PubMed Health⌕ Search

Biomedical subjects

J Apostolopoulos

Publications and source records attributed to J Apostolopoulos.

13 recordsLinked to original sources
PubMed2003

ORP3 splice variants and their expression in human tissues and hematopoietic cells.

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34(+) hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.

Alternative Splicing↗
PubMed2001

Identification and characterization of a novel family of mammalian ependymin-related proteins (MERPs) in hematopoietic, nonhematopoietic, and malignant tissues.

Evidence is presented for a family of mammalian homologs of ependymin, which we have termed the mammalian ependymin-related proteins (MERPs). Ependymins are secreted glycoproteins that form the major component of the cerebrospinal fluid in many teleost fish. We have cloned the entire coding region of human MERP-1 and mapped the gene to chromosome 7p14.1 by fluorescence in situ hybridization. In addition, three human MERP pseudogenes were identified on chromosomes 8, 16, and X. We have also cloned the mouse MERP-1 homolog and an additional family member, mouse MERP-2. Then, using bioinformatics, the mouse MERP-2 gene was localized to chromosome 13, and we identified the monkey MERP-1 homolog and frog ependymin-related protein (ERP). Despite relatively low amino acid sequence conservation between piscine ependymins, toad ERP, and MERPs, several amino acids (including four key cysteine residues) are strictly conserved, and the hydropathy profiles are remarkably alike, suggesting the possibilities of similar protein conformation and function. As with fish ependymins, frog ERP and MERPs contain a signal peptide typical of secreted proteins. The MERPs were found to be expressed at high levels in several hematopoietic cell lines and in nonhematopoietic tissues such as brain, heart, and skeletal muscle, as well as several malignant tissues and malignant cell lines. These findings suggest that MERPs have several potential roles in a range of cells and tissues.

Amino Acid Sequence↗
PubMed2000

Ly6d-L, a cell surface ligand for mouse Ly6d.

The mouse Ly6 gene family encodes proteins found in lymphocytes and other cells. Some are involved in cell activation; no ligands have been found. A ligand for Ly6d (ThB) was identified on lymphocytes using microspheres loaded with Ly6d and the cDNA isolated from a spleen/thymus library by panning on Ly6d. The Ly6d ligand (Ly6d-L) is a nonglycosylated protein of 9 kDa of broad distribution, rich in cysteine, with no discernable transmembrane sequence. Its N and C termini are on the cell surface, where it associates with a 30 kDa protein. Ly6d-L is homologous with an EGF repeat of Notch.

Amino Acid Sequence↗
PubMed1997

Mechanisms of T cell-induced glomerular injury in anti-glomerular basement membrane (GBM) glomerulonephritis in rats.

The effector mechanisms of T cell-dependent acute glomerular injury were studied in autologous phase anti-GBM glomerulonephritis (GN) in rats. Acute proliferative GN was induced in sensitized rats by a subnephritogenic dose of sheep anti-rat GBM antibody. Injury was manifested by proteinuria and glomerular leucocyte infiltration composed predominantly of macrophages but also CD4+ and CD8+ T cells. T cell depletion, using an anti-CD5 MoAb, demonstrated that glomerular leucocyte infiltration and proteinuria were T cell-dependent. Inhibition of T helper cell function using an anti-CD4 MoAb prevented proteinuria and glomerular macrophage and CD4+ T cell influx, but not accumulation of CD8+ T cells. Depletion of CD8+ T cells also prevented proteinuria and the influx of macrophages and CD8+ T cells, but not accumulation of CD4+ T cells. Macrophage depletion, using micro-encapsulated clodronate, prevented proteinuria and glomerular macrophage infiltration, but not the accumulation of CD4+ or CD8+ T cells, indicating that macrophages are the common cellular effectors for both CD4 and CD8 T cell-dependent injury. Evidence for cytotoxic mechanisms of injury (increased numbers of apoptotic cells or accumulation of natural killer (NK) cells in glomeruli) could not be demonstrated. These data suggest that acute glomerular injury in anti-GBM GN is the result of macrophage recruitment, which is dependent on both CD4 and CD8 T cells, and that direct T cell-mediated injury (cellular cytotoxicity) is not involved.

