet the mechanisms by which these particles initiate inflammation of endothelium are unknown. TGRL isolated from human plasma during the postprandial state was examined for its capacity to bind to cultured human aortic endothelial cells (HAECs) and alter the acute inflammatory response to tumor necrosis factor-alpha. HAECs were repetitively incubated with dietary levels of freshly isolated TGRL for 2 hours per day for 1 to 3 days to mimic postprandial lipidemia. TGRL induced membrane upregulation of the low-density lipoprotein family receptors LRP and LR11, which was inhibited by the low-density lipoprotein receptor-associated protein-1. TGRLs alone did not elicit inflammation in HAECs but enhanced the inflammatory response via a 10-fold increase in sensitivity to cytokine stimulation. This was reflected by increased mitogen-activated protein kinase activation, nuclear translocation of NF-kappaB, amplified expression of endothelial selectin and VCAM-1, and a subsequent increase in monocyte-specific recruitment under shear flow as quantified in a microfabricated vascular mimetic device.

Imaging-assisted nanoimmunotherapy for atherosclerosis in multiple species.

Nanomedicine research produces hundreds of studies every year, yet very few formulations have been approved for clinical use. This is due in part to a reliance on murine studies, which have limited value in accurately predicting translational efficacy in larger animal models and humans. Here, we report the scale-up of a nanoimmunotherapy from mouse to large rabbit and porcine atherosclerosis models, with an emphasis on the solutions we implemented to overcome production and evaluation challenges. Specifically, we integrated translational imaging readouts within our workflow to both analyze the nanoimmunotherapeutic's in vivo behavior and assess treatment response in larger animals. We observed our nanoimmunotherapeutic's anti-inflammatory efficacy in mice, as well as rabbits and pigs. Nanoimmunotherapy-mediated reduction of inflammation in the large animal models halted plaque progression, supporting the approach's translatability and potential to acutely treat atherosclerosis.

Scavenger Receptor Type B1 and Lipoprotein Nanoparticle Inhibit Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSC) are innate immune cells that potently inhibit T cells. In cancer, novel therapies aimed to activate T cells can be rendered ineffective due to the activity of MDSCs. Thus, targeted inhibition of MDSCs may greatly enhance T-cell-mediated antitumor immunity, but mechanisms remain obscure. Here we show, for the first time, that scavenger receptor type B-1 (SCARB1), a high-affinity receptor for spherical high-density lipoprotein (HDL), is expressed by MDSCs. Furthermore, we demonstrate that SCARB1 is specifically targeted by synthetic high-density lipoprotein-like nanoparticles (HDL NP), which reduce MDSC activity. Using in vitro T-cell proliferation assays, data show that HDL NPs specifically bind SCARB1 to inhibit MDSC activity. In murine cancer models, HDL NP treatment significantly reduces tumor growth, metastatic tumor burden, and increases survival due to enhanced adaptive immunity. Flow cytometry and IHC demonstrate that HDL NP-mediated suppression of MDSCs increased CD8+ T cells and reduced Treg cells in the metastatic tumor microenvironment. Using transgenic mice lacking SCARB1, in vivo data clearly show that the HDL NPs specifically target this receptor for suppressing MDSCs. Ultimately, our data provide a new mechanism and targeted therapy, HDL NPs, to modulate a critical innate immune cell checkpoint to enhance the immune response to cancer. Mol Cancer Ther; 17(3); 686-97. ©2017 AACR.

Oxidized HDL are much less cytotoxic to lymphoblastoid cells than oxidized LDL.

The possible effect of oxidized HDL was investigated on lymphoblastoid cells, in comparison to the cytotoxic effect of oxidized LDL. Oxidation of HDL was promoted by UV-C irradiation, or by copper ion (5 microM) or the combination of the two treatments. HDL extensively treated by UV-C for 20 h did not exhibit any cytotoxic effect on cultured lymphoblastoid cells even at a concentration of 500 micrograms apolipoprotein A-I/ml. In contrast to UV-treated (2 h) LDL, which were highly cytotoxic (already at a concentration of 100 micrograms apolipoprotein B/ml), HDL treated by copper or copper + UV were oxidized, as shown by TBARS formation and PUFA content decrease, but were slightly cytotoxic.

