miR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) conveys a high risk of premature atherosclerosis as a result of lifelong exposure to high LDL cholesterol levels that are not fully reduced by standard-of-care lipid-lowering treatment. Inflammatory mediators have played a role in the progression of atherosclerotic lesions. Here, we investigated whether innate immunity cells in patients with FH have a specific proinflammatory phenotype that is distinct from that of cells in normal participants. To this end, miR-505-3p-a microRNA related to chronic inflammation-and its target genes were investigated in monocyte-derived macrophages (MACs) of patients with FH (FH-MACs) and non-FH controls (co-MACs). On the basis of the profiler PCR array analysis of agomiR-505-3p-transfected MACs, we identified the chemokine receptors, CCR3, CCR4, and CXCR1, as genes that are regulated by miR-505-3p via the transcription factor, RUNX1. miR-505-3p was significantly down-regulated, whereas CCR3, CCR4, CXCR, and RUNX1 were increased in FH-MAC compared with co-MAC, with the increase being more evident in the proinflammatory M1-like FH-MAC. Chemokine receptor levels were unrelated to LDL plasma levels at entry, but correlated with age in patients with FH, not in controls. In summary, we demonstrate for first time to our knowledge that MACs from FH-MACs have an inflammatory phenotype that is characterized by the up-regulation of CCR3, CCR4, and CXCR1 under the control of miR-505-3p. These results suggest a chronic inflammatory condition in FH innate immunity cells that is not reverted by standard lipid-lowering treatment.-Escate, R., Mata, P., Cepeda, J. M., Padró, T., Badimon, L. miR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia.

Preparation and comparative properties of antisera against glyco-3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid linked to bovine serum albumin at the C-3, C-15 alpha, and C-24 positions.

Anti glyco-3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid antisera were prepared by immunizing rabbits with hapten-bovine serum albumin (BSA) conjugates coupled at the C-3, C-15 alpha, and C-24 positions on the bile acid molecule, and their properties were investigated by heterologous combination assay using 125I-labeled tracer. The antiserum raised against the C-3 BSA conjugate showed poor titer and specificity, while the antisera from the other two conjugates showed satisfactorily high affinity constants (Ka = 5.0 x 10(8) and 7.0 x 10(8) M-1) and reasonable specificity, exhibiting negligible cross-reactivities with other major human bile acids and cholesterol. Among the unsaturated bile acids tested, high reactivity was observed with tauro-3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid, which suggested that bridge phenomena were significant in this assay system.

Preparation and antigenic property of methyl 3 beta-hydroxy-19-oxo-5-cholen-24-oate 19-(O-carboxymethyl) oxime-bovine serum albumin conjugate.

The preparation and antigenic property of 3 beta-hydroxy-5-cholen-24-oic acid-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through an (O-carboxymethyl) oxime bridge at the C-19 position on the steroid nucleus is described. Antibody raised against antigen in the rabbit possessed high titer and specificity to 3 beta-hydroxy-5-cholen-24-oic acid, exhibiting no significant cross-reactions with various bile acids.

A highly specific 125I-radioimmunoassay for cholic acid conjugates.

Several modifications of the immunization procedure permitted development of a highly specific radioimmunoassay (RIA) for cholic acid conjugates. Antiserum was produced in guinea pigs using cholic acid-thyroglobulin complex as immunogen. 125-I-Cholyglycylhistamine was prepared as radioactive ligand according to a modification of the method of Spenney et al. (Spenney, J.G., Johnson, B.J., Hirschowitz, B.I., Mihas, A.A. and Gibson, R. (1977) Gastroenterology 72, 305--311). The association constant of the antisera to taurocholic acid was 1.8 x 10(7) l/mol, the working range of the assay between 9.5--890 pmol. Cross-reactivities of the antiserum to bile acids other than cholic acid species were less than 3%, which is lower than for any published bile acid RIA. Concentrations of cholic acid conjugates in sera obtained from 17 healthy fasting volunteers ranged from 0.4--1.9/mumul/l.

Investigations into the choice of immunogen, ligand, antiserum and assay conditions for the radioimmunoassay of conjugated cholic acid.

Investigations into the choice of immunogen, ligand, antiserum and assay conditions for the radioimmunoassay of conjugated cholic acid have been performed with a view to producing optimal assay conditions. Cholic acid-BSA was found to be the best immunogen to produce antibodies to conjugated cholic acid and the response was of an IgG type. Incorporating a spacer (hexanoic acid) between hapten and carrier protein resulted in a decrease in antiserum titre. Optimal conditions for the assay were found using [125I]histamine-glycocholic acid as ligand with a dilution of antiserum to produce 60% binding of ligand and a pH of 7.4. Using these assay conditions no serum effects were found; extraction of serum prior to assay was therefore unnecessary. The assay was sensitive enough to detect post-prandial increased in serum bile acid concentrations following a liquid test meal; no increase was observed throughout the same time period in a fasting control.

A critical evaluation of a procedure for measurement of serum bile acids by radioimmunioassay.

A radioimmunoassay for the quantitative determination of cholic acid conjugates has been developed. Antisera were raised in rabbits injected with cholylglycine coupled by amide linkage to bovine serum albumin. The antibodies reported in this communication were not found to be mono-specific in the strictest sense. This assay is relatively simple, rapid, sensitive, reliable, and most importantly, specific for bile acids; the specificity of each antibody, however, must be thoroughly characterized at the dilution in routine use. The range of normal values for cholic acid conjugates in serum was found to be 0.1--1.6 micronmol/l (mean: 0.62, +/- S.D.: 0.4).

An 125I radioimmunoassay for primary conjugated bile salts.

Cholylglycylhistamine, a derivative of cholic acid, has been synthesized and characterized. This derivative has been iodinated using Na125I and chloramine-T and purified free from unlabeled cholylglycylhistamine. Application of this iodinated bile salt derivative to radioimmunoassay of bile salts in human serum is reported. Antibody titers have uniformly increased over titers used in tritium-based assays; some antibodies are usable in dilutions of 1:80,000. The radioimmunoassay described here was found to measure predominantly the primary conjugated bile salts. Sensitivity has been maintained, with the least detectable amount being 0.5 pmoles per assay tube. Normal values in human serum are 3.47 +/- 2.16 (SD) nmoles per ml.