Isolation and partial characterization of lipoprotein A-II (LP-A-II) particles of human plasma.

High density lipoproteins (HDL) consist of a mixture of chemically and functionally distinct families of particles defined by their characteristic apolipoprotein (Apo) composition. The two major lipoprotein families are lipoprotein A-I (LP-A-I) and lipoprotein A-I:A-II (LP-A-I:A-II). This study describes the isolation of a third minor HDL family of particles referred to as lipoprotein A-II (LP-A-II) because it lacks ApoA-I and contains ApoA-II as its main or sole apolipoprotein constituent. Because ApoA-II is an integral protein constituent of three distinct lipoprotein families (LP-A-I:A-II, LP-A-II: B:C:D:E and LP-A-II), LP-A-II particles were isolated from whole plasma by sequential immunoaffinity chromatography on immunosorbers with antisera to ApoA-II, ApoB and ApoA-I, respectively. In normolipidemic subjects, the concentration of LP-A-II particles, based on ApoA-II content, is 4-18 mg/dl accounting for 5-20% of the total ApoA-II not associated with ApoB-containing lipoproteins. The lipid composition of LP-A-II particles is characterized by low percentage of triglycerides and cholesterol esters and a high percentage of phospholipids in comparison with lipid composition of LP-A-I and LP-A-II: A-II. The major part of LP-A-II particles contain ApoA-II as the sole apolipoprotein constituent; however, small subsets of LP-A-II particles may also contain ApoD and other minor apolipoproteins. The lipid/protein ratio of LP-A-II is higher than those of LP-A-I and LP-A-I:A-II. In homozygous ApoA-I and ApoA-I/ApoC-III deficiencies, LP-A-II particles are the only ApoA-containing high density lipoprotein with levels found to be within the same range (7-13 mg/dl) as those of normolipidemic subjects. However, in contrast to normal LP-A-II, their lipid composition is characterized by higher percentages of triglycerides and cholesterol esters and a lower percentage of phospholipids and their apolipoprotein composition by the presence of ApoC-peptides and ApoE in addition to ApoA-II and ApoD. These results show that LP-A-II particles are a minor HDL family and suggest that, in the absence of ApoA-I-containing lipoproteins, they become an efficient acceptor/donor of ApoC-peptides and ApoE required for a normal metabolism of triglyceride-rich lipoproteins. Their other possible functional roles in lipid transport remain to be established in future experiments.

Similarity between in vitro and in vivo cellular aging.

In vivo and in vitro cellular aging were compared by determining the division capacity of individual, cloned cells of (1) the primary stromal population of human bone marrow, obtained from donors of various ages, and (2) the population at various passage levels of an in vitro subcultivated culture of the same origin. We find a strong similarity between the two series of data. This observation provides a further argument that cellular aging in vitro represents a biologically relevant phenomenon.

[Human apolipoprotein E: polymorphism and binding domain of the receptors]. / Apolipoproteine E humaine: polymorphisme et domaine de fixation aux récepteurs.

This paper summarizes present knowledge of the LDL receptor-binding domain of apolipoprotein E, with special emphasis on the influence of apolipoprotein polymorphism on the interaction with apo B/E receptors.

Crohn's disease with adenocarcinoma and dysplasia. Macroscopical, histological, and immunohistochemical aspects of two cases.

We present two cases of small-bowel adenocarcinoma and dysplasia in patients with longstanding Crohn's disease. In one case, the dysplasia and cancer were exclusively located in the terminal ileum, whereas in the other case, several cancers were found from the ileum toward the transverse colon. In both cases, we found a clinically unsuspected Dukes C1 mucinous adenocarcinoma together with large foci of polypoid villous dysplasia or with multifocal high-grade dysplasia and intramucosal carcinoma. Immunohistochemical staining for carcinoembryonic antigen (CEA) revealed a different staining pattern in various diseased areas. The intensity of CEA staining paralleled the histologic degrees of dysplasia and neoplasia. Cytokeratin expression was disturbed in inflamed mucosa, and it was more pronounced in high-grade dysplasia and invasive carcinoma. We conclude that the presence of dysplasia in an intestinal biopsy of a patient with Crohn's disease should arouse the pathologist's suspicion of carcinoma and force him or her to take multiple sections from strictures and polypoid lesions, especially since the clinical symptoms of a carcinoma may be obscured by the symptoms of inflammatory bowel disease. Immunohistochemical staining with CEA and cytokeratin are useful in the objectivation of dysplasia.

Electroimmunoassay of human serum lipoproteins containing Apo A-I by monoclonal antibodies.

