辛伐他汀对IFN-γ 诱导的人外周血单个核细胞OX40L 表达的影响

目的:探讨经IFN-γ刺激后,人外周血单个核细胞OX40L表达的变化,以及辛伐他汀对单个核细胞OX40L表达的影响.方法:将实验标本随机分为2组,分别干扰素-γ(IFN-γ)刺激组、辛伐他汀干预组.应用RT-PCR及Western blotting技术,观察IFN-γ诱导的人外周血单个核细胞OX40L表达情况及辛伐他汀对人单个核细胞OX40L表达的影响.结果:1.1000U/mlIFN-与人单个核细胞共同培养24h后,OX40L mRNA和蛋白水平的表达明显增加.2.预先给予10 mol/L的辛伐他汀干预1h可以明显降低IFN-诱导的OX40L表达.结论:IFN-可诱导人单个核细胞OX40L表达.辛伐他汀可以抑制IFN-诱导人单个核细胞OX40L表达的增强,从而可能抑制了OX40L信号通路介导的与炎症有关的血管损伤,延缓动脉粥样硬化的进程.     

Repression of alpha 2-macroglobulin and stimulation of alpha 1-proteinase inhibitor synthesis in human mononuclear phagocytes by endotoxin

Mononuclear phagocytes are a bone-marrow-derived subgroup of white blood cells which circulate as monocytes and, after differentiation into macrophages, become resident in many tissues. By synthesizing the important proteinase inhibitors alpha 2-macroglobulin and alpha 1-proteinase inhibitor mononuclear phagocytes contribute to the control of proteolysis both in blood and tissues. Applying a culture system which enables human blood monocytes to differentiate into macrophages in vitro, synthesis of alpha 2-macroglobulin and alpha 1-proteinase inhibitor was studied. The normal course of monocyte-macrophage maturation is accompanied by a strong increase of specific alpha 2-macroglobulin synthesis and a concomitant slight decrease of alpha 1-proteinase inhibitor. alpha 2-Macroglobulin can be designated as a marker protein of the monocyte/macrophage differentiation. Endotoxin (Salmonella typhi) in a concentration as low as 100 ng/ml strongly represses alpha 2-macroglobulin synthesis both in monocytes and macrophages. Furthermore, endotoxin completely abolishes the induction of alpha 2-macroglobulin synthesis during the course of normal monocyte in vitro cultivation, indicating that endotoxin is a strong inhibitor of the monocyte-macrophage maturation. In contrast to alpha 2-macroglobulin, alpha 1-proteinase inhibitor synthesis is strongly stimulated by endotoxin in monocytes as well as in macrophages.     

Sequence and acute phase regulation of rat alpha 1-inhibitor III messenger RNA

cDNA clones coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from an acute phase rat liver library. The isolates could be divided into four groups with characteristic BamHI restriction fragment patterns. The identity of the prototype clone pRLA1I3/2J was established by comparison with the published amino acid sequence of the purified protein. It codes for a 1477-amino acid precursor polypeptide with a 24-residue signal peptide. The mature protein shares 58% overall sequence identity with rat alpha 2-macroglobulin and contains a typical internal thiolester sequence. Twenty-two of its twenty-three cysteinyl residues are conserved with alpha 2-macroglobulin implying similar tertiary structure. However, the prototype alpha 1-inhibitor III sequence differed significantly from the rat and human alpha 2-macroglobulin sequences in its bait region suggesting alpha 1-inhibitor III possesses proteinase inhibitory specificities different from those of alpha 2-macroglobulin. The variant alpha 1-inhibitor III clone pRLA1I3/2J from a second cDNA group also differed from the prototype in the bait region coding sequence, although both specify similar signal peptides and NH2 termini. The observation of variant cDNA classes suggests that acute phase rat livers produce a heterogeneous mixture of alpha 1-inhibitor III mRNA molecules. Evidence was obtained for the presence of at least four different alpha 1-inhibitor III-related genes in the rat genome. During the first 24 h of an acute phase response the abundance of hepatic alpha 1-inhibitor III mRNA was decreased 3-4-fold. This decrease was of the same order of magnitude as the reported reduction of the corresponding plasma protein concentration, suggesting that in the early phase of the acute inflammatory response the plasma concentration of this protein is mainly controlled through the abundance of its hepatic mRNA.     