Animals↗
PubMed1997

Tissue factor pathway inhibitor expression in atherosclerosis.

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor (TF) -initiated coagulation and may play a role in regulating coagulation in atherosclerotic plaques. The expression of TFPI protein and mRNA was examined by immunohistology and in situ hybridization in normal human and rabbit arteries, in human carotid arteries with advanced atherosclerosis, and in atherosclerotic aortas from cholesterol-fed rabbits. In normal human and rabbit arteries, TFPI protein and mRNA were detected in the adventitial layer but were undetectable in the luminal endothelium. In the medial smooth muscle layer of rabbits, weak expression of TFPI mRNA, but not protein, was detected; in that of humans, neither TFPI mRNA nor protein was detectable. In atherosclerotic arteries, TFPI protein and mRNA were detected in three of six internal carotid plaques from patients undergoing endarterectomy, and mRNA alone was detected in one further specimen. TFPI protein was found in areas of the plaque where TF was abundant and colocalized with macrophages, suggesting that these cells are responsible for TFPI synthesis. TFPI protein and mRNA were also detected in fatty-streak lesions in 18 of 19 rabbits fed a high-cholesterol diet for periods between 4 and 16 weeks. In these macrophage-rich lesions, expression of TFPI protein and mRNA was most intense at the base of the plaques. These studies suggest that TFPI is expressed in the adventitial layer of large arteries and that in atherosclerotic vessels, TFPI is expressed by macrophages in focal areas throughout the plaque. Local production of TFPI may regulate procoagulant activity and thrombotic events within atherosclerotic plaques.

Aged↗
PubMed1996

Interleukin-1 receptor antagonist: characterisation of its gene expression in rabbit tissues and large-scale expression in eucaryotic cells using a baculovirus expression system.

The gene expression of rabbit interleukin-1 receptor antagonist (RbIL-lra) was examined in rabbit tissues. RNA was isolated from heart, lung, kidney, muscle, liver, spleen, brain, and peripheral blood monocytes (PBMs), and RbIL-lra mRNA was identified as a single species by Northern analysis using a RbIL-lra probe. RbIL-lra was abundantly expressed in lung, brain, heart, and liver, expressed at low levels in spleen, and undetectable in kidney and unstimulated PBMs. Expression of large scale recombinant production of RbIL-lra was achieved by subcloning the cDNA into a baculovirus expression vector. Recombination of this vector was completed with the BacPAK6 baculovirus genome. The recombinant virus, containing the RbIL-lra cDNA, was used to infect Spodoptera frugiperda (Sf21) insect cells in a spinner flask system and in monolayers in cell culture flasks. Recombinant rabbit IL-lra (rRbIL-lra) was secreted into the culture medium in this system at very high levels (35 mg/l). The protein was identified by reducing SDS-PAGE electrophoresis, was variably glycosylated and had a molecular weight between 19-25 kDa. It was then purified by size exclusion HPLC on a Du Pont Gf-250 column. The rRbIL-lra was demonstrated to be functionally active by inhibiting recombinant human IL-1 alpha in a mouse thymocyte proliferation assay. 20 ng/ml (6.7 U/ml) of rRbIL-lra inhibited 95% of the activity of 2 ng/ml IL-1 alpha.

Animals↗
PubMed1996

Renal expression of tissue factor pathway inhibitor and evidence for a role in crescentic glomerulonephritis in rabbits.

