'Off-the-shelf' allogeneic CAR T cells: development and challenges.

Autologous chimeric antigen receptor (CAR) T cells have changed the therapeutic landscape in haematological malignancies. Nevertheless, the use of allogeneic CAR T cells from donors has many potential advantages over autologous approaches, such as the immediate availability of cryopreserved batches for patient treatment, possible standardization of the CAR-T cell product, time for multiple cell modifications, redosing or combination of CAR T cells directed against different targets, and decreased cost using an industrialized process. However, allogeneic CAR T cells may cause life-threatening graft-versus-host disease and may be rapidly eliminated by the host immune system. The development of next-generation allogeneic CAR T cells to address these issues is an active area of research. In this Review, we analyse the different sources of T cells for optimal allogeneic CAR-T cell therapy and describe the different technological approaches, mainly based on gene editing, to produce allogeneic CAR T cells with limited potential for graft-versus-host disease. These improved allogeneic CAR-T cell products will pave the way for further breakthroughs in the treatment of cancer.

A methodological framework to assess the socio-economic impact of underground quarries: A case study from Belgian Limburg.

This study developed a methodology to assess the socio-economic impact of the presence and collapse of underground limestone quarries. For this we rely on case study evidence from Riemst, a village located in Eastern Belgium and use both secondary and primary data sources. A sinkhole inventory as well as data about the prevention costs provided by the municipality was used. To estimate the recreational values of the quarries, visitor data was obtained from the tourist office of Riemst. Next, two surveys were conducted among inhabitants and four real estate agents and one notary. The direct and indirect damages were assessed using respectively the repair cost and production and real estate value losses. The total yearly direct and indirect damage equals €415000 (±€85000) and more than half of it can be attributed to the depreciation of real estate (€230000). The quarries have recreational, cultural-historical and ecological values and thus generate societal benefits. The yearly recreational value was at least €613000 in 2012 values. The ecological and cultural-historical values augment to €180000 per year (in 2012 values). Further, our study indicates that the gains from filling up the quarries below the houses located above an underground limestone quarry outweigh the costs in the case study area. The net gain from filling up the underground quarry ranges €38700 to €101700 per house. This is only the lower bound of the net gain from filling up these underground quarries since preventive filling makes future collapses less likely so that future direct repair costs will be most likely smaller.

Lentiviral protein delivery of meganucleases in human cells mediates gene targeting and alleviates toxicity.

Site-specific endonucleases can be engineered for custom recognition of any genetic locus and used for gene targeting. Yet, the prolonged expression and accumulation of these nucleases in cells lead to toxic effect. Here we describe an efficient and quantitative method for introducing nucleases into cells as proteins packaged within lentiviral vector particles. I-CreI-derived meganucleases, which can be engineered as single-chain proteins, were incorporated into lentiviral vector particles either without modification or as fusions with cyclophilin A. The small amount of nuclease delivered by the viral particles is sufficient to induce efficient targeted mutagenesis in human HEK293H and primary T cells. When a repair template sequence was packaged in the lentiviral vector, high levels of homologous gene targeting were obtained and toxicity was markedly reduced.

Apolipoprotein L gene family: tissue-specific expression, splicing, promoter regions; discovery of a new gene.

Previously we identified and cloned the cDNA for a new protein, apolipoprotein L (apoL), present in plasma and mainly associated with large high density lipoprotein particles. Using 5' rapid amplification of cDNA ends, RT-PCR and comparison with three Human Genome Project and three expressed sequence tag sequences, we have characterized the gene for apoL and for three additional, highly homologous proteins that constitute a new family of proteins that display no homology with previously described apolipoproteins. The genes for all four proteins, apoL-I, apoL-II, apoL-III, and apoL-IV, are located at chromosome 22q12.1-13.1 within a 127,000-bp region. The apoL-I gene is in the opposite orientation to the other three. All four genes have TATA-less promoters, which contain putative sterol regulatory elements, suggesting that transcription of these genes may be coordinated with that of the low density lipoprotein receptor and genes in pathways involving the synthesis of triglycerides and cholesterol. The gene family has a consensus eight-exon structure with alternative splice sites that could produce as many as eight distinct gene products. The apoL-II and apoL-III genes have alternative transcriptional start sites as a result of additional 5' exons. apoL-I, apoL-II, and apoL-III are expressed to the highest degree in the lung. Other tissues with high expression are the pancreas, prostate, spleen, liver, and placenta. Four clustered common polymorphisms, three of which altered the protein sequence, were found in apoL-I, all in linkage disequilibrium, and describing two haplotypes: the more common Lys166/Ile244/Lys271 and the rarer Glu166/Met244/Arg271.

