Cultivation and Immortalization of Human B-Cells Producing a Human Monoclonal IgM Antibody Binding to MDA-LDL: Further Evidence for Formation of Atherogenic MDA-LDL Adducts in HumansIn Vivo

Oxidatively modified low-density lipoprotein (oLDL) is firmly believed to play an important role in the initiation and development of atherosclerosis, and malonic dialdehyde (MDA) is one of the major lipid peroxidation breakdown products involved in this process. In recent decades, antibodies against MDA-LDL have been detected in human and animal sera. In our study, human B-cells from the peripheral blood of a healthy female donor were fused with the SP2/0 mouse myeloma cell line. Antibody-producing hybridomas were detected by MDA-LDL-IgG/IgM enzyme-linked immunosorbent assays (ELISA) and Cu++-oxidized LDL IgG/IgM (oLAb) ELISA. Cells with supernatants emitting positive signals for antibodies were then cloned and after sufficient multiplication frozen and stored under liquid nitrogen. Due to the loss of antibody-producing ability, we established an MDA-LDL-IgM-producing cell line by recloning. This allowed isolation and immortalization of several human B-cells. The human donor had not been immunized with MDA-modified proteins, thus obviously producing MDA-LDL antibodiesin vivo. Furthermore, using these antibodies forin vitroexperiments, we were able to demonstrate that MDA epitopes are among the epitopes generated during Cu++-LDL oxidation as well. Finally, these antibodies compete in ELISA and cell culture experiments with MDA as a challenging toxin or ligand.

Download Full-text

Protective effect of dietary fenugreek (Trigonella foenum-graecum) seeds and garlic (Allium sativum) on induced oxidation of low-density lipoprotein in rats

Journal of Basic and Clinical Physiology and Pharmacology ◽

10.1515/jbcpp-2015-0037 ◽

2016 ◽

Vol 27 (1) ◽

Cited By ~ 11

Author(s):

Puttaswamy Mukthamba ◽

Krishnapura Srinivasan

Keyword(s):

Protective Effect ◽

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Lipid Peroxide ◽

Ldl Oxidation ◽

High Cholesterol Diet ◽

Fenugreek Seeds

AbstractDietary fenugreek seeds (Fenugreek seeds, garlic, and their combination were included along with a high-cholesterol diet for 8 weeks.Iron-induced oxidation of LDL in vivo was considerably lowered by dietary fenugreek and garlic. The extent of copper-induced oxidation of isolated LDL in vitro was also signiﬁcantly lesser in fenugreek-fed or fenugreek+garlic-fed rats. Anodic electrophoretic mobility of the oxidized LDL on agarose gel in case of spice-fed animals was decreased and hence consistent with the observed protective inﬂuence on LDL oxidation. Dietary fenugreek, garlic, and their combination significantly lowered lipid peroxide levels in plasma, liver, and heart in iron (II)-administered rats. The results suggest that these two dietary spices have protective effect on LDL oxidation under normal situation as well as in hypercholesterolemic situation. The protective effect of the combination of dietary fenugreek and garlic on LDL oxidation both in vivo and in vitro was greater than that of the individual spices.The protective effect of dietary fenugreek and garlic on LDL oxidation both in vivo and in vitro as evidenced in the present study is suggestive of their cardioprotective potential since LDL oxidation is a key factor in the arteriosclerotic process.

Download Full-text

Inhibition of in vitro macrophage-induced low density lipoprotein oxidation by thyroid compounds

Journal of Endocrinology ◽

10.1677/joe.0.1770137 ◽

2003 ◽

Vol 177 (1) ◽

pp. 137-146 ◽

Cited By ~ 11

Author(s):

L Oziol ◽

P Faure ◽

N Bertrand ◽

P Chomard

Keyword(s):