Three monoclonal antibodies to human serum apolipoprotein (Apo) A-I (4A12, 4B11 and 2G11) were produced by Sanofi. They were directed to three distinct epitopes of the Apo A-I molecule, present on the surface of the lipoprotein particles. They were able to precipitate individually some lipoprotein units from the serum. Only the mixture of the three monoclonal antibodies could allow a precipitation of all Apo A-I containing particles. An electroimmunoassay using this oligoclonal mixture was assessed to standardize the Apo A-I measurement in serum. Results agreed well with those obtained by electroimmunoassays using polyclonal antisera. Moreover no pretreatment of serum samples with dissociating agents or detergents was required. Therefore, this specific, rapid (7-8 h), precise (within- and between-assay precisions were 4.25 and 3.1%, respectively) immunoassay is routinely available for apolipoprotein A-I measurement in serum.

Plasma lipoproteins in infantile visceral leishmaniasis: deficiency of apolipoproteins A-I and A-II.

This study describes the plasma lipoprotein system of young children with visceral Leishmaniasis (Kala-azar disease). In addition to the presence of amastigote forms in the sternal aspirates of bone marrow, the patients exhibited fever, anemia, hepatosplenomegaly, various degrees of pancytopenia and a slight liver cytolysis. Patients had normal total cholesterol levels and increased triglyceride levels in the plasma. The concentrations of HDL and LDL were 30% and 50% of these reported for normolipemic subjects, respectively. In contrast, there was a three-fold increase in the concentration of VLDL. The ratio of free to total cholesterol was high; this was further substantiated by electron microscopy of HDL showing the presence of disc-like particles. Quantitative determination of apolipoproteins revealed a three- and seven-fold decrease of apolipoproteins (Apo) A-I and A-II, respectively, whereas Apo B levels were within the normal range. The presence of LP-A-II particles was demonstrated by two-dimensional immunoelectrophoresis in most of the patients' plasma during the acute phase of disease.

Competitive enzyme inhibition immunoassay of apolipoprotein A-I: use of monoclonal antibodies.

An original competitive enzyme inhibition immunoassay has been developed for determination of total apolipoprotein A-I (apo A-I) in serum and in plasma. This specific assay involves a single monoclonal antibody (F59 4A12 2F4, directed to the -COOH terminal region of the apo A-I molecule) and a stable secondary plasma standard. Delipidation of serum samples exposed no additional antigenic sites, which suggests that all the apo A-I molecules express the epitope detected by Mab 4A12 on the surface of the apo A-I-containing particles. Within- and between-run CVs were, respectively, 6.1% and 7.5% at a 1.47 g/L concentration of apo A-I, 7.1% and 8.2% at 1.21 g/L, and 6.5% and 7.3% at 1.80 g/L. Results (y) correlated with those obtained with an electroimmunoassay (x), in which we used a mixture of three monoclonal antibodies, including Mab 4A12, as follows: y = 250x - 7, r = 0.744, P less than 0.001.

Composition of plasma ApoA-I-containing lipoprotein particles in children and adults.

The purpose of this study was to examine sex- and age-related differences in the concentration and composition of lipoprotein particles containing apoA-I (LP-A-I) and those containing apoA-I and apoA-II (LP-I:A-II), the main HDL as defined by their apolipoprotein composition. Lipoproteins were isolated by immunoaffinity chromatography of whole plasma from 16 normal prepubertal children and 15 normal male and female adults using "pan"-MAb to apoA-I and apoA-II. Although there was no difference between children and adults in the concentration of LP-A-I:A-II, adult females had significantly higher levels of LP-A-I than either children or adult males. Main differences between children and adults as well as between adult males and females were in the apolipoprotein composition of the lipoprotein particles; children had the highest content of minor apolipoproteins (apoC and apoE) in LP-A-I but the lowest in LP-A-I:A-II. The lipid/apolipoprotein ratios of LP-A-I and LP-A-I:A-II were significantly higher in children and women than in men. The LP-A-I and LP-A-I:A-II contained 75% of the total plasma apoC and apoE in women and children but only 50% in men. However, in all three groups, 70-90% of the minor HDL apolipoproteins were associated with LP-A-I:A-II. The nonmolar ratios of minor apolipoproteins in LP-A-I and LP-A-I:A-II and the sex- and age-related differences in apoA-I/apoA-II ratios of LP-A-I:A-II suggest that both lipoproteins may consist of a spectrum of lipoprotein subfamilies differing in their apolipoprotein composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-I Milano: sex-related differences in the concentration and composition of apoA-I- and apoB-containing lipoprotein particles.

