Vitamin A-sensitive tissues in transgenic mice expressing high levels of human cellular retinol-binding protein type I are not altered phenotypically.

The suggested function of cellular retinol-binding protein type I [CRBP(I)] is to carry retinol to esterifying or oxidizing enzymes. The retinyl esters are used in storage or transport, whereas oxidized forms such as all-trans or 9-cis retinoic acid are metabolites used in the mechanism of action of vitamin A. Thus, high expression of human CRBP(I) [hCRBP(I)] in transgenic mice might be expected to increase the production of retinoic acid in tissues, thereby inducing a phenotype resembling vitamin A toxicity. Alternatively, a vitamin A-deficient phenotype could also be envisioned as a result of an increased accumulation of vitamin A in storage cells induced by a high hCRBP(I) level. Signs of vitamin A toxicity or deficiency were therefore examined in tissues from transgenic mice with ectopic expression of hCRBP(I). Testis and intestine, the tissues with the highest expression of the transgene, showed normal gross morphology. Similarly, no abnormalities were observed in other tissues known to be sensitive to vitamin A status such as cornea and retina, and the epithelia in the cervix, trachea and skin. Furthermore, hematologic variables known to be influenced by vitamin A status such as the hemoglobin concentration, hematocrits and the number of red blood cells were within normal ranges in the transgenic mice. In conclusion, these transgenic mice have normal function of vitamin A despite high expression of hCRBP(I) in several tissues.     

Low density lipoprotein receptor determination in peripheral blood mononuclear cells: influence of differences in cell concentration

Low density lipoprotein (LDL) receptor determination in peripheral blood mononuclear cells (PBMCs) is influenced by differences in cell concentration. As the cell concentration increases, measured LDL receptor activity decreases. This inter-relationship is caused by a PBMC-induced modification of 125I-LDL. The PBMC-modified 125I-LDL results from shedding of polyanionic cell membrane constituents that subsequently bind to 125I-LDL, and has reduced capacity of binding to the LDL receptors, to the cell membrane independent of the receptors and even to plastic. The cell membrane constituents contain sulphate, have a MW = 200,000-300,000, are heat stable and are rapidly released at 37 degrees C as well as at 4 degrees C. They probably represent a heterogeneous group of proteoglycans, glycoproteins and glycolipids. The higher the cell concentration is, the more polyanionic cell membrane constituents are released, and at high concentrations they may even form aggregates of LDL. We conclude that differences in PBMC concentration interfere with LDL receptor analyses through shedding of different amounts of polyanionic cell membrane constituents into the medium. Thus, standardisation of the experimental procedures with respect to cell number is of great importance in LDL receptor determination in PBMCs.     

Plasma cholesterol esterification and plasma lipoproteins in bile-duct-ligated dogs.

To study the lipoprotein changes in cholestasis while the capacity for plasma cholesterol esterification was normal, the common bile duct was ligated in dogs and plasma investigated 8 h and 48 h later. The plasma concentration of cholesteryl esters was slightly increased, concomitant with a tendency toward an increase in the activity of lecithin:cholesterol acyltransferase (LCAT). The content of cholesteryl esters in the main lipoprotein classes was normal. Marked alteration in the low density lipoproteins (LDL) and the high density lipoproteins (HDL) took place and were essentially similar 8 h and 48 h after bile duct ligation. In LDL, (density 1.006--1.019 g/ml) and LDL2 (density 1.019--1.063 g/ml) an increase in the content of polar lipids was observed, and in LDL2 heterogeneity in particle size was demonstrated by gelfiltration on 2% agarose and by electron microsopcy. Large myelin structures, flattened disc-shaped particles, and particles with the appearance of normal LDL2 were present. HDL isolated after operation was characterized by a decreased protein/lipid ratio and an increased content of phospholipids. By gelfiltration on Sephadex G-200 and by electron microscopy changes in particle size were observed, with the presence of disc-shaped particles with a tendency in form rouleaux. These results demonstrate that marked lipoprotein changes occur as early as 8 h after bile duct-ligation in dogs and indicate that a deficient LCAT mechanism is present in cholestasis even with normal or high plasma LCAT activity.     

Survival of activated human T lymphocytes is promoted by retinoic acid via induction of IL-2

At the end of an immune response, most activated T cells spontaneously undergo programmed cell death (apoptosis). In the present study we show that all-trans retinoic acid (atRA), a major vitamin A metabolite, can inhibit the spontaneous apoptosis of activated human T lymphocytes in vitro. Isolated peripheral blood T lymphocytes were activated by 12-O-tetradecanoyl phorbol 13-acetate and cultured for up to 11 days without any further stimuli. With time, a gradual increase in cell death was observed. This spontaneous death of activated T cells was apoptotic, as demonstrated by cell shrinkage, DNA fragmentation and depolarization of the mitochondrial membrane. In the presence of physiological concentrations of atRA, the percentage of T cells exhibiting these apoptotic features was significantly reduced. After 5 days of stimulation, the percentage of TUNEL+ T cells decreased from 28 to 12% in the presence of atRA. The anti-apoptotic effect of atRA was mimicked by the retinoic acid receptor (RAR)-selective agonists 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and AM-580, and totally abrogated by the RAR-selective antagonist Ro 41-5253. Cytokines of the IL-2 family have been shown to improve the survival of activated T cells. Strikingly, we found that the ability of atRA to inhibit apoptosis was significantly correlated with its ability to increase the production of IL-2. Furthermore, a blocking anti-IL-2 receptor antibody completely abrogated the anti-apoptotic effect of atRA. Together, these results suggest that retinoic acid inhibits spontaneous apoptosis of activated T lymphocytes through a RAR-dependent increase in IL-2 production.