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CD4+ T cells from young and aged mice were sorted into Mel-14+ cells which are regarded as naive cells and Mel-14- cells which are regarded as memory cells. These subsets were stimulated in short-time cultures with anti-CD3 or anti-CD3/anti-CD28 in order to determine the presence of Th1 and/or Th2 cytokines. Based on the simultaneous production of IL-2, IL-4, IL-10, and IFN-γ upon anti-CD3 stimulation by Mel-14- cells from young and aged mice, it is concluded that this cell population comprises Th1, Th2, and/or Th0 cells. Mel-14+ cells from young mice only secrete substantial amounts of IL-2 in the presence of anti-CD28 as a costimulatory signal and can therefore be regarded as Th precursor cells. By contrast, Mel-14+ cells from aged mice responded to anti-CD3 alone, not only by the production of IL-2 but also by the production of high amounts of IFN-γ and minute amounts of IL-4 and IL-10, suggesting that these 'naive' cells in aged mice are enriched for Th1 cells. This was not due to lack of CD28 triggering since anti-CD28 enhanced IFN-γ as well as IL-4 and IL-10 to a similar extent. Our data therefore indicate that Mel-14 is not exclusively expressed on naive CD4+ T cells.

The aim of this study was to evaluate the tissue infiltration and phenotypic adhesion profile of 5T2 multiple myeloma (MM) and 5T33 MM cells and to correlate it with that observed in human disease. For each line, 30 mice were intravenously inoculated with myeloma cells and at a clear-cut demonstrable serum paraprotein concentration; mice were sacrificed and a number of organs removed. The haematoxylin-eosin stainings on paraffin sections were complemented with immunohistochemistry using monoclonal antibodies developed against the specific MM idiotype. When analysed over time, 5T2 MM cells could be observed in bone marrow samples from week 9 after transfer of the cells. For the 5T33 MM, a simultaneous infiltration was observed in bone marrow, spleen and liver 2 weeks after inoculation. Osteolytic lesions consistently developed in the 5T2 MM, but this was not consistent for 5T33 MM. PCNA staining showed a higher proliferative index for the 5T33 MM cells. The expression of adhesion molecules was analysed by immunohistochemistry on cytosmears: both 5T2 MM and 5T33 MM cells were LFA-1, CD44, VLA-4 and VLA-5 positive. We conclude that both lines have a phenotypic adhesion profile analogous to that of human MM cells. As the 5T2 MM cells are less aggressive than the 5T33 MM cells, their organ distribution is more restricted to the bone marrow and osteolytic lesions are consistently present, the former cell line induces myeloma development similar to the human disease.

Scavenger receptors, which include various classes, play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins, resulting in the massive accumulation of cholesteryl esters. Because macrophage-derived foam cells are considered to be an important feature in early atherogenesis, we investigated the role of scavenger receptor class A (SR-A) overexpression, especially on macrophages in lipoprotein metabolism and atherosclerosis. Bone marrow from human SR-A (MSR1)-overexpressing mice was transplanted into irradiated low density lipoprotein receptor knockout [LDLR(-/-)] mice. The transplantation resulted in an increase in total serum cholesterol (approximately 15 to 25%), especially in the VLDL fraction, when compared with LDLR(-/-) mice that were transplanted with bone marrow of wild-type littermates. Quantification of atherosclerotic lesions in the mice that were fed a 'Western-type' diet for 3 months revealed that there were no differences in mean lesion area between LDLR(-/-) mice transplanted with MSR1 overexpressing and wild-type littermate bone marrow, despite increased scavenger receptor activity in vitro. The presence or absence of the LDLR in the transplanted bone marrow did not influence these results. In conclusion, introduction of MSR1-overexpressing bone marrow in LDLR(-/-) mice via bone marrow transplantation resulted in a slight increase in lipoprotein levels, but had no effect on the atherosclerotic lesion area, despite increased scavenger receptor activity in vitro. - Herijgers, N., M. P. J. de Winther, M. Van Eck, L. M. Havekes, M. H. Hofker, P. M. Hoogerbrugge, and T. J. C. Van Berkel. Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knockout mice. Chemicals/CAS: acetyl-LDL; Cell Adhesion Molecules; Lipoproteins; Lipoproteins, LDL; MSR1 protein, human; Msr1 protein, mouse; Receptors, Immunologic; Receptors, LDL; Receptors, Scavenger; Scavenger Receptors, Class A