The presence of apolipoprotein (apo) A-IMilano (A-IM) mutant of apoA-I has a marked effect on plasma lipoproteins of A-IM carriers including variable hypertriglyceridemia, increased levels of very low density lipoproteins (VLDL), slightly elevated levels of triglyceride-enriched low density lipoproteins (LDL) and greatly reduced levels of high density lipoproteins (HDL). To gain further insight into this dyslipoproteinemic syndrome characterized clinically by the absence of coronary artery disease, we have determined the concentration and composition of apoA- and apoB-containing lipoprotein families in four male and four female carriers and corresponding normal controls. Results have shown that A-IM carriers have significantly reduced levels of lipoprotein (LP) A-I (45%), LP-A-I:A-II (60%), and LP-A-II (70%) and significantly increased levels of cholesterol-rich LP-B (67%) and triglyceride-rich LP-B:C, LP-B:C:E, and LP-A-II:B:C:D:E (65%) particles compared to controls. However, there were significant sex-related differences in the levels of apoA-and apoB-containing lipoproteins. Female carriers had significantly higher concentrations of LP-A-I (39 +/- 10 vs. 12 +/- 6 mg/dl) and LP-A-I:A-II (48 +/- 11 vs. 30 +/- 6 mg/dl) than male carriers. Furthermore, female carriers had higher levels of LP-B:C (23 +/- 18 vs. 6 +/- 5 mg/dl) and LP-A-II:B:C:D:E (13 +/- 6 vs. 2.3 +/- 0.8 mg/dl) but lower concentrations of LP-B (103 +/- 52 vs. 152 +/- 54 mg/dl) and LP-B:C:E (5 +/- 2.5 vs. 13 +/- 8 mg/dl) than male carriers. In general, the levels of LP-A-I and LP-A-I:A-II particles correlated positively with the levels of all three types of triglyceride-rich lipoproteins (LP-Bc) and negatively with the levels of LP-B particles. A comparative study of lipoprotein families in several dyslipoproteinemic states characterized by low levels of HDL has indicated that the characteristic lipoprotein particle profile of A-IM carriers results most probably from the selective effect of apoA-IM mutant rather than a general reduction in HDL levels. It appears that increased levels of LP-A-II:B:C:D:E particles, an inefficient substrate for lipoprotein lipase, and structurally defective LP-A-I:A-II particles, the normal acceptors of minor apolipoproteins released during lipolysis of triglyceride-rich lipoproteins, may be the main contributing factors to moderate hypertriglyceridemia characteristic of A-IM carriers.

Characterization of apoA- and apoB-containing lipoprotein particles in a variant of familial apoA-I deficiency with planar xanthoma: the metabolic significance of LP-A-II particles.

This study describes a variant of familial apoA-I deficiency associated with a moderate risk for premature coronary artery disease. The proband, a 25-year-old man of Philippine origin, and his 62-year-old maternal aunt had peripheral corneal opacification, xanthelasma, and planar xanthoma; the aunt had coronary artery bypass surgery at 61 years of age. Proband's parents and three brothers were asymptomatic and apparently healthy. The characteristic apolipoprotein features of affected patients were the immunochemically and chemically undetectable apoA-I, reduced levels of apoA-II, apoC-II, apoC-III, and apoD, and normal levels of apoB and apoE; except for negligible levels of high density lipoprotein (HDL)-cholesterol (2-3 mg/dl), their plasma lipid profile was normal. The apoA-I levels in all five unaffected relatives were more than one SD below the normal mean values for their age and sex; the HDL-cholesterol levels of proband's unaffected brothers were below the 10th percentile of normal control values. Patient's very low density lipoprotein (VLDL), low density lipoprotein (LDL), and HDL contained 1.4, 80.4, and 18.1%, whereas those of control subjects contained 2.7, 28.8, and 68.1% of the total apolipoprotein mass, respectively. In unaffected relatives, the levels of LP-A-I, but not LP-A-I:A-II, were significantly lower than in controls. Neither of the two patients had detectable concentrations of LP-A-I or LP-A-I:A-II. Their HDL only consisted of LP-A-II particles, the levels of which (7-13 mg/dl) were similar to those of unaffected relatives or controls. There was no difference in the lipid composition of LP-A-II between patients and their relatives. However, LP-A-II from patients contained substantial amounts of apoC-peptides and apoE (0.40-0.98 mg/mg apoA-II), whereas those from unaffected relatives were free of these minor apolipoproteins. In patients, among all four major apoB-containing lipoproteins, only the levels of LP-B and LP-B:C were slightly higher than those in controls. Results of this study suggest a genetic cause for this variant of apoA-I deficiency characterized most probably by autosomal recessive inheritance. It appears that patients are likely to be homozygous for a gene present in single dose in the parents and brothers of the affected proband.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute phase proteins and plasma lipoproteins during antimony treatment in infantile visceral leishmaniasis.