Reconstitution of the trypanolytic factor from components of a subspecies of human high-density lipoproteins.

Trypanosoma brucei brucei is non-infectious to man due to the sensitivity of these parasites to the lytic activity of normal human serum. Apolipoproteins (apo) have been purified, under non-denaturing conditions, from the subclass of human high-density lipoprotein (HDL), termed trypanosome lytic factor (TLF), which is responsible for the cytotoxicity of human serum to T. b. brucei. The TLF apolipoproteins were purified by anion exchange chromatography in the presence of the nonionic detergent octylglucoside and a reconstitution method was developed which allowed the role of the individual apolipoproteins and different lipids to be assessed. The results suggest that the TLF lipids do not have a direct role in lysis but are necessary for the correct assembly of the lytic HDL particle. Apo A-I, apo L-III and apo L-I contribute to lysis in reconstituted particles but individually they are not cytotoxic. Apo A-II was not required in the reconstituted TLF particle for trypanosome lysis. Formation of a lytic HDL particle required apo L-III suggesting its potential role as a toxin. Thermal inactivation of TLF activity correlated with the amount of denatured apo L-I, indicating that apo L-I was involved in lysis of T. b. brucei by native TLF.

Photosensitizer targeting in photodynamic therapy. II. Conjugates of haematoporphyrin with serum lipoproteins.

Conjugates between haematoporphyrin (HP) and human low-density lipoprotein (LDL), human high-density lipoprotein (HDL) and bovine HDL have been prepared, purified and characterized. HP-LDL is aggregated possibly via interparticle apoB protein cross-linking. HP-HDL human and bovine conjugates show different degrees of intraparticle apoA polypeptide cross-linking. Receptor-mediated endocytosis of HP-LDL by NIH 3T3 cells is inferred from the increased uptake observed when LDL receptors are upregulated. HP-LDL uptake into HT29 cells faces competition from unlabelled LDL, albeit at rather high doses. HP-HDL uptake is also inhibited by LDL, suggesting that both lipoprotein conjugates may have cell-surface binding sites in addition to the specific LDL (apoB) receptor. J774.2 macrophages avidly accumulate HP-LDL, retaining most of the fluorescence and some of the protein while degrading the remainder. Oxidized LDL species compete in these processes, with the major effect on protein degradation. Chloroquine has little effect on the fluorescence uptake but inhibits protein degradation (and hence enhances protein accumulation). HP-HDL is also avidly taken up by J774.2 cells, but in the case of the bovine material with a sigmoidal concentration dependence. This is consistent with prior aggregation before the particles can be endocytosed. P388.D1 cells, which appear to be less activated than the J774.2 line, take up less fluorescence and retain and degrade less protein, but still to higher extents than observed for non-phagocytic cells. We conclude that photosensitizer-lipoprotein conjugates can be taken up in large amounts by cells possessing scavenger receptors and/or phagocytic activity, and that this may be a means of targeting photodynamic therapy.

Lysis of Trypanosoma brucei by a toxic subspecies of human high density lipoprotein.

Trypanosoma brucei brucei is an important pathogen of domestic cattle in sub-Saharan Africa and is closely related to the human sleeping sickness parasites, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, T. b. brucei is non-infectious to humans. The restriction of the host range of T. b. brucei results from the sensitivity of the parasite to lysis by toxic human high density lipoproteins (HDL) (Rifkin, M. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3450-3454). We show in this report that trypanosome lytic activity is not a universal feature of all human HDL particles but rather that it is associated with a minor subclass of HDL. We have purified the lytic activity about 8,000-fold and have identified and characterized the subspecies of HDL responsible for trypanosome lysis. This class of HDL has a relative molecular weight of 490,000, a buoyant density of 1.21-1.24 g/ml, and a particle diameter of 150-210 A. It contains apolipoproteins AI, AII, CI, CII, and CIII, and monoclonal antibodies against apo-AI and apo-AII inhibit trypanocidal activity. In addition to these common apolipoproteins, the particles also contain at least three unique proteins, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Treatment of the particles with dithiothreitol resulted in the disappearance of two of the proteins and abolished trypanocidal activity. Two-dimensional gel electrophoresis showed that these proteins were a disulfide-linked trimer of 45,000, 36,000, and 13,500-Da polypeptides and dimers of the 36,000- and 13,500-Da polypeptides or of 65,000- and 8,500-Da polypeptides. Studies on the lysis of T. b. brucei by the purified particle suggest that the lytic pathway may involve the uptake of the trypanocidal subspecies of HDL by endocytosis.