Tissue factor pathway inhibitor (TFPI) was demonstrated in the kidneys of normal rabbits and in a crescentic model of glomerulonephritis (GN), where fibrin is a key mediator of injury. In normal kidneys, TFPI was expressed in glomeruli, in intrarenal arteries and the interstitial capillary network. Evidence for TFPI synthesis in vivo was provided by in situ demonstration of TFPI mRNA in glomeruli and intrarenal vessels and by biosynthetic labeling of TFPI released from glomeruli in vitro. In fibrin-dependent crescentic GN, glomerular TFPI synthesis and expression was initially decreased (TFPI antigen at 24 h, 7.5 +/- 0.7 ng/10(3) glomeruli; normal, 11.1 +/- 0.9 ng/10(3) glomeruli, P < 0.02) and subsequently returned to normal values. Plasma TFPI levels increased progressively throughout the evolution of disease. In vivo inhibition of TFPI using an anti-TFPI antibody during the development of GN significantly increased glomerular fibrin deposition (GFD) and exacerbated renal impairment. Infusion of recombinant human TFPI significantly reduced development of GFD (fibrin scores, TFPI treated 0.82 +/- 0.11, control 1.49 +/- 0.14, P < 0.01), proteinuria and renal impairment. This data indicates that TFPI is synthesized and expressed in normal glomeruli and is down regulated in the early response to glomerular injury. Endogenous glomerular TFPI and treatment with recombinant TFPI reduces GFD and injury in fibrin dependent GN. TFPI has the potential to be of therapeutic benefit in the management of fibrin dependent human GN.

Animals↗
PubMed1996

Interleukin-8 production by macrophages from atheromatous plaques.

Interleukin-8 (IL-8) is a chemotactic peptide produced by macrophages that may be involved in the recruitment of inflammatory cells into atherosclerotic plaques. In vitro, IL-8 production by macrophages isolated from carotid plaques (1240 +/- 510 pg.10(5) cells-1.24h-1, mean +/- SEM, n = 6) and noncarotid plaques (4312 +/- 1588 pg.10(5) cells-1.24 h-1, n = 9) was significantly greater than IL-8 production by blood monocytes isolated from the same patients (526 +/- 278 pg.10(5) cells-1.24 h-1, n = 6, P < .05 and 726 +/- 384 pg.10(5) cells-1.24 h-1, n = 9, P < .01, respectively). IL-8 produced by atherosclerotic macrophages was demonstrated to be biologically active in a neutrophil chemotaxis assay. IL-8 mRNA was detectable in plaque macrophages and blood monocytes from these patients, but blood monocytes from normal donors did not exhibit detectable IL-8 mRNA. IL-8 mRNA was localized in macrophage-rich areas of atherosclerotic plaques by in situ hybridization. These studies demonstrate that macrophages from atherosclerotic plaques show an enhanced capacity to produce IL-8 compared with normal and patient blood monocytes and that macrophages are a major site of IL-8 mRNA production in atherosclerotic plaques. These results provide further evidence for a proinflammatory role for macrophages in atherosclerosis.

Aged↗
PubMed1995

Glomerular tissue factor expression in crescentic glomerulonephritis. Correlations between antigen, activity, and mRNA.

Correlations between glomerular expression of tissue factor (TF) activity and antigen and cellular localization of TF mRNA was studied in crescentic glomerulonephritis (GN) in rabbits. Glomerular TF activity increased 8.7-fold 24 hours after initiation of GN (234 +/- 49 mU/10(3) glomeruli; normal, 27 +/- 10 mU/10(3) glomeruli; P = 0.003) in association with a 2.1-fold increase in TF antigen (154 +/- 34 ng/10(3) glomeruli; normal, 72 +/- 10 ng/10(3) glomeruli; P = 0.055), early macrophage infiltration, and no significant increase in TF mRNA. At the peak glomerular macrophage infiltration (day 4), TF activity remained augmented (230 +/- 63 mU/10(3) glomeruli) and TF mRNA, colocalized within macrophages, was significantly increased compared with normal (267 +/- 42%; P = 0.001). TF antigen was not increased in glomeruli (114 +/- 17 ng/10(3) glomeruli), although significant urinary excretion of TF antigen was detectable (478 +/- 121 ng/24 hours; normal, < 1 ng/24 hours; P = 0.032). At this time, the M(r) of glomerular TF (49 to 61 kd) was increased compared with TF in normal glomeruli (49 to 58 kd) as a result of increased glycosylation. At day 7, TF activity and antigen within glomeruli had decreased, although urinary excretion of TF antigen and glomerular TF mRNA remained elevated. These studies suggest that early up-regulation of TF activity is largely a result of functional up-regulation of constitutive TF in intrinsic glomerular cells. In more advanced disease, infiltrating macrophages are the major site of TF synthesis. The increased M(r) of glomerular TF, as a result of synthesis of more highly glycosylated protein by macrophages and the shedding of TF into the urine, suggests that substantial turnover of glomerular TF occurs at this stage.