The purpose of this study was to determine the effects of antimony treatment on the altered lipoprotein system of four young Tunisians with visceral Leishmaniasis and to further investigate possible relationships between acute phase proteins (C-reactive protein and apolipoprotein SAA)-known to be increased in this disease-and lipid or apolipoprotein (apo) parameters measured before and throughout the treatment. A marked improvement of all investigated parameters was already observed after the first cure: the lecithin-cholesterol acyltransferase activity was the only parameter remaining deficient even at the end of the second cure. Statistical analysis reveals a significant inverse relationship between changes in C-reactive protein and changes in the plasma ratio of esterified to total cholesterol (r = -0.78; p < 0.001) as well as in plasma apoA-I concentration (r = -0.64; p < 0.01). Because no such correlation could be observed with apoSAA, results of this study suggest that the major mechanism of lowering the plasma levels of apoA-I and the plasma-cholesteryl content could be closely related to the appearance of large amounts of C-reactive protein in plasma or more likely to the process inducing the hepatic synthesis of this acute phase protein.

Epitope mapping of the human biliary amphipathic, anionic polypeptide: similarity with a calcium-binding protein isolated from gallstones and bile, and immunologic cross-reactivity with apolipoprotein A-I.

Biliary amphipathic anionic polypeptide (APF) the major protein of the pigment-lipoprotein complex in bile, and calcium-binding protein (CBP) from gallstones are both small (less than 10 kDa), highly acidic, amphipathic proteins present in bile and closely associated also with pigmented areas in human gallstones. Polyclonal antibodies against APF have shown cross-reactivity with plasma high density lipoproteins (HDL). This study examines the hypothesis that APF and CBP might be closely related or even identical, and might also share common epitopes with the larger apoA-I (23 kDa). To assess this, immunoreactivity of the three delipidated, highly purified proteins was determined against a panel of 12 monoclonal antibodies (MAbs) prepared against APF and a panel of 4 MAbs against apoA-I. APF was isolated from bile by zonal ultracentrifugation. CBP was isolated from proteins precipitated from bile by CaCl2, as well as from the calcium bilirubinate shells of cholesterol gallstones, by extraction successively with methyl-t-butyl ether, methanol, and Na2EDTA, followed by Sephadex G-25 chromatography and two-stage preparative SDS-PAGE. ApoA-I was prepared by two types of chromatography: Sephacryl S200 chromatography and heparin-chromatographic immunoaffinity. Specific polyclonal antibodies to APF and apoA-I were prepared from immunized rabbits. MAbs to APF and apoA-I were prepared by immunization of mice, using standard hybridoma technique. Western blotting of APF and CBP in 15% SDS-PAGE yielded one band with an apparent molecular weight of 6.5 kDa, which, along with apoA-I, was immunostained by polyclonal antibodies to APF and apoA-I. Using 12 MAbs against APF with three types of ELISA (direct antigen binding, competitive antigen displacement, and epitope competition between antibodies), it was shown that APF and delipidated apoA-I shared six epitopes, three of which were detected also on the surface of intact HDL particles. Six other epitopes were present in APF but not apoA-I, four of which were exposed on the surface of HDL. Four MAbs against apoA-I reacted with APF and CBP. Amino acid analyses of APF and CBP were similar with 20-23% acidic and 7-11% basic amino acids and low contents of cysteine, methionine, and tyrosine; both differed from apoA-I in containing isoleucine and cysteine. Using ELISA and one MAb (no. 32) against APF, this polypeptide was detected in human plasma HDL, the pigment-lipoprotein complex in the bile of humans, dogs, and rats, and in both pigment and cholesterol gallstones.(ABSTRACT TRUNCATED AT 400 WORDS)

Alterations in lipoprotein density classes in infantile visceral leishmaniasis: presence of apolipoprotein SAA.

This study describes the alterations in the plasma lipoproteins from nine young Tunisian children with active visceral Leishmaniasis. The plasma lipid profile from affected patients was characterized by a marked hypertriglyceridaemia associated with reduced levels of total and high density lipoprotein (HDL)-cholesterol and a significant increase in the plasma ratio of unesterified to total cholesterol. Quantitative determination of plasma apolipoproteins revealed significantly decreased levels of all measured apolipoproteins, especially of apolipoproteins A-I and A-II, with the exception of apolipoprotein E, the levels of which were markedly increased. Moreover, at least two isoforms of the apolipoprotein serum amyloid A (SAA), an acute phase protein, were detected in all patients' plasma using two-dimensional electrophoresis. Immunochemical evidence was presented that apolipoproteins E and SAA, although both primarily associated with apolipoprotein A- (A-I and A-II) as well as with apolipoprotein B-containing lipoproteins, could occur as LP-E and LP-SAA subspecies, devoid of apolipoproteins A and B. However, it should be pointed out that LP-SAA particles were found in HDL2 from only two patients whereas the abnormal LP-E particles were detected in LDL and HDL2 from all investigated patients. The polydispersity and heterogeneity of patients' HDL3 were assessed by electron microscopy. It was further suggested that the profound changes in the lipoprotein metabolism of these young patients may be due to the increased hepatic synthesis of apolipoprotein SAA and/or to their altered immune function during active visceral Leishmaniasis.