Inflammatory mediators and soluble cell adhesion molecules predict cardiovascular events. It is not clear whether they reflect the severity of underlying atherosclerotic disease. Within the Rotterdam Study, we investigated the associations of C-reactive protein (CRP), interleukin-6 (IL-6), soluble intercellular adhesion molecule-1, and soluble vascular cell adhesion molecule-1 with noninvasive measures of atherosclerosis. Levels of CRP were assessed in a random sample of 1317 participants, and levels of IL-6 and soluble cell adhesion molecules were assessed in a subsample of 714 participants. In multivariate analyses, logarithmically transformed CRP (regression coefficient [β]=-0.023, 95% CI -0.033 to -0.012) and IL-6 (β=-0.025, 95% CI -0.049 to -0.001) were inversely associated with the ankle-arm index. Only CRP was associated with carotid intima-media thickness (β=0.018, 95% CI 0.010 to 0.027). Compared with the lowest tertile, the odds ratio for moderate to severe carotid plaques associated with levels of CRP in the highest tertile was 2.0 (95% CI 1.3 to 3.0). Soluble intercellular adhesion molecule-1 levels were strongly associated with carotid plaques (odds ratio 2.5, 95% CI 1.5 to 4.4 [highest versus lowest tertile]). Soluble vascular cell adhesion molecule-1 was not significantly associated with any of the measures of atherosclerosis. This study indicates that CRP is associated with the severity of atherosclerosis measured at various sites. Associations of the other markers with atherosclerosis were less consistent. Chemicals/CAS: C-Reactive Protein, 9007-41-4; Cell Adhesion Molecules; Inflammation Mediators; Intercellular Adhesion Molecule-1, 126547-89-5; Interleukin-6

Apolipoprotein E3-Leiden (APOE3-Leiden) transgenic mice develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. We have studied the progression and regression of atherosclerosis using immunohistochemistry. Female transgenic mice were fed a moderate fat diet to study atherosclerosis over a longer time period. Fatty streaks arose in the intima and consisted of lipid filled macrophages which differed in origin. All macrophages expressed the macrophage scavenger receptor while two thirds expressed sialoadhesin and were positive for an antibody recognizing marginal zone macrophages (MOMA-1). All macrophages were negative for the scavenger receptor MARCO and 50% were positive for CD4. Small fatty streaks contained CD-3 positive T-lymphocytes which were for more than 70% CD4-positive. ICAM- 1 was positive both in atherosclerotic and control mice. In early plaques, fibrosis was observed on the luminal and medial site of the foam cells while smooth muscle cells were only observed in the fibrous cap. To study regression, we used a high fat, high cholesterol diet to rapidly induce atherosclerosis (14 weeks). The animals were then fed normal chow. Subsequently, atherosclerosis was assayed over time (4, 8, 16 weeks). Cholesterol levels dropped in 4 weeks to control levels. The animals did not show a significantly decrease in plaque size over time, but the percentage macrophages was significantly smaller in the animals after 4 weeks. In conclusion, the APOE3-Leiden mouse is a useful model to study the progression and regression of atherosclerosis.

CD56 is a member of the neural cell adhesion molecule family expressed on cells of the central nervous system and also on NK cells. Previous studies suggest the involvement of CD56 in effector-to-target cell conjugation mediated by NK cells. It was shown recently that CD56 is also expressed by subpopulations of CD8+ and CD4+ T cells. The present study describes the functional characteristics of CD4+CD56+ T cell lines established from blood of multiple sclerosis patients by stimulation with myelin basic protein (MBP). CD4+CD56+, MBP-specific T cell lines were able to lyse MBP-pulsed target cells in an HLA class II-restricted fashion. At the same time, they mediated MHC-unrestricted lysis of CD56+ target cells such as CD56+ lymphoid or glial tumor cells, but not of the typical NK target, K562. A number of experimental results including separation of CD4+CD56+ T cells into CD56 high and low expressing populations, cold target inhibition, as well as killing of CD56-transfected cells indicate that homotypic CD56 interactions are involved in the MHC-unrestricted lysis. CD56 interactions are not sufficient but are required for effector/target interaction. Our findings raise the possibility that CD4+CD56+ T cells sharing properties of both typical Ag-specific Th0-like T cells and NK cells might be involved in damage of tissues expressing CD56/neural cell adhesion molecule, such as the central nervous system. Thus, we provide evidence for a novel mechanism that could lead to organ-specific autoreactivity. Chemicals/CAS: Antigens, CD4; Antigens, CD56; Biological Markers; Epitopes; Histocompatibility Antigens Class II; Myelin Basic Proteins; Neural Cell Adhesion Molecules

