Hepatitis C virus: Enslavement of host factors.

Hepatitis C virus (HCV) has infected over 170 million people world-wide. This infection causes severe liver damage that can progress to hepatocellular carcinoma leading to death of the infected patients. Development of a cell culture model system for the study of HCV infection in the recent past has helped the researchers world-wide to understand the biology of this virus. Studies over the past decade have revealed the tricks played by the virus to sustain itself, for as long as 40 years, in the host setup without being eliminated by the immune system. Today we understand that the host organelles and different cellular proteins are affected during HCV infection. This cytoplasmic virus has all the cellular organelles at its disposal to successfully replicate, from ribosomes and intracellular membranous structures to the nucleus. It modulates these organelles at both the structural and the functional levels. The vast knowledge about the viral genome and viral proteins has also helped in the development of drugs against the virus. Despite the achieved success rate to cure the infected patients, we struggle to eliminate the cases of recurrence and the non-responders. Such cases might emerge owing to the property of the viral genome to accumulate mutations during its succeeding replication cycles which favours its survival. The current situation calls an urgent need for alternate therapeutic strategies to counter this major problem of human health. © 2017 IUBMB Life, 70(1):41-49, 2018.

Impact of maternal cholesterol metabolism on ovarian follicle development and fertility.

The relationship among maternal lipid metabolism, fetal development, and adult disease of the offspring represents an emerging topic of high epidemiological relevance. The present review highlights the very early aspects of this process. Recent data suggest a link between lipid metabolism and reproduction/fertility, not only on the systemic level, but also locally on the level of the ovary that maintains its own sterol metabolism, likely in a self-regulated fashion. Follicular fluid - which surrounds oocytes in a developing follicle - contains all relevant lipoprotein subclasses that reach the follicular fluid either by diffusion, in the case of high-density lipoproteins (HDL), or by local production within the granulosa cells, in the case of very low-density lipoproteins (VLDL). Here, we summarize current knowledge on lipoprotein metabolism in the ovary in the context of fertility, and hypothesize that lipoproteins within follicular fluid are relevant to the development of the early embryo and thereby putatively also to the programming of metabolic disease later in life.

The potential role of preventing atherosclerosis by induction of neonatal tolerance to VLDL.

Induction of immune tolerance to ox-LDL could reduce atherosclerosis by modulation immune response. We suppose that very low density lipoprotein (VLDL) may have a similar role to ox-LDL in autoimmune response of atherosclerosis. In this study, neonatal rats were injected with ox-LDL, VLDL, or equal-volume saline, respectively. Vaccination with ox-LDL reduced the level of specific antibody, T cells proliferation response, and the level of endothelins. The method also had a tendency of reducing blood lipids. Vaccination with VLDL obviously reduced the level of specific antibody and T cells proliferation. Though there was also a tendency of reducing blood lipids and endothelins, the effect was less prominent than that with ox-LDL. We conclude that, although the effect was less obvious, vaccination with VLDL to induce neonatal tolerance had an effect on modulating immune response, protecting endothelial cells, and reducing blood lipids.

High-density lipoprotein phospholipids interfere with dendritic cell Th1 functional maturation.

Lipoproteins are both lipid carriers in the blood and regulators of essential biological processes. Several studies demonstrated that lipoproteins modified during pathological conditions could alter dendritic cell (DC) maturation. Here the immune function of non-pathological lipoproteins is addressed by analysing their impact on human DC maturation triggered by TLR ligands. Upon TLR4 stimulation, low- and high-density lipoproteins (LDL and HDL) strongly inhibited the ability of DC to induce a Th1 response of T cells, characterized by high levels of IFNγ secretion, whereas the effect of very low-density lipoprotein was subject to variations. HDL also inhibited the Th1 function of DC stimulated by TLR1/2 and TLR2/6 ligands. The phospholipid fraction from HDL retained the inhibitory activity of the lipoprotein. We identified the 1-palmitoyl-2-linoleyl-phosphatidylcholine (PLPC) as one active phospholipid that inhibited the Th1 function of mature DCs whereas the dipalmitoyl-phosphatidylcholine had no significant effect. The treatment of DC by PLPC, 24h before TLR4 stimulation, resulted in reduced activation of NF-κB. This study shows that some HDL phospholipids have a direct immunoregulatory function, by modulating DC ability to activate a Th1 response of T cells.

[Purification of human VLDL apolipoproteins by middle pressure liquid chromatography].