Cell Viability ◽

Antioxidant Capacity ◽

Density Lipoprotein ◽

Thiobarbituric Acid ◽

Human Monocyte ◽

Ldl Oxidation ◽

Low Density ◽

Redox Systems

Oxidized low density lipoproteins (LDL) are highly suspected of initiating the atherosclerosis process. Thyroid hormones and structural analogues have been reported to protect LDL from lipid peroxidation induced by Cu2+ or the free radical generator 2,2'-azobis-'2-amidinopropane' dihydrochloride in vitro. We have examined the effects of thyroid compounds on macrophage-induced LDL oxidation. Human monocyte-derived macrophages (differentiated U937 cells) were incubated for 24 h with LDL and different concentrations (0-20 microM) of 3,5,3'-triiodo-l -thyronine (T3), 3,5,3',5'-tetraiodo-L-thyronine (T4), 3,3',5'-tri-iodo-l -thyronine (rT3), the T3 acetic derivative (3,5,3'-tri-iodothyroacetic acid; TA3) or L-thyronine (T0) (experiment 1). Cells were also preincubated for 24 h with 1 or 10 microM of the compounds, washed twice, then incubated again for 24 h with LDL (experiment 2). Oxidation was evaluated by measurement of thiobarbituric acid-reactive substances (TBARS) and cell viability by lactate deshydrogenase release. In experiment 1, T0 had no effect, whereas the other compounds decreased LDL TBARS production, but T3 and TA3 were less active than T4 and rT3 (IC50: 11.0 +/- 2.6 and 8.1 +/- 0.8 vs 1.4 +/- 0.5 and 0.9 +/- 0.3 microM respectively). In experiment 2, the compounds at 1 microM had no effect; at 10 microM, T3 and rT3 slightly reduced LDL TBARS production, whereas TA3 and T4 inhibited it by about 50% and 70% respectively. TBARS released by the cells were also highly decreased by T3, T4, rT3 and TA3 in experiment 1, but only by T3 (30%) and T4 (70%) in experiment 2. Cell viability was not affected by the compounds except slightly by TA3 at 10 microM. The data suggested that the physico-chemical antioxidant capacity of thyroid compounds was modulated by their action on the intracellular redox systems of macrophage. Overall cellular effects of T3 led to a reduction of its antioxidant capacity whereas those of T4 increased it. Thus T4 might protect LDL against cellular oxidation in vivo more than T3.

Download Full-text

B lymphocyte binding to E- and P-selectins is mediated through the de novo expression of carbohydrates on in vitro and in vivo activated human B cells.

Journal of Clinical Investigation ◽

10.1172/jci117500 ◽

1994 ◽

Vol 94 (4) ◽

pp. 1585-1596 ◽

Cited By ~ 24

Author(s):

A A Postigo ◽

M Marazuela ◽

F Sánchez-Madrid ◽

M O de Landázuri

Keyword(s):

B Cells ◽

B Lymphocyte ◽

De Novo ◽

Human B Cells

Download Full-text

Folate: In Vitro and in Vivo Effects on VLDL and LDL Oxidation

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831.77.1.66 ◽

2007 ◽

Vol 77 (1) ◽

pp. 66-72 ◽

Cited By ~ 7

Author(s):

McEneny ◽

Couston ◽

McKibben ◽

Young ◽

Woodside

Keyword(s):

Folic Acid ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Folic Acid Supplementation ◽

Serum Folate ◽

Ldl Oxidation ◽

Low Density ◽

Acid Supplementation

Raised total homocysteine (tHcy) levels may be involved in the etiology of cardiovascular disease and can lead to damage of vascular endothelial cells and arterial wall matrix. Folic acid supplementation can help negate these detrimental effects by reducing tHcy. Recent evidence has suggested an additional anti-atherogenic property of folate in protecting lipoproteins against oxidation. This study utilized both an in vitro and in vivo approach. In vitro: Very-low-density lipoprotein (VLDL) and low density lipoprotein (LDL) were isolated by rapid ultracentrifugation and then oxidized in the presence of increasing concentrations (0→ μmol/L) of either folic acid or 5-methyltetrahydrofolate (5-MTHF). In vivo: Twelve female subjects were supplemented with folic acid (1 mg/day), and the pre- and post-VLDL and LDL isolates subjected to oxidation. In vitro: 5-MTHF, but not folic acid, significantly increased the resistance of VLDL and LDL to oxidation. In vivo: Following folic acid supplementation, tHcy decreased, serum folate increased, and both VLDL and LDL displayed a significant increase in their resistance to oxidation. These results indicated that in vitro, only the active form of folate, 5-MTHF, had antioxidant properties. In vivo results demonstrated that folic acid supplementation reduced tHcy and protected both VLDL and LDL against oxidation. These findings provide further support for the use of folic acid supplements to aid in the prevention of atherosclerosis.

Download Full-text

Evidence for the role of high density lipoproteins in mediating the antioxidant effect of estrogens

Acta Endocrinologica ◽

10.1530/eje.0.1420079 ◽

2000 ◽

pp. 79-83 ◽

Cited By ~ 15

Author(s):

W Abplanalp ◽

MD Scheiber ◽

K Moon ◽

B Kessel ◽

JH Liu ◽

...