Background: Hyperhomocysteinaemia may constitute an independent risk factor for cardiovascular disease, but it is still unclear by which pathophysiological mechanisms homocysteine (tHcy) may promote atherothrombosis. The aim of this study was firstly to examine whether tHcy is associated with endothelial dysfunction, increased adherence of leukocytes, and/or chronic low-grade inflammation, as estimated from plasma levels of von Willebrand factor (vWf), soluble vascular cell adhesion molecule 1 (sVCAM-1) and C-reactive protein (CRP), respectively. Secondly we investigated whether the presence of type 2 diabetes modifies these associations. Materials and Methods: Six hundred and ten subjects of a general population of middle-aged and elderly subjects, 170 of whom had type 2 diabetes, participated in this cross-sectional study. Linear regression analyses were used to study whether tHcy was associated with vWf, sVCAM-1 and CRP, and whether the presence of diabetes modified these associations. Results: After adjustment for confounders, tHcy was significantly but weakly associated with vWf (β=0·15, P=0·05) and sVCAM-1 (β=0·082, P=0·04). tHcy was not significantly associated with CRP (β=0·02, P=0·91). The presence of diabetes did not significantly modify these associations. Conclusions: This study provides evidence that tHcy is, at most, weakly associated with endothelial dysfunction as estimated from plasma vWf, and with leukocyte adhesion as estimated from plasma sVCAM-1. tHcy was not significantly associated with chronic low-grade inflammation as estimated from plasma CRP. Our data thus suggest that the link between tHcy and atherothrombosis cannot be explained by associations of tHcy with vWf, sVCAM-1 or CRP. Chemicals/CAS: C-Reactive Protein, 9007-41-4; Cell Adhesion Molecules; Homocysteine, 454-28-4; von Willebrand Factor

Background - Statins can exert anti-inflammatory antiatherosclerotic effects through an anti-inflammatory action, independent of lowering cholesterol. We addressed the question whether the anti-inflammatory activities of statins can reduce atherosclerosis beyond the reduction achieved by cholesterol lowering per se. Methods and Results - Two groups of 20 female APOE*3-Leiden mice received either a high-cholesterol diet (HC) or a high-cholesterol diet supplemented with 0.005% (wt/wt) rosuvastatin (HC+R). The HC diet alone resulted in a plasma cholesterol concentration of 18.9±1.4 mmol/L, and administration of rosuvastatin lowered plasma cholesterol to 14.1±0.7 mmol/L. In a separate low-cholesterol (LC) control group, the dietary cholesterol intake was reduced, which resulted in plasma cholesterol levels that were comparable to the HC+R group (13.4±0.8 mmol/L). Atherosclerosis in the aortic root area was quantified after 24 weeks. As compared with the HC group, the LC group had a 62% (P<0.001) reduction in cross-sectional lesion area. When compared with the LC group, the HC+R group showed a further decrease in cross-sectional lesion area (80%, P<0.001), size of individual lesions (63%, P<0.05), lesion number (58%, P<0.001), monocyte adherence (24%, P<0.05), and macrophage-containing area (60%, P<0.001). Furthermore, rosuvastatin specifically suppressed the expression of the inflammation parameters MCP-1 and TNF-α in the vessel wall and lowered plasma concentrations of serum amyloid A and fibrinogen, independent of its cholesterol-lowering effect. Conclusions - Rosuvastatin reduces atherosclerosis beyond and independent of the reduction achieved by cholesterol lowering alone. This additional beneficial effect of rosuvastatin may be explained, at least partly, by its antiinflammatory activity. Chemicals/CAS: amyloid A protein, 59165-71-8; fibrinogen, 9001-32-5; rosuvastatin, 147098-18-8, 147098-20-2; cholesterol, 57-88-5; fluorobenzene, 2367-82-0, 327-54-8, 363-72-4, 367-23-7, 372-38-3, 462-06-6, 540-36-3; lipid, 66455-18-3; Anti-Inflammatory Agents; apolipoprotein E3 (Leidein); Apolipoprotein E3; Apolipoproteins E; Cholesterol, 57-88-5; Cytokines; Fluorobenzenes; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lipids; Lipoproteins; Pyrimidines; rosuvastatin, 287714-41-4; Sulfonamides

