The effects of low-density lipoprotein (LDL) and chylomicron remnants on lipid accumulation in human monocyte-derived macrophages (HMDMs) and in macrophages derived from the human monocyte cell line THP-1 were compared. The HMDMs or THP-1 macrophages were incubated with LDL, oxidized LDL (oxLDL), chylomicron remnant-like particles (CMR-LPs), or oxidized CMR-LPs (oxCMR-LPs), and the amount and type of lipid accumulated were determined. As expected, the lipid content of both cell types was increased markedly by oxLDL but not LDL, and this was due to a rise in cholesterol, cholesteryl ester (CE), and triacylglycerol (TG) levels. In contrast, both CMR-LPs and oxCMR-LPs caused a considerable increase in cellular lipid in HMDMs and THP-1 macrophages, but in this case there was a greater rise in the TG than in the cholesterol or CE content. Lipid accumulation in response to oxLDL, CMR-LPs, and oxCMR-LPs was prevented by the ACAT inhibitor CI976 in HMDMs but not in THP-1 macrophages, where TG levels remained markedly elevated. The rate of incorporation of [(3)H]oleate into CE and TG in THP-1 macrophages was increased by oxLDL, CMR-LPs, and oxCMR-LPs, but incorporation into TG was increased to a greater extent with CMR-LPs and oxCMR-LPs compared with oxLDL. These results demonstrate that both CMR-LPs and oxCMR-LPs cause lipid accumulation in human macrophages comparable to that seen with oxLDL and that oxidation of the remnant particles does not enhance this effect. They also demonstrate that a greater proportion of the lipid accumulated in response to CMR-LPs compared with oxLDL is TG rather than cholesterol or CE and that this is associated with a higher rate of TG synthesis. This study, therefore, provides further evidence to suggest that chylomicron remnants have a role in foam cell formation that is distinct from that of oxLDL.
The effects of chylomicron remnants (non-oxidised or oxidised) and oxidised low density lipoprotein (oxLDL) on the expression of mRNA for a wide range of genes believed to play a role in macrophage foam cell formation were compared using macrophages derived from the human monocyte cell line THP-1. Chylomicron remnant-like particles (CMR-LPs), oxidised CMR-LPs (oxCMR-LPs) and oxLDL were incubated with THP-1 macrophages, and the relative abundance of mRNA transcripts for genes involved in lipoprotein uptake, intracellular lipid metabolism, transport and storage and cholesterol efflux from macrophages was determined. The results show that CMR-LPs and oxLDL differ markedly in their effects on the expression of mRNA for a number of the genes tested. OxLDL increased mRNA levels for the scavenger receptors CD36 (x3.2) and lectin-like oxLDL receptor 1 (x2.1), and peroxisome proliferator-activated receptor gamma while CMR-LPs did not. In contrast, the expression of mRNA for the LDL receptor-like protein was raised by CMR-LPs (x1.8) but not oxLDL. Furthermore, down-regulation of mRNA levels for the ATP-binding cassette transporter (ABC) A1 was observed with CMR-LPs (x0.6), compared to the up-regulation found with oxLDL (x4.4). In addition, a number of significant differences were found between the effects of CMR-LPs and oxCMR-LPs, with the oxidised particles causing a striking rise in mRNA expression for the multi-drug resistance 1 gene (x13.7), but otherwise showing pattern more similar to that seen with oxLDL. These findings provide evidence to indicate that chylomicron remnants cause lipid accumulation in macrophages by influencing the expression of genes which regulate lipid metabolism at the transcriptional level, and that the mechanisms involved differ in important respects from those triggered by oxLDL.
Bispecific antibodies (BsAbs) are emerging as an important class of biopharmaceutical. The majority of BsAbs are created from conventional antibodies or fragments engineered into more complex configurations. A recurring challenge in their development, however, is the identification of components that are optimised for inclusion in the final format in order to deliver both efficacy and robust biophysical properties. Using a modular BsAb format, the mAb-dAb, we assessed whether an 'in-format' screening approach, designed to select format-compatible domain antibodies, could expedite lead discovery. Human nerve growth factor (NGF) was selected as an antigen to validate the approach; domain antibody (dAb) libraries were screened, panels of binders identified, and binding affinities and potencies compared for selected dAbs and corresponding mAb-dAbs. A number of dAbs that exhibited high potency (IC 50 ) when assessed in-format were identified. In contrast, the corresponding dAb monomers had »1000-fold lower potency than the formatted dAbs; such dAb monomers would therefore have been omitted from further characterization. Subsequent stoichiometric analyses of mAb-dAbs bound to NGF, or an additional target antigen (vascular endothelial growth factor), suggested different target binding modes; this indicates that the observed potency improvements cannot be attributed simply to an avidity effect offered by the mAb-dAb format. We conclude that, for certain antigens, screening na€ ıve selection outputs directly in-format enables the identification of a subset of format-compatible dAbs, and that this offers substantial benefits in terms of molecular properties and development time.
The effects of native and oxidized chylomicron remnants on the synthesis of cholesteryl ester and triacylglycerol in macrophages, and the way that this is influenced by exposure of the cells to oestrogen, was investigated using the human monocyte cell line THP-1 and chylomicron-remnant-like particles containing human apolipoprotein E (CRLPs). Synthesis of the lipids was measured by the incorporation of [(3)H]oleate into cholesteryl ester and triacylglycerol. CRLPs (5-40 microgram of cholesterol/ml) containing either trilinolein or triolein as the triacylglycerol component caused a dose-dependent decrease in cholesteryl ester formation, while triacylglycerol production was unchanged. After oxidation of the CRLPs, the level of thiobarbituric acid-reactive substances was increased by 6.3-fold and 2.2-fold in particles containing trilinolein and triolein respectively. Furthermore, CRLPs containing oxidized trilinolein lost their ability to down-regulate cholesterol esterification, while CRLPs containing oxidized triolein did not. Both types of oxidized CRLPs decreased triacylglycerol synthesis. Treatment of the macrophages with 17beta-oestradiol caused increases of approx. 94% and 34% in the synthesis of cholesteryl ester and triacylglycerol respectively in the absence of CRLPs. The differences between control and oestrogen-treated cells were abolished, however, when CRLPs (40 microgram of cholesterol/ml) were added to the incubations. In addition, in contrast with their lack of effect in control cells, CRLPs containing oxidized trilinolein decreased cholesterol esterification in oestrogen-treated cells by approx. 48%. These findings with CRLPs suggest that chylomicron remnants have significant effects on cholesteryl ester and triacylglycerol synthesis in macrophages, which may be modulated both by the oxidation state of the particles and by oestrogen.
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