Plasma apolipoprotein L concentrations correlate with plasma triglycerides and cholesterol levels in normolipidemic, hyperlipidemic, and diabetic subjects.

Apolipoprotein L is a newly recognized component of human plasma lipoproteins. Mainly associated with apoA-I-containing lipoproteins, it is a marker of distinct HDL subpopulations. In an effort to gain inference as to its as yet unknown function, we studied biological determinants of apoL levels in human plasma. The distribution of apoL in normal subjects is asymmetric, with marked skewing toward higher values. No difference was found in apoL concentrations between males and females, but we observed an elevation of apoL in primary hypercholesterolemia (10.1 vs. 8.5 microgram/mL in control), in endogenous hypertriglyceridemia (13.8 microgram/mL, P < 0.001), combined hyperlipidemia phenotype (18.7 g/mL, P < 0.0001), and in patients with type II diabetes (16.2 microgram/mL, P < 0.02) who were hyperlipidemic. Significant positive correlations were observed between apoL and the log of plasma triglycerides in normolipidemia (0.446, P < 0.0001), endogenous hypertriglyceridemia (0.435, P < 0.01), primary hypercholesterolemia (0.66, P < 0.02), combined hyperlipidemia (0.396, P < 0.04), hypo-alphalipoproteinemia (0.701, P < 0.005), and type II diabetes with hyperlipidemia (0.602, P < 0. 01). Apolipoprotein L levels were also correlated with total cholesterol in normolipidemia (0.257, P < 0.004), endogenous hypertriglyceridemia (0.446, P = 0.001), and non-insulin-dependent diabetes mellitus (NIDDM) (0.548, P < 0.02). No significant correlation was found between apoL and body mass index, age, sex, HDL-cholesterol or fasting glucose and glycohemoglobin levels. ApoL levels in plasma of patients with primary cholesteryl ester transfer protein deficiency significantly increased (7.1 +/- 0.5 vs. 5.47 +/- 0.27, P < 0.006).

Analysis of hABC1 gene 5' end: additional peptide sequence, promoter region, and four polymorphisms.

Evidence linking mutations in ATP-binding-cassette transporter gene 1 (ABC1) to Tangier disease suggests it functions in the active transport of free cholesterol out of cells. Since its mRNA level is regulated in response to cellular cholesterol stores it is of interest to explore its promoter response elements, and to investigate polymorphisms for their contributions to the prevalence of low levels of HDL in the population that promotes premature coronary heart disease. Investigation of the 5' end of the gene by 5' RACE analysis revealed 455 nucleotides additional to published sequences, and predicts another 60 amino acid N-terminal residues, resulting in a 2261-residue protein. Protein sequence analysis predicts a membrane-spanning region and possible signal peptide. The 5' flanking region was located by a Human Research Project BLAST search. This region contains regulatory elements that potentially control ABC1 gene expression. In addition to numerous SP1 binding sites there are four putative sterol regulatory elements (SREs). Our studies uncovered three single nucleotide substitution polymorphisms, one in the promoter region and two in the 5' untranslated region (5'UTR), plus an insertion/deletion polymorphism.

Prebeta-1 HDL in plasma of normolipidemic individuals: influences of plasma lipoproteins, age, and gender.