OBJECTIVE: To purify human VLDL apolipoproteins by middle-pressure liquid chromatography. METHODS: Human VLDLs were isolated by one step density ultracentrifugation. Delipided human VLDL was separated by Sephacryl S-200 molecular sieve chromatography. ApoE was purified by heparin Sepharose CL-6B affinity chromatography. ApoC I ,C II and C III were purified from apoC. fraction by DEAE-Sephacel ion exchange chromatography: RESULTS: Purified apoE, apoC I, apoC II and apoC III were obtained. SDS-PAGE and immunodiffusion tests indicated the isolated proteins were pure. CONCLUSION: We have established a purification procedure for human VLDL apolipoproteins with highly efficiency and simplicity by MPLC.

Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions.

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3beta-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum.

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5 percent and 47.6 percent less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

[Protective immunity of the recombinant Schistosoma japonicum specific very low density lipoprotein binding protein as a vaccine candidate].

OBJECTIVE: To investigate the protective immunity against Schistosoma japonicum in mice immunized with recombinant specific very low density lipoprotein binding protein (SVLBP) and its potential as vaccine candidate. METHODS: Recombinant SVLBP antigen was over-expressed under IPTG induction and purified by Ni-NTA affinity chromatography. C57BL/6 mice were immunized three times with purified reSVLBP complexed with Freund's adjuvant, at biweekly intervals. Then 35+/-1 cercariae of S. japonicum were given to each mouse by abdominal skin 10 days after the 3rd immunization. 45 days later, all mice were sacrificed to collect adult worms and count liver eggs. serum samples were collected before immunization and after challenge respectively, and were probed the antigen-specific antibodies using a panel of ELISAs. RESULTS: The worm burden and the egg deposition in liver tissue were reduced by 33.4% and 47.6% respectively in the immunized group, in comparison with the adjuvant control group (P<0.05). Higher titer (>1:6 400) of total IgG was observed after challenge infection. The vaccinated mice developed significantly higher levels of IgG2a, IgG2b, IgG1 than those of control mice. CONCLUSION: The recombinant tegumental SVLBP antigen could induce partial protection against S. japonicum infection. These data demonstrate the potential of SVLBP as a schistosome vaccine candidate.

Elevation of ceramide in serum lipoproteins during acute phase response in humans and mice: role of serine-palmitoyl transferase.

Recent studies have indicated that ceramide generated in the liver is secreted into the bloodstream as component of very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). This manuscript investigates the effect of host acute phase response to inflammation on lipoprotein ceramide levels. In humans, two different patterns of responses were found. One group of volunteers experienced transient increases in serum ceramide at 1.5h after LPS administration. Second group showed prolonged increases that reached up to 10-fold above the basal level and continued for up to 24h. Increases in ceramide were found only in VLDL and LDL particles. LPS administration induced similar increases in mice. These increases were accompanied by activation of secreted sphingomyelinase in serum and serine-palmitoyl transferase in liver. ASMase knockout mice retained LPS-induced increases in serum ceramide, thus suggesting that the elevation of VLDL and LDL ceramide content is attributed at least in part to activation of de novo synthesis of ceramide in the liver.

Instability of crab vitellogenin and its immunological relatedness with mammalian atherogenic lipoproteins.

Vitellogenesis is the process of accumulation of vitellogenin (Vg) in rapidly growing oocytes of oviparous animals and its' subsequent transformation into lipovitellin (Lv). Lipovitellin, which forms the major yolk protein, serves as a principal nutrient reserve for the developing embryo. In the present study, Vg and Lv were purified from the hemolymph and ovary, respectively of the crab Scylla serrata by gel filtration followed by preparative gel electrophoresis. It was observed that purified Vg, but not Lv, possessed an intrinsic protease activity with which it underwent autoproteolysis giving rise to several smaller proteins. Furthermore, urea-mediated unfolding studies by UV-spectral analysis revealed clearly that Vg was easily disrupted by urea whereas Lv was resistant. Taken together, these results suggest that although Lv had a stable conformation, its precursor Vg was labile and highly sensitive to degradation. Another aspect that was investigated in the present study was the immunological kinship of crab Vg and Lv to mammalian atherogenic lipoproteins, the low density lipoprotein (LDL), very low density lipoprotein (VLDL), and apolipoprotein B (apoB). By Western blot analysis, it was demonstrated that crab Vg and Lv were immunoreactive to antibodies to human LDL, VLDL, and apoB. These observations suggest the existence of common epitope recognition sites in crab Vg and mammalian lipid transferring proteins. This corroborates well with our earlier study on the recognition of crab Vg receptor by mammalian lipoproteins.