Keyword(s):

Density Lipoprotein ◽

Antioxidant Effect ◽

High Density ◽

In Vivo Studies ◽

Ldl Oxidation ◽

High Density Lipoproteins ◽

Oh Groups

Estrogens possess strong antioxidant effects in vitro, but in vivo studies in humans have yielded conflicting results. Little is known regarding factors that mediate the antioxidant effect of estrogens in vivo. In this study the potential role of high density lipoprotein (HDL) was examined. The antioxidant effect of estradiol-17beta (E2) added to low density lipoprotein (LDL) was lost after dialysis. In contrast, the antioxidant effect of E2 added to HDL was conserved after dialysis, suggesting that E2 was bound to HDL. Binding of E2 to LDL increased after esterification (especially to long chain fatty acids). In the presence of HDL, an increased amount of E2 was transferred to LDL. E2-17 ester was as potent as E2 in preventing LDL oxidation in vitro, but 3,17-diesters were not as effective (E2=E2-17 ester>E2-3 ester>E2-3,17 diester). This was also supported by experiments which showed that estrogens with masked 3-OH groups were not effective as antioxidants. These studies provide evidence that HDL could facilitate the antioxidant effect of E2 through initial association, esterification and eventual transfer of E2 esters to LDL. Therefore it is critical that HDL peroxidation parameters be evaluated in subjects receiving estrogen replacement therapy.

Download Full-text

Paraoxonase-1 (PON-1) genotype and activity and in vivo oxidized plasma low-density lipoprotein in Type II diabetes

Clinical Science ◽

10.1042/cs20050089 ◽

2005 ◽

Vol 109 (2) ◽

pp. 189-197 ◽

Cited By ~ 26

Author(s):

Mike J. Sampson ◽

Simon Braschi ◽

Gavin Willis ◽

Sian B. Astley

Keyword(s):

Type Ii Diabetes ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Paraoxonase 1 ◽

Ldl Oxidation ◽

Phenyl Acetate ◽

Low Density ◽

Type Ii

The HDL (high-density lipoprotein)-associated enzyme PON (paraoxonase)-1 protects LDL (low-density lipoprotein) from oxidative modification in vitro, although it is unknown if this anti-atherogenic action occurs in vivo. In a cross-sectional study of 58 Type II diabetic subjects and 50 controls, we examined the fasting plasma LDL basal conjugated diene concentration [a direct measurement of circulating oxLDL (oxidatively modified LDL)], lipoprotein particle size by NMR spectroscopy, PON-1 polymorphisms (coding region polymorphisms Q192R and L55M, and gene promoter polymorphisms −108C/T and −162G/A), PON activity (with paraoxon or phenyl acetate as the substrates) and dietary antioxidant intake. Plasma oxLDL concentrations were higher in Type II diabetic patients (males, P=0.048; females, P=0.009) and unrelated to NMR lipoprotein size, PON-1 polymorphisms or PON activity (with paraoxon as the substrate) in any group. In men with Type II diabetes, however, there was a direct relationship between oxLDL concentrations and PON activity (with phenyl acetate as the substrate; r=0.611, P=0.0001) and an atherogenic NMR lipid profile in those who were PON-1 55LL homozygotes. Circulating oxLDL concentrations in vivo were unrelated to PON-1 genotypes or activity, except in male Type II diabetics where there was a direct association between PON activity (with phenyl acetate as the substrate) and oxLDL levels. These in vivo data contrast with in vitro data, and may be due to confounding by dietary fat intake. Male Type II diabetic subjects with PON-1 55LL homozygosity have an atherogenic NMR lipid profile independent of LDL oxidation. These data do not support an in vivo action of PON on LDL oxidation.

Download Full-text

Vascular responses and ion exchange in diabetes: Authors' reply

Clinical Science ◽

10.1042/cs0820339a ◽

1992 ◽

Vol 82 (3) ◽

pp. 339-339

Author(s):

J. M. Ritter ◽

G. C. Viberti

Keyword(s):

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Large Body ◽

Plasma Lipid ◽

Density Lipoprotein ◽

High Plasma ◽

Diabetic Patients ◽

Vascular Sensitivity

1. Na+/Li+ countertransport is not a gold standard, or indeed any other kind of standard. It is a measure of the activity of one particular cation exchanger. 2. There is a large body of literature regarding the effects of oxidized low-density lipoprotein (LDL) in experimental animals and in vitro. Whether abnormal oxidized LDL or one of many other possible mechanisms underlies the inverse relationship that we observed between vascular sensitivity in vivo to nitroprusside or carbachol with erythrocyte Na+/Li+ countertransport in diabetic patients remains to be seen. 3. We caution against post hoc subgroup analysis (smokers versus non-smokers, low versus high plasma lipid levels, etc.) in studies of this size.