The acute-phase induction of alpha 2-macroglobulin in rat hepatocyte primary cultures: action of a hepatocyte-stimulating factor, triiodothyronine and dexamethasone

During inflammation a number of liver-derived plasma proteins increases in concentration. In the rat these so-called acute-phase proteins are mainly proteinase inhibitors, such as alpha 1-proteinase inhibitor, alpha 1-acute-phase globulin and alpha 2-macroglobulin. At present, the mechanisms responsible for the enhanced synthesis of acute-phase proteins are poorly understood. Therefore, we have studied the induction of alpha 2-macroglobulin synthesis in rat hepatocyte primary cultures. Adrenaline, triiodothyronine, estradiol and progesterone were tested for their ability to stimulate alpha 2-macroglobulin synthesis. Only triiodothyronine induced alpha 2-macroglobulin synthesis markedly. However, the presence of dexamethasone was a prerequisite for alpha 2-macroglobulin induction indicating a permissive action of glucocorticoids. Besides glucocorticoids and triiodothyronine a non-dialyzable factor (HSF) derived from rat Kupffer cells or human peripheral blood monocytes was found to be able to stimulate alpha 2-macroglobulin synthesis in hepatocytes. Equal amounts of HSF activity were found in conditioned media from lipopolysaccharide-stimulated and unstimulated rat Kupffer cells as well as in human monocytes. Since the supernatants of unstimulated rat Kupffer cells or human monocytes did not exhibit interleukin 1 activity, HSF activity distinct from interleukin 1 must exist. No HSF activity was found in media conditioned by rat Kupffer cells which had been treated with dexamethasone. Hepatocyte primary cultures were incubated with [35S]methionine-labeled proteins secreted by rat Kupffer cells. A 30 kDa polypeptide was found to be bound to or internalized by rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

辛伐他汀对IFN-y诱导的人外周血单个核细胞OX40L表达的影响

目的：探讨经IFN-y刺激后,人外周血单个核细胞OX40L表达的变化,以及辛伐他汀对单个核细胞OX40L表达的影响。方法：将实验标本随机分为2组,分别干扰素-y（IFN-y）刺激组、辛伐他汀干预组。应用RT—PCR及Western blotting技术,观察IFN-y诱导的人外周血单个核细胞OX40L表达情况及辛伐他汀对人单个核细胞OX40L表达的影响。结果：1．1000U／ml IFN-与人单个核细胞共同培养24h后,OX40LmRNA和蛋白水平的表达明显增加。2．预先给予10mol／L的辛伐他汀干预1h可以明显降低IFN-诱导的OX40L表达。结论：IFN-可诱导人单个核细胞OX40L表达。辛伐他汀可以抑制IFN-诱导人单个核细胞OX40L表达的增强,从而可能抑制了OX40L信号通路介导的与炎症有关的血管损伤,延缓动脉粥样硬化的进程。     

Elevated plasma concentrations of haptoglobin in European brown bears during hibernation

Haptoglobin (Hp), a hemoglobin-binding protein, is known as an acute phase protein and increases during the acute phase of inflammation in most mammals. We reported previously in brown bears that the mean Hp concentrations were higher in blood samples obtained in winter than those in spring. To examine a possible relation of the seasonal variations of Hp to hibernation, in the present study, we measured the plasma concentrations of Hp as well as some other acute phase proteins (alpha(2)-macroglobulin, alpha(1)-antitrypsin, C-reactive protein) in 6 European brown bears (Ursus arctos), from which blood samples were obtained at 5-6 different months of year including February, the time of hibernation. The Hp concentrations showed clear seasonal variations, being highest in February. The alpha(2)-macroglobulin concentrations also showed a similar but much smaller rise in February, but those of alpha(1)-antitrypsin and C-reactive protein did not show any seasonal variations. Our results suggest that the seasonal variation of plasma Hp concentration in brown bears is associated with a hibernation-specific mechanism more than that of acute phase response.     

The identification of distinctive forms of human alpha 2-macroglobulin by using the numerical relationship between trypsin binding in alpha- and beta-modes.

Interactions between the serine proteinase trypsin and the protein proteinase inhibitors in human blood were expressed in terms of a coupled set of non-linear differential equations, which has been solved for each of 110 samples of serum obtained from colleagues and from a variety of hospital sources. Optimization of nine unknown theoretical parameters and 21 experimental rate measurements of the hydrolytic activity of trypsin in free and bound states after admixture with various amounts of a given serum was achieved by an iterative procedure using initial estimates of the parameters derived from the "four-straight-line" model described in the preceding paper [Topping & Seilman (1979) Biochem. J. 177, 493--499.] Such a procedure yielded the following information for each sample of serum examined: (a) the concentrations of alpha 1-antitrypsin and alpha 2-macroglobulin; (b) the unequivocal assignment of alpha 2-macroglobulin into one of seven categories on the basis of trypsin binding in two kinetically differentiated modes (alpha and beta); (c) the hydrolytic activities of trypsin (versus Bz-Arg-OEt) when bound to alpha 1-antitrypsin, and to alpha 2-macroglobulin in the alpha- and beta-modes. Molecular interpretations of the binding of trypsin to alpha 2-macroglobulin are discussed and the potential clinical value of recognizing the nature of such binding is reported.     