Assays for acetaldehyde-derived adducts in blood proteins based on antibodies against acetaldehyde/lipoprotein condensates.

BACKGROUND: Acetaldehyde-derived protein condensates (adducts) have been suggested as promising biological markers of alcohol abuse because they represent actual metabolites of ethanol. However, the detection of such condensates in vivo has been hampered by a lack of sensitive and specific methods. METHODS: To develop new approaches for the detection of acetaldehyde adducts, we have raised antibodies against condensates with acetaldehyde and lipoproteins, which have previously been shown to be readily modified by acetaldehyde in vitro. The characteristics of these antibodies were compared with those raised against bovine serum albumin/acetaldehyde adduct and against other types of lipoprotein modifications, as induced by malondialdehyde, oxidation, and acetylation. RESULTS: The antibodies raised against low-density lipoprotein (LDL)/acetaldehyde, very low density lipoprotein (VLDL)/acetaldehyde, and bovine serum albumin/acetaldehyde all reacted with protein adducts generated at physiologically relevant concentrations of acetaldehyde in vitro, whereas the antibodies raised against malondialdehyde/LDL, oxidized LDL, or acetylated LDL were not found to cross-react with the acetaldehyde-derived adducts. In assays for acetaldehyde adducts from erythrocyte and serum proteins of patients with excessive ethanol consumption (n = 32) and healthy control individuals (n = 22), the antibody prepared against the acetaldehyde/VLDL condensate was found to provide the most effective detection of acetaldehyde adducts in vivo. CONCLUSIONS: Current data indicate that acetaldehyde generates immunogenic adducts with lipoproteins in vivo. Antibodies raised against the VLDL/acetaldehyde may provide a basis for new diagnostic assays to examine excessive alcohol consumption.

LDL immunization induces T-cell-dependent antibody formation and protection against atherosclerosis.

Atherosclerosis is an inflammatory disease, and the involvement of immune mechanisms in disease progression is increasingly recognized. Immunization with oxidized low density lipoprotein (LDL) decreases atherosclerosis in several animal models. To explore humoral and cellular immune reactions involved in this protection, we immunized apolipoprotein E knockout mice with either homologous plaque homogenates or homologous malondialdehyde (MDA)-LDL. Immunization with both these antigen preparations reduced lesion development. The plaques contained immunogen(s) sharing epitopes on MDA-LDL, MDA-very low density lipoprotein, and oxidized cardiolipin. This shows that a T-cell-dependent antibody response was associated with protection against atherosclerosis. The protection was associated with specific T-cell-dependent elevation of IgG antibodies against MDA-LDL and oxidized phospholipids, and the increased titers of IgG antibodies were correlated with decreased lesion formation and lower serum cholesterol levels.

Sphingomyelinase converts lipoproteins from apolipoprotein E knockout mice into potent inducers of macrophage foam cell formation.

The apoE knockout (E0) mouse is one of the most widely used animal models of atherosclerosis, and there may be similarities to chylomicron remnant-induced atherosclerosis in humans. Although the lesions of these mice contain large numbers of cholesteryl ester (CE)-laden macrophages (foam cells), E0 plasma lipoproteins are relatively weak inducers of cholesterol esterification in macrophages. Previous in vivo work has suggested that arterial wall sphingomyelinase (SMase) may promote atherogenesis in the E0 mouse, perhaps by inducing subendothelial lipoprotein aggregation and subsequent foam cell formation. The goal of the present study was to test the hypothesis that the modification of E0 lipoproteins by SMase converts these lipoproteins into potent inducers of macrophage foam cell formation. When d<1.063 E0 lipoproteins were pretreated with SMase and then incubated with E0 macrophages, cellular CE mass and stimulation of the cholesterol esterification pathway were increased approximately 5-fold compared with untreated lipoproteins. SMase-treated E0 lipoproteins were more potent stimulators of cholesterol esterification than either E0 lipoproteins in the presence of lipoprotein lipases or oxidized E0 lipoproteins. The uptake and degradation of SMase-treated E0 lipoproteins by macrophages were saturable and specific and substantially inhibited by partial proteolysis of cell-surface proteins. Uptake and degradation were diminished by an anti-apoB antibody and by competition with human S(f) 100-400 hypertriglyceridemic VLDL, raising the possibility that a receptor that recognizes apoB-48 might be involved. In conclusion, SMase-modification of E0 lipoproteins, a process previously shown to occur in lesions, may be an important mechanism for foam cell formation in this widely studied model of atherosclerosis. Moreover, the findings in this report may provide important clues regarding the atherogenicity of chylomicron remnants in humans.