Prebeta-1 HDL is a molecular species of plasma HDL of approximately 67 kDa mass that contains apolipoprotein A-I, phospholipids, and unesterified cholesterol. It participates in a cyclic process involved in the retrieval of cholesterol from peripheral tissues. In this cycle, unesterified cholesterol from cells is incorporated into prebeta-1 HDL, providing a substrate for esterification of cholesterol by lecithin:cholesterol acyltransferase. Prebeta-1 HDL then becomes incorporated into larger HDL species of alpha mobility as esterification proceeds and is regenerated during the transfer of cholesteryl esters from alpha HDL particles to acceptor lipoproteins. Thus the steady state level of prebeta-1 HDL in plasma reflects the relative efficiencies of the major metabolic processes involved in its generation and removal. We have used an isotope dilution technique to measure prebeta-1 HDL levels in the plasmas of 136 normolipidemic individuals (46 M, 90 F). The mean absolute concentration of prebeta-1 HDL as apolipoprotein A-I was 68 +/- 40 microg/ml for women, and 84 +/- 49 m/ml for men. Prebeta-1 HDL represented 5.5 +/- 3.3% of total apolipoprotein A-I in women, and 7.2 +/- 4.0% in men. The distributions of both absolute and percent prebeta-1 HDL are highly asymmetric, with skew toward higher values. However, the skew appears not to be attributable to either plasma cholesterol or triglyceride levels which are also skewed in population samples. The percent prebeta-1 HDL was negatively correlated with HDL cholesterol levels (P < 0.0001), whereas absolute levels of prebeta-1 HDL were positively correlated with apolipoprotein A-I and negatively correlated with HDL cholesterol (P, for both, < 0.0001). Multiple linear regression analysis revealed effects of age and gender, but no association with lipoprotein fractions other than HDL. Lower levels of prebeta-1 HDL were associated with female gender in all models.

Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.

In this study, we have identified and characterized a new protein present in human high density lipoprotein that we have designated apolipoprotein L. Using a combination of liquid-phase isoelectrophoresis and high resolution two-dimensional gel electrophoresis, apolipoprotein L was identified and partially sequenced from immunoisolated high density lipoprotein (Lp(A-I)). Expression was only detected in the pancreas. The cDNA sequence encoding the full-length protein was cloned using reverse transcription-polymerase chain reaction. The deduced amino acid sequence contains 383 residues, including a typical signal peptide of 12 amino acids. No significant homology was found with known sequences. The plasma protein is a single chain polypeptide with an apparent molecular mass of 42 kDa. Antibodies raised against this protein detected a truncated form with a molecular mass of 39 kDa. Both forms were predominantly associated with immunoaffinity-isolated apoA-I-containing lipoproteins and detected mainly in the density range 1.123 < d < 1.21 g/ml. Free apoL was not detected in plasma. Anti-apoL immunoaffinity chromatography was used to purify apoL-containing lipoproteins (Lp(L)) directly from plasma. Nondenaturing gel electrophoresis of Lp(L) showed two major molecular species with apparent diameters of 12.2-17 and 10.4-12.2 nm. Moreover, Lp(L) exhibited both pre-beta and alpha electromobility. Apolipoproteins A-I, A-II, A-IV, and C-III were also detected in the apoL-containing lipoprotein particles.

A novel apolipoprotein C-III variant, apoC-III(Gln38-->Lys), associated with moderate hypertriglyceridemia in a large kindred of Mexican origin.

Apolipoprotein C-III (apoC-III) is a major protein component of very low density lipoproteins (VLDL), chylomicrons, and a minor component of high density lipoproteins (HDL). Studies of naturally occurring human variants of apoC-III will help in adding to our understanding of the physiological function of this apolipoprotein. Using isoelectric focusing (IEF) of VLDL fractions we screened over 2500 lipid clinic patients and have identified an individual with a novel apoC-III variant. DNA sequencing revealed the variant to be a Lys for GIn exchange at amino acid residue 38 due to an A for C substitution in exon 3. This was confirmed by NH2-terminal protein sequence analysis. The mutant Lys38 variant was present in VLDL at about the same level as the normal form although the total amount of apoC-III was increased by 34%. The proband, a 16-year-old boy of Mexican origin, had a plasma level of total triglycerides above the 95th percentile for his age. Family studies revealed a further 16 individuals who were heterozygous for this apoC-III (Gln38-->Lys) variant. Compared to 21 unaffected relatives, the 17 heterozygous subjects had a statistically significant 32% elevation of their plasma levels of triglycerides when adjusted for age, sex, body mass index, and lifestyle. Other lipid and lipoprotein values were unaffected. The presence of an additional positive charge at residue 38 suggests that this residue is involved in the function of apoC-III. The elevation of plasma levels of triglycerides supports the view that apoC-III is involved in the regulation of the catabolism of triglyceride-rich lipoproteins.