Cloning,sequencing and expression of porcine CD40 ligand in Escherichia coli and human and porcine cells

The CD40L ligand (CD40L) plays an important role in the interaction between antigen-specific T lymphocytes and antigen-presenting cells. The porcine CD40L encoding gene was isolated from porcine peripheral blood mononuclear cells (PBMC) using RT-PCR. Sequence analysis of the cloned CD40L gene showed an open reading frame of 786 base pairs encoding a 262 amino acid protein with a predicted molecular mass of 29 kD. The deduced amino acid sequence of the porcine CD40L shared 82%, 88% and 93% similarity with the CD40L protein of mouse, human and cattle. The isolated CD40L sequence was expressed as a hexahistidine fusion protein in Escherichia coli and purified by affinity chromatography. The analysis of the CD40L-expression in human 293 and porcine MAX cells by immunofluorescence showed its location on the cell surface.     

The primary sequence and the subunit structure of mouse alpha-2-macroglobulin, deduced from protein sequencing of the isolated subunits and from molecular cloning of the cDNA.

Mouse plasma alpha-2-macroglobulin (m alpha 2M) was isolated and the N-terminal amino-acid sequences determined after separation of the 165-kDa and 35-kDa subunits. These sequences were compared to the protein sequence predicted by the cDNA, which was cloned from a mouse liver library and sequenced. From these data it is evident that both subunits are encoded by one mRNA of approximately 5 kb expressed predominantly in liver. The smaller subunit, with the N-terminal sequence DLSSSDLT, comprises the C-terminal 257 residues of m alpha 2M and is derived from a single-chain precursor probably by proteolytic processing at an arginine residue in the sequence PTRDLSS. Analysis of the predicted protein further showed all the salient features of a proteinase inhibitor of the macroglobulin family: a bait region that deviates from all known sequences in this family, a very conserved internal thiolester site and conserved cysteine residues and putative N-glycosylation sites. The synthesis of m alpha 2M in adult liver was demonstrated by Northern blotting and in fetal liver by in-situ hybridization. Transient transfection of COS cells with the cDNA under control of a viral promoter demonstrated the secretion and partial processing of m alpha 2M in the culture medium. In plasma the level of m alpha 2M was found to be stable as expected for the murine counterpart of human plasma alpha-2-macroglobulin. The possibilities of using the mouse as a genetic model to study this proteinase inhibitor in vivo are discussed.     

Monoclonal antibody OKT11A inhibits and recombinant interleukin 2 (IL 2) augments expression of IL 2 receptors at a pretranslational level

The expression of receptors for interleukin 2 (IL 2) represents a critical event regulating the growth of normal T lymphocytes. We investigated the effects of the inhibitory monoclonal antibody OKT11A (anti-sheep erythrocyte receptor) and of purified recombinant IL 2 (rIL 2) on the expression of IL 2 receptors by activated T cells at both the protein and the mRNA levels. Adding OKT11A antibody (0.5 microgram/ml) to phytohemagglutinin (PHA)-stimulated cultures of human peripheral blood mononuclear cells (PBMC) markedly suppressed cellular proliferation (assessed by [3H]thymidine incorporation) and IL 2 receptor expression (determined by immunofluorescence assay by using the anti-IL 2-receptor antibody, anti-Tac). Northern blot analysis performed with the use of a cDNA probe specific for the human IL 2 receptor gene demonstrated that OKT11A antibody also decreased the accumulation of IL 2 receptor mRNA induced by PHA in PBMC. Purified rIL 2 (10 U/ml) alone had little effect on the expression of IL 2 receptors in unstimulated PBMC cultures. In combination with PHA or with PHA plus OKT11A, however, rIL 2 augmented both the expression of IL 2 receptor protein on PBMC and the accumulation of IL 2 receptor mRNA in PBMC. Adding anti-Tac antibody to PBMC cultures to block the interaction of IL 2 with its receptor diminished the accumulation of IL 2 receptor mRNA induced by PHA. Taken together, these data demonstrate that OKT11A antibody inhibits and IL 2 augments expression of IL 2 receptors on PHA-stimulated T cells, at least in part, at a pretranslational level.