Very low-density lipoprotein receptor in fetal intestine and gastric adenocarcinoma cells.

BACKGROUND: A very low-density lipoprotein receptor (VLDLR) was recently identified. This receptor reportedly binds specifically to very low-density lipoproteins; however, its distribution and functions in vivo have yet to be elucidated. We investigated the expression and regulation of VLDLR in fetal and carcinoma cells. OBJECTIVE: The expression of VLDLR was examined by immunohistochemistry and reverse-transcriptase polymerase chain reaction using several specimens, including a fetus of 12 to 15 weeks' gestation, various tumors, AGS cells, and INT407 cells. RESULTS: Immunoreactive VLDLR was abundantly present in human fetal intestinal epithelial and gastric adenocarcinoma cells. This receptor was also noted in the intestinal cell line, INT407, and gastric cancer cell line, AGS. In addition, the VLDLR that was expressed in INT407 cells, AGS cells, and gastric adenocarcinoma tissue was present mainly in a variant form lacking the O-linked sugar domain. CONCLUSIONS: These data suggest that an important function of VLDLR may be the mediation of cell growth in developing tissues, such as fetal intestinal and cancer cells. The INT407 and AGS cell lines appear to be useful for examining the regulation of VLDLR expression.

Very low density lipoproteins and interleukin 2 enhance the immunogenicity of 9-O-acetyl-GD3 ganglioside in BALB/c mice.

Gangliosides expressed by tumor cells constitute potential targets for immunotherapy. A major limitation of protocols aiming to immunize patients against tumor gangliosides is the weak immunogenicity of these molecules. We have previously shown that exogenous gangliosides are essentially bound to serum lipoproteins. In this study we have analyzed the influence of human serum lipoproteins on the immunogenicity of purified human ganglioside 9-O-acetyl-GD3 in BALB/c mice. Although expressed at very low levels in mice, this ganglioside was not immunogenic when administered in the form of micelles. However 9-O-acetyl-GD3 adsorbed onto Very Low Density Lipoproteins (VLDL) was strongly and reproducibly immunogenic, inducing both an IgM and an IgG response, with higher titers than those obtained with total serum. The IgM antibody response appeared after a single injection whereas the IgG response was observed after 3 weeks but was stronger and more durable. The antibody response to 9-O-acetyl-GD3 bound to other serum fractions was weak or absent. The addition of recombinant interleukin 2 (IL-2) enhanced weak antibody responses to 9-O-acetyl-GD3 thereby facilitating responses to ganglioside in micelles and in protein-free Very Low Density Particles. Using in vitro assays, we demonstrated that VLDL-bound ganglioside 14C-GM3 was more sensitive to the effect of neuraminidase than gangliosides bound to other lipoprotein fractions, suggesting greater accessibility of VLDL-bound gangliosides. These results indicate that VLDL-bound gangliosides are the most immunologically active fraction of serum gangliosides. VLDL or similar particles and recombinant IL-2 may be useful adjuvants for immunization with gangliosides.

In the absence of the low density lipoprotein receptor, human apolipoprotein C1 overexpression in transgenic mice inhibits the hepatic uptake of very low density lipoproteins via a receptor-associated protein-sensitive pathway.