Measurement of prebeta-1 HDL in human plasma by an ultrafiltration-isotope dilution technique.

Prebeta-1 HDL is a 67-kDa species of plasma high-density lipoproteins (HDL) that contains two copies of apolipoprotein A-I. It functions in a metabolic cycle of cholesterol retrieval and may be formed during lipolysis in plasma. We have found that centrifugal ultrafiltration using a membrane with a permeability limit of 100 kDa discriminates categorically between the 67-kDa species and larger HDL particle species. Thus, the ultrafiltrate samples the pool of prebeta-1 HDL in plasma. We have developed a technique using the dispersal of purified prebeta-1 HDL, labeled covalently with tritium, in plasma samples, to label the prebeta-1 HDL pool. Subsequent determination of the specific activity of prebeta-1 HDL in the ultrafiltrate provides a means of calculating the content of prebeta-1 HDL in plasma by the isotope dilution principle. We employ a modification of an enzyme-linked immunosorbent assay technique for apolipoprotein A-I that allows the equal detection of that protein in prebeta-1 HDL and in other HDL particle species for determination of the fraction of total apolipoprotein A-I that is present in the prebeta-1 HDL particle species. The mean level of prebeta-1 HDL-associated apolipoprotein A-I in plasma samples from 86 normolipidemic adults was 74 +/- 43 microg/ml (+/-SD), representing an average of 6.6% of the total apolipoprotein A-I in plasma.

Conformational differences in human apolipoprotein B-100 among subspecies of low density lipoproteins (LDL). Association of altered proteolytic accessibility with decreased receptor binding of LDL subspecies from hypertriglyceridemic subjects.

We asked at what point in the metabolic cascade of very low density lipoproteins (VLDL) to low density lipoproteins (LDL) the accessibility of proteolytic cleavage sites in B-100 changes, and we evaluated the effect of hypertriglyceridemia on the proteolytic accessibility, secondary structure, and receptor-binding affinity of B-100 in LDL subspecies of varying density. Limited proteolysis with Staphylococcus aureus V8 protease and cathepsin D identified the density (about 1.033 g/ml) between two LDL subspecies, designated LDL-1 and -2, as the transition point during VLDL metabolism of both normolipidemic (N-) and hypertriglyceridemic (HTG-) subjects at which accessibility to protease attack changed in three peptide regions of B-100. Hypertriglyceridemia greatly altered proteolytic accessibility of B-100 in the denser LDL subspecies. Specifically, B-100 in HTG-LDL exposed more cleavage sites than in N-LDL, including two novel sites, approximately 120 and approximately 130 kDa from the NH2 terminus in the small and dense subspecies (designated LDL-4, -4.5 or -5, d = 1.048-1.062 g/ml). Analysis of circular dichroic spectra indicated no difference in helical content between B-100 in N- and HTG-LDL but showed a greater content of beta-structure in HTG-LDL. Binding affinity for the LDL receptor of human fibroblasts decreased markedly with increasing density among HTG-LDL subspecies (by approximately 50% for LDL-4.5 or -5). We conclude that the changes in proteolytic accessibility observed between LDL-1 and -2 and in LDL-4, -4.5, or -5 indicate significant differences in local conformation of B-100 at specific peptide regions. The association of exposure of more cleavage sites, especially novel sites in the NH2-terminal regions, with greatly decreased receptor-binding affinity in LDL-4.5 or -5 suggests that altered local conformation in B-100 apart from the putative receptor-binding domain might affect interaction with the receptor.