Download Full-text

Low-density lipoprotein oxidation, antioxidants, and atherosclerosis: a clinical biochemistry perspective

Clinical Chemistry ◽

10.1093/clinchem/42.4.498 ◽

1996 ◽

Vol 42 (4) ◽

pp. 498-506 ◽

Cited By ~ 134

Author(s):

I Jialal ◽

S Devaraj

Keyword(s):

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Atherosclerotic Lesion ◽

Density Lipoprotein ◽

Direct Methods ◽

Ldl Oxidation ◽

Low Density ◽

Indirect Measures ◽

Oxidatively Modified

Abstract Cardiovascular disease is the leading cause of mortality in westernized populations. An increased concentration of plasma low-density lipoprotein (LDL) cholesterol constitutes a major risk factor for atherosclerosis. Several lines of evidence support a role for oxidatively modified LDL in atherosclerosis and for its in vivo existence. Antioxidants have been shown to decrease atherosclerotic lesion formation in animal models and decrease LDL oxidation; the evaluation of LDL oxidation in vivo is therefore very important. However, there is a paucity of methods for direct measurement of LDL oxidation. Of the direct methods currently available, the preferred ones seem to be the measurement of F2-isoprostanes, autoantibodies to epitopes on oxidized LDL, and the assessment of antioxidant status. Of the indirect measures, the most uniformly accepted procedure is examining the oxidative susceptibility of isolated LDL by monitoring conjugated diene formation.

Download Full-text

An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo

Journal of Cell Science ◽

10.1242/jcs.107.4.813 ◽

1994 ◽

Vol 107 (4) ◽

pp. 813-825 ◽

Cited By ~ 8

Author(s):

M.R. Shanks ◽

D. Cassio ◽

O. Lecoq ◽

A.L. Hubbard

Keyword(s):

Plasma Membrane ◽

B Cells ◽

Membrane Proteins ◽

Cell Line ◽

Hybrid Cell ◽

The Other ◽

Hepatocyte Polarity ◽

Rat Hepatoma

Studies of hepatocyte polarity, an important property of liver epithelial cells, have been hampered by the lack of valid in vitro models. We report here that a new polarized hepatoma-derived hybrid cell line, called WIF-B, has improved characteristics to those of its parent, WIF12-1. This latter line originated from the fusion of non-polarized rat hepatoma Fao cells with human fibroblasts (WI-38) and selection for a polarized phenotype. We generated the WIF-B line by growing WIF12-1 cells as unattached aggregates for three weeks and selecting for survivors. Karyotype analysis showed a broad chromosome pattern in the initial WIF-B population, but this pattern stabilized after a few passages. The growth and phenotypic properties of these cells were quite different from those of their polarized WIF12-1 parent. WIF-B cells attained a 4-fold higher maximal density in monolayer culture, survived at this density for > 5 days rather than 1 day, and exhibited two to three times more apical structures during this period (80 to 95%). We compared several parameters of liver differentiation in the WIF-B cells with those of a related hybrid clone, WIF12-E, which is extinguished for most liver-specific functions, and with the common hepatoma parent, Fao. By immunoblot analysis, the levels of expression of eight plasma membrane proteins were higher in the WIF-B cells than in either of the other two cell lines and ranged from 10 to 200% of those in vivo. Two plasma membrane proteins were not detected in WIF12-E cells. By immunofluorescence, the apical membrane proteins in WIF-B displayed different cellular localizations than in either of the other two cell lines. In WIF-B cells, apical proteins were confined to a plasma membrane region that we have identified as the apical domain by several criteria (Ihrke, G., Neufeld, E.D., Meads, T., Shanks, M.R., Cassio, D., Laurent, M., Schroer, T.A., Pagano, R. E. and Hubbard, A. L. J. Cell Biol., 123, 1761–1765). The same molecules were distributed over the entire plasma membrane of Fao and WIF12-E cells and also (for Fao cells) in intracellular punctate structures that did not colocalize with the majority of structures containing a secretory protein, albumin. Our results indicate that the WIF-B cells are more highly differentiated than any of their ancestors (Fao or WIF12-1 cells) and thus, are promising candidates for in vitro studies of hepatocyte polarity.

Download Full-text