To study the role of apoC1 in lipoprotein metabolism, we have generated transgenic mice expressing the human APOC1 gene. On a sucrose-rich diet, male transgenic mice with high APOC1 expression in the liver showed elevated levels of serum cholesterol and triglyceride compared with control mice (5.7+/-0.7 and 3.3+/-2.1 vs. 2.7+/-0.1 and 0.4+/-0.1 mmol/liter, respectively). These elevated levels were mainly confined to the VLDL fraction. Female APOC1 transgenic mice showed less pronounced elevated serum lipid levels. In vivo VLDL turnover studies revealed that, in hyperlipidemic APOC1 transgenic mice, VLDL particles are cleared less efficiently from the circulation as compared with control mice. No differences were observed in the hepatic production and extrahepatic lipolysis of VLDL-triglyceride. Also, VLDL isolated from control and APOC1 transgenic mice were found to be equally good substrates for bovine lipoprotein lipase in vitro. These data indicate that the hyperlipidemia in APOC1 transgenic mice results primarily from impaired hepatic VLDL particle clearance, rather than a defect in the hydrolysis of VLDL-triglyceride. To investigate which hepatic receptor is involved in the apoC1-mediated inhibition of VLDL clearance, APOC1 transgenic mice were bred with an LDL receptor-deficient (LDLR(-/-)) background. In addition, control, LDLR(-/-), and LDLR(-/-)/APOC1 mice were transfected with adenovirus carrying the gene for the receptor-associated protein (Ad-RAP). Both serum cholesterol and triglyceride levels were strongly elevated in LDLR(-/-)/APOC1 mice compared with LDLR(-/-) mice (52+/-19 and 36+/-19 vs. 8.4+/-0.9 and 0.5+/-0.2 mmol/liter, respectively), indicating that apoC1 inhibits the alternative VLDL clearance pathway via the remnant receptor. Transfection of LDLR(-/-) mice with Ad-RAP strongly increased serum cholesterol and triglyceride levels, but to a lesser extent than those found in LDLR(-/-)/APOC1 mice (39+/-8 and 17+/-8 vs. 52+/-19 and 36+/-19 mmol/liter, respectively). However, in LDLR(-/-)/APOC1 mice the transfection with Ad-RAP did not further increase serum cholesterol and triglyceride levels (52+/-19 and 36+/-19 vs. 60+/-10 and 38+/-7 mmol/liter, respectively). From these studies we conclude that, in the absence of the LDLR, apoC1 inhibits the hepatic uptake of VLDL via a RAP-sensitive pathway.

Immunization with cholesterol-rich liposomes induces anti-cholesterol antibodies and reduces diet-induced hypercholesterolemia and plaque formation.

Immunization of rabbits with a protein-free formulation consisting of liposomes containing 71% cholesterol and lipid A as an adjuvant induced anticholesterol antibodies that caused complement-dependent lysis of liposomes lacking lipid A. The antibodies, immunoglobulin G (IgG) and immunoglobulin M (IgM), also recognized nonoxidized crystalline cholesterol as an antigen by enzyme-linked immunosorbent assay (ELISA). The effects of immunization against cholesterol on elevations in serum cholesterol and development of atherosclerosis were examined in rabbits fed a diet containing 0.5% to 1.0% cholesterol. Although the mean serum cholesterol level, mainly in the form of very-low-density lipoprotein cholesterol, rose as much as 60-fold in the nonimmunized rabbits, the elevation was significantly less--as much as 35% lower--in the immunized rabbits. Elevation of serum cholesterol was accompanied by an apparent drop in the level of antibodies on initiating the diet, followed by a rebound on stopping the diet, thus suggesting that the antibodies were adsorbed to cholesterol that was present in circulating lipoproteins. When lipoprotein fractions--composed of either very-low-density and intermediate-density lipoproteins derived from cholesterol-fed nonimmunized rabbits or human low-density lipoproteins--were tested as capture antigens by solid-phase ELISA, reactivity was observed with IgG and IgM antibodies present in the serum of immunized rabbits. Immunization also resulted in a marked decrease in the risk of developing atherosclerosis. Analysis of aortic atherosclerosis by quantitative histologic examination and fatty streaks by automated morphometric probability-of-occurrence mapping showed diminished atherosclerosis in most areas of the aorta in vaccine recipients. It is proposed that immunization with liposomes containing 71% cholesterol and lipid A can reduce diet-induced hypercholesterolemia and atherosclerosis.

Challenges for development of hepatitis C virus vaccines.

Impediments to the development of a hepatitis C virus (HCV) vaccine are reviewed. Foremost is the perception that the limited transmissability of HCV, and reduced spread by blood-associated routes, make this a low priority target. It is argued that such a vaccine may have an important therapeutic use in the treatment of chronic HCV carriers of which an estimated 30 million exist worldwide. An HCV vaccine would also have prophylactic use in multivalent (hepatitis) vaccines, and in the developing world. An effective HCV vaccine will not be easy to develop. The high variability of the viral proteins, especially that of the envelope proteins, provide a major challenge. The association of HCV with very low density lipoproteins renders a major proportion of the virions non-neutralizable, a further challenge. It may be necessary to design an HCV vaccine which acts primarily through the generation of cytotoxic lymphocytes reactive with conserved epitopes displayed on the surface of infected cells.