Structural domain of apolipoprotein A-I involved in its interaction with cells.

Apolipoprotein A-I (apo A-I) is the major protein constituent of high-density lipoprotein (HDL), the lipoprotein fraction which mediates the reverse cholesterol transport. This apolipoprotein plays an important role in the binding of HDL to cells and participates in the efflux of cellular cholesterol. We have recently compared six different genetic variants of apo A-I and found that the apo A-I (Pro 165-->Arg) mutant is defective in promoting cellular cholesterol efflux from murine adipocytes and peritoneal macrophages and we have proposed that this region of apo A-I may be involved in their interaction with cells. To confirm this hypothesis, four monoclonal antibodies (mAbs) specific for apo A-I were used to study the inhibition of the interaction of palmitoyloleoylphosphatidylcholine (POPC): apoA-I complexes with HeLa cells and adipocytes. Among these antibodies, the apo A-I epitope recognized by the A44 mAb lies in the COOH terminal region (amino acid residues 149-186) including the proposed region. The antibodies A05, and A03 react with residues 25-82, 135-140, respectively and the A11 mAb corresponds to a discontinuous epitope at residues 99-105 and 126-132. Our results show clearly that the A44 and A05 mAbs reduce both the binding to HeLa cells and the cholesterol efflux from adipocytes. The inhibition of POPC: apoA-I complexes binding to both cell types is more strictly observed with the Fab fragments of monoclonal antibodies A44 and A05. Partial cotitration curves of these mAbs in a solid phase assay (RIA), indicated partial competition between these two antibodies. We propose a structural model for the POPC: apoA-I complexes where the N-terminal domain of one apo A-I molecule is in close spatial relationship with the C-terminal domain of the adjacent apo A-I molecule. We therefore suggest that the domain around amino acid 165 of apo A-I and which is recognized by mAb A44 (149-186) forms or contains some specific regions which mediate selectively the interaction with the binding site of cells and is involved in the efflux of cellular cholesterol.

Transfer of cholesterol from Ob1771 cells or LDL to reconstituted, defined high density lipoproteins.

We used defined, reconstituted high density lipoproteins (rHDL) to study the effects of structure and composition of these particles on their role as cholesterol acceptors from cell membranes or from low density lipoproteins (LDL). Three discoidal rHDL and one spherical rHDL with distinct apolipoprotein A-I conformations, diameters and compositions were used in conjunction with Ob1771 cells to measure the rate of [3H]cholesterol efflux from the cells, direct binding to the cells, and competition with native HDL3 for binding. In addition, the same rHDL particles were used to study the kinetics of cholesterol mass transfer from LDL. The results show that the rates of cholesterol transfer depend on the nature of the donor (t1/2 11-19 min from LDL, and t1/2 5 h from the cells), on the phosphatidylcholine/cholesterol ratio in the acceptors (the closer this ratio is to the equilibrium value, the slower is the rate), and on the diameter of the acceptors (the smallest particles have the lowest t1/2 for cholesterol uptake from LDL, and are the most effective acceptors of [3H]cholesterol from cells after their phospholipid content is taken into account). The cholesterol uptake by the rHDL, both from the cells and from LDL, is determined mostly by the phospholipid pool available in the acceptors. Binding to the cells was equivalent for all the rHDL (Kd = 38-67 micrograms/ml) and comparable to HDL3, suggesting that the differences in apoA-I conformation have no effect on the binding to cells. Finally we observed that exposure of rHDL to cells may lead to remodeling of some of the lipoprotein particles.

Biochemical characterization of the three major subclasses of lipoprotein A-I preparatively isolated from human plasma.