Cell-transforming potential of low-density lipoproteins.

Arterial smooth muscle cell proliferation is thought to be essential for the development of atherosclerotic lesions. The monoclonal hypothesis of atherogenesis proposes that the proliferative smooth muscle cells are derived from a stable transformed cell population. The present study demonstrates for the first time evidence that, in addition to carcinogens such as 3-methylcholanthrene (MCA), 'atherogenic' low-density lipoproteins (LDL) also possess cell-transforming potential. LDL caused dose-dependent cytotoxicity and transformation of C3H/10T1/2 cells of type II and type III morphology of up to six transformed cell clones per 10(4) survivors in the concentration range of 50-200 micrograms cholesterol/ml after 72 h treatments. MCA (0.1 micrograms/ml) induced morphological transformation of 2.6 foci per 10(4) survivors. In a two-stage in vitro transformation assay LDL (5-25 micrograms cholesterol/ml) enhanced MCA-induced cell transformation 2- to 2.4-fold in a dose-dependent way. 'Non-atherogenic' high-density lipoproteins did not induce cell transformation by themselves or in an initiation-promotion model. These results show that LDL could act as (co)carcinogens.

Interaction of high-density lipoprotein with Trypanosoma brucei: effect of membrane stabilizers.

The specific lysis of bloodstream trypanosomes by serum from a nonpermissive mammalian host is the result of interaction between the serum trypanocidal factor (high-density lipoprotein) and the trypanosome surface. The studies described in this paper attempt to define further the mode of action of this cytotoxic lipoprotein. The binding of high-density lipoprotein to Trypanosoma brucei was instantaneous at 4 degrees C and readily reversible. Binding was not mediated by the surface glycoprotein as removal of the surface coat enhanced binding at 4 degrees C, and no stable glycoprotein-lipoprotein complex could be detected. Pretreatment of trypanosomes with the cross linker dimethylsuberimidate rendered cells resistant to lysis. Addition of membrane-stabilizing drugs, such as cytochalasins C, D, and E, and local anesthetics (dibucaine, tetracaine, and procaine), also inhibited high-density lipoprotein-induced cell lysis. The data presented support the idea that at 37 degrees C lateral diffusion of the variant surface glycoprotein, an integral membrane protein, allows maximal high-density lipoprotein-cell interaction in serum-sensitive cells, and that altered properties of the plasma membrane induced by low temperature or the addition of cytochalasins, local anesthetics, or zinc inhibit this interaction, possibly by increasing the shielding of the plasma membrane by more rigidly anchored surface glycoprotein molecules.

Role of phospholipids in the cytotoxic action of high density lipoprotein on trypanosomes.

Host range among the African trypanosomes, protozoa that cause fatal diseases both in humans and livestock, may be, in part, regulated by toxic properties associated with host high density lipoproteins (HDL). High density lipoproteins from hosts resistant (baboon, human) or susceptible (rabbit, rat) to Trypanosoma brucei infection were isolated and their trypanocidal activity was determined in in vitro cell lysis assays. Rabbit and rat HDL were not cytotoxic while baboon and human HDL rapidly lysed trypanosomes within 2 h at 37 degrees C. Analysis of the phospholipid composition of HDL preparations from these species suggested a correlation between trypanocidal activity and low phosphatidylinositol content. Phospholipase digestion of HDL resulted in a loss of trypanocidal activity, indicating the importance of native phospholipids in maintaining this biological activity of HDL. Cell lysis and loss of trypanosome infectivity induced by baboon HDL could be inhibited either by addition of rabbit or rat HDL to the incubation medium or by addition of purified phospholipids, phosphatidylinositol being the most effective inhibitor. Although the mechanism by which HDL lyses trypanosomes remains to be elucidated, these results suggest an important role for phospholipids in determining the specificity of this cytotoxic property of HDL.