Animals↗
PubMed1993

Atheromatous plaque macrophages produce plasminogen activator inhibitor type-1 and stimulate its production by endothelial cells and vascular smooth muscle cells.

The capacity of macrophages to influence directly and indirectly fibrinolytic processes in atherosclerosis was studied using macrophages isolated from atherosclerotic plaques of patients undergoing surgical repair of distal aortic and femoral arteries. These cells were characterized by their morphology, adherence, esterase positivity, and expression of CD14 antigen. Production of plasminogen activator inhibitor type-1 (PAI-1) by plaque macrophages (6.7 +/- 2.7 ng/10(5) cells/24 hours [mean +/- SEM]) was significantly greater than PAI-1 production by blood monocytes isolated simultaneously from the same patients (1.8 +/- 1.5 ng/10(5) cells/24 hours). Production of tissue type plasminogen activator and urokinase type was not augmented compared to blood monocytes. Conditioned medium from cultured plaque macrophages significantly increased production of PAI-1 by endothelial cells (85 +/- 11% above basal) and vascular smooth muscle cells (25 +/- 10%) in vitro. This response was significantly greater than the response to monocyte-conditioned medium (endothelial cells 38 +/- 11%, vascular smooth muscle cells 2.5 +/- 2.0%). Stimulation of endothelial cell PAI-1 production by macrophage-conditioned medium was partially inhibitable by a monoclonal antibody to transforming growth factor-beta. Tissue type plasminogen activator production by endothelial cells and vascular smooth muscle cells was not affected by plaque macrophage- or monocyte-conditioned medium. Urokinase type plasminogen activator production by endothelial cells and vascular smooth muscle cells was undetectable in control medium and was augmented to similar levels in response to plaque macrophage- and monocyte-conditioned media. These results demonstrate upregulation of PAI-1 production by macrophages in atheromatous plaques and the capacity of soluble products from plaque macrophages to upregulate PAI-1 production by endothelial cells and vascular smooth muscle cells in vitro. These data suggest that macrophages in atherosclerotic plaques may inhibit thrombolysis both directly and indirectly by effects of their soluble products on endothelial cells and vascular smooth muscle cells.

Aged↗
PubMed1987

Effect of acute inflammation on rat apolipoprotein mRNA levels.

Hybridization studies using specific cDNA have been used to determine the mRNA levels for rat apolipoproteins AI, AII, AIV, and E in extracts of rat liver and intestine. The ratios of intestinal mRNA/liver mRNA for apolipoprotein AI (apo AI), apo AIV, and apo E were 1.3, 1.7, and 0.1, respectively. Apo AII mRNA was detected in the liver but not in the intestine. The mRNA levels for apo AII and apo AIV in rat liver decreased during inflammation to minimums of 40% and 25% of normal, respectively. The mRNA levels for apo AIV in the intestine, apo E in the liver and for apo AI in both the liver and intestine did not change significantly during inflammation. The time course for the decrease in the hepatic mRNA levels for apo AIV was similar to those previously observed for the negative acute-phase proteins albumin and transthyretin. The serum levels for apo AIV were not affected by inflammation.

Animals↗