Apolipoprotein (apo) A-I is the major protein constituent of plasma high-density lipoproteins (HDL). HDL consist of two major classes of apoA-I-containing lipoproteins: LpA-I and LpA-I:A-II. LpA-I includes heterogeneous lipoprotein particles that differ in size and hydrated density. LpA-I was isolated by immunoaffinity chromatography from the fasting plasma of 24 normal human subjects and separated by gel filtration chromatography. Three major subclasses of LpA-I were eluted: large (Lg-LpA-I), medium (Md-LpA-I), and small LpA-I (Sm-LpA-I). By nondenaturing gradient PAGE, Lg-LpA-I, Md-LpA-I, and Sm-LpA-I had mean Strokes diameters of 10.8 +/- 0.5, 8.9 +/- 0.5, and 7.5 +/- 0.3 nm, respectively. The lipid/protein ratios were 1.25 +/- 0.12 for Lg-LpA-I, 0.75 +/- 0.10 for Md-LpA-I, and 0.38 +/- 0.08 for Sm-LpA-I. Lg-LpA-I was relatively lipid and cholesteryl ester rich compared with Md-LpA-I and Sm-LpA-I. Sm-LpA-I contained phospholipids as the major lipid component. ApoA-I was the major apolipoprotein in all LpA-I subfractions, whereas apoE was present only in Lg-LpA-I and apoA-IV was associated with both Md-LpA-I and Sm-LpA-I. All three LpA-I subclasses exhibited predominantly alpha mobility on agarose electrophoresis. Lg-LpA-I migrated as a diffuse band in the fast alpha position, whereas Md-LpA-I and Sm-LpA-I migrated to the slow alpha position.(ABSTRACT TRUNCATED AT 250 WORDS)

Tangier disease: isolation and characterization of LpA-I, LpA-II, LpA-I: A-II and LpA-IV particles from plasma.

Tangier disease (TD) is characterized by extremely low plasma levels of HDL, apoA-I and apoA-II due to very rapid catabolism. However, the risk of premature coronary heart disease (CHD) is not markedly increased in TD. In order to gain insight into reverse cholesterol transport in TD, we isolated LpA-I, LpA-I:A-II, LpA-II and LpA-IV particles from fasting plasma of 5 TD patients. LpA-I composition was similar to control LpA-I, but TD LpA-I had more LCAT and CETP activity (respectively, 0.35 +/- 0.14 and 0.14 +/- 0.04 mumol of cholesterol esterified/h/micrograms of protein, and 7 +/- 2.5 and 1.4 +/- 0.3 mumol of cholesteryl ester transferred/h/micrograms of protein). In contrast, TD LpA-I:A-II had abnormal composition, with a low molar ratio of apoA-I to apoA-II (0.2-1.33). In addition, LpA-I:A-II in TD contained a substantial amount of apoA-IV compared with control, making this particle an LpA-I:A-II:A-IV complex. LpA-I:A-II from normal plasma do not promote cholesterol efflux from adipocytes cells, whereas TD LpA-I:A-II:A-IV complexes promoted cholesterol efflux from these cells. Moreover LpA-I:A-II:A-IV complexes have more LCAT and CETP activity than control (respectively 1.2 +/- 0.16 and 0.05 +/- 0.01 mumol of cholesterol esterified/h/micrograms of protein and, 41 +/- 3.7 and 1 +/- 0.4 mumol of cholesteryl ester transferred/h/micrograms of protein). The LpA-II particle in TD represented in fact an LpA-II:A-IV complex (75% mol apoA-II and 22% mol apoA-IV).(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol esters selectively delivered in vivo by high-density-lipoprotein subclass LpA-I to rat liver are processed faster into bile acids than are LpA-I/A-II-derived cholesterol esters.

High-density lipoprotein (HDL) subclass LpA-I has been reported to promote cholesterol efflux from mouse adipose cells in vitro, whereas subclass LpA-I/A-II has no effect. To investigate whether the apolipoprotein composition of HDL plays a role in the selective delivery of cholesterol esters to the liver in vivo, we labelled HDL in its cholesterol ester moiety and separated [3H]cholesterol oleate-labelled HDL into subclasses LpA-I and LpA-I/A-II by immuno-affinity chromatography. Serum decay and liver association of LpA-I and LpA-I/A-II were compared for the apoprotein and cholesterol ester moieties. Both LpA-I and LpA-I/A-II selectively delivered cholesterol esters to the liver with similar kinetics. The kinetics of biliary secretion of processed cholesterol esters, initially associated with LpA-I or LpA-I/A-II, were studied in rats equipped with permanent catheters in bile, duodenum and heart. For both LpA-I and LpA-I/A-II, liver association was coupled to bile acid synthesis, with an increase in secretion rate during the night. During the first night period, the biliary secretion of LpA-I-derived radio-activity was significantly greater than for LpA-I/A-II. The data indicate that with both LpA-I and LpA-I/A-II selective delivery of cholesterol esters from HDL to the liver occurs, but that cholesterol esters delivered by LpA-I are more efficiently coupled to bile acid synthesis.

Interaction of reconstituted high density lipoprotein discs containing human apolipoprotein A-I (ApoA-I) variants with murine adipocytes and macrophages. Evidence for reduced cholesterol efflux promotion by apoA-I(Pro165-->Arg).

Interaction of cells with both native and reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I mediates efflux of cellular cholesterol. To characterize structural requirements for this activity in apoA-I, six different genetic apoA-I variants and the corresponding normal allele products were isolated from heterozygous carriers, reconstituted with dimyristoylphosphatidylcholine (DMPC) into discoidal HDL and compared with regard to their ability to release intracellular cholesterol from murine adipocytes and peritoneal macrophages. In previous studies we determined the amino acid changes of these variants: Pro3-->Arg, Pro4-->Arg, Lys107-->0, Lys107-->Met, Pro165-->Arg, and Glu198-->Lys (von Eckardstein, A., Funke, H., Walter, M., Altland, K., Benninghoven, A., and Assmann, G. (1990) J. Biol. Chem. 265, 8610-8617) and demonstrated that all apoA-I variants except apoA-I(Lys107-->0) form discoidal HDL particles with phosphatidylcholine analogues indistinguishable from normal apoA-I (Jonas, A., von Eckardstein, A., Kezdy, K. E., Steinmetz, A., and Assmann, G. (1991) J. Lipid Res. 32, 95-106). In the present study, all apoA-I.DMPC complexes except those containing apoA-I(Pro165-->Arg) promoted cholesterol efflux as effectively as those containing normal apoA-I. Cholesterol efflux from adipocytes obtained after 180 min of incubation with apoA-I(Pro165-->Arg).DMPC complexes was decreased by 30% although this variant was bound to adipocytes with normal affinity. By contrast, apoA-I(Lys107-->Met).DMPC complexes were decreased in their binding affinity compared to normal apoA-I.DMPC complexes but normally promoted cholesterol efflux. Incubation of mouse peritoneal macrophages with apoA-I(Pro165-->Arg).DMPC complexes did also result in a 30% decreased efflux of radiolabeled cholesterol if compared to rHDL with the normal allele product from the same donor. Together the observations suggest that the substitution of proline at residue 165 interferes with the formation of a structural domain in apoA-I that is crucial for cellular cholesterol efflux stimulation. Furthermore, our results suggest that binding of HDL to adipocytes and cholesterol efflux promotion by HDL requires different structural domains.

Apolipoprotein A-I-containing particles and reverse cholesterol transport in IDDM.

Lipoprotein particles containing apoA-I but not apoA-II are, among high-density lipoproteins, effective protectors against atherosclerosis that act by promoting the efflux of cellular cholesterol and the reverse cholesterol transport process. Because previous studies showed that in vitro nonenzymatic glycosylation of HDL impairs HDL receptor-mediated cholesterol efflux, we isolated Lp A-I from two poorly controlled insulin-dependent diabetic patients and compared the chemical composition and ability to promote cholesterol efflux with the same particles purified from two matched nondiabetic control subjects. No differences in lipid composition or in the ability to promote cholesterol efflux from cultured adipose cells were noted between the two types of Lp A-I preparations. However, when we separated Lp A-I from diabetic subjects by degree of glycosylation, the specifically glycosylated subfractions were about 50% less effective in producing cholesterol efflux than the nonglycosylated particles.