Infections with verocytotoxin (VT) producing Escherichia coil have been strongly implicated in the epidemic form of hemolytic uremic syndrome (HUS). Endothelial damage plays a central role in the pathogenesis of HUS. In vitro studies have shown that VT can damage endothelial cells after interaction with its cellular receptor globotriaosylceramide (GbOse3cer). Cytokines, such as tumor necrosis factor α (TNFα) and interleukin-1 (IL-1) can potentiate the toxic effect of VT by inducing a protein-synthesis dependent increase in VT receptors on endothelial cells. In this study, the mechanisms underlying the increase in endothelial VT receptors induced by TNFα were studied in more detail. To investigate which proteins were involved in this induction, endothelial cells were incubated with and without TNFα in the presence of 14C-galactose or 14C-glucose. Thin-layer chromatography (TLC) analysis of the glycolipid extracts of these cells demonstrated a markedly enhanced incorporation of 14C-galactose in GbOse3cer and other galactose- containing glycolipids, suggesting that TNFα enhanced galactosyl-transferase activity. To examine the role of the two recently cloned TNF-receptors (TNFR- p75 and TNFR-p55) in the TNFα-induced increase in GbOse3cer in human endothelial cells, cells were incubated with TNFα, the TNFR-p55 selective R32W-S86T-TNFα-mutant, or the TNFR-p75 selective D143N-A145R-TNFα-mutant. The effect of TNFα activation,determined by binding-experiments with 125I-VT-1, could be largely, but not completely mimicked by R32W-S86T- TNFα. Although incubation of cells with D143N-A145R-TNFα did not show an increase in VT-1 binding, the monoclonal antibody utr-1, which prevents binding to TNFR-p75, decreased the TNFα-induced VT-1 binding. Activation of protein kinase C (PKC) by phorbol ester increases the expression of VT-1 receptors; this effect was prevented by the PKC inhibitor Ro31-8220 and by homologous desensitization by pretreatment with phorbol ester. In contrast, the presence of the protein kinase inhibitor Ro31-8220 or desensitization of PKC activity reduced the TNFα-induced increase in VT-1 receptors maximally by 50% end 24%, respectively. Comparable reductions in overall protein synthesis and the synthesis of E-selectin and plasminogen activator inhibitor-1 (PAI-1) were observed. This suggests an effect on general protein synthesis rather than a specific effect of PKC in the signal transduction pathway, by which TNFα induces VT-1 receptors. Our results indicate that TNFα can increase the VT-1 receptors on endothelial cells by inducing galactosyl-transferase activity, that this action of TNFα mainly occurs via the TNFR-p55; and that PKC activation increases expression of VT-1 receptors by a separate mechanism that acts additively to the TNFα-induced increase in VT-1 receptors.

Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT- cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin- stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca2+](i) was strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca2+ and endothelial permeability. These data indicate that cGMP-PK type I is expressed in various types of human macrovascular and microvascular ECs but is absent or expressed in very low amounts in umbilical vein ECs. cGMP-PK type I expression in ECs may be important in the regulation of endothelial permeability and the release of factors involved in vasoregulation and hemostasis. Chemicals/CAS: 8-((4-chlorophenyl)thio)cyclic-3',5'-GMP, 54364-02-2; Calcium Channels; Cell Adhesion Molecules; Cyclic GMP, 7665-99-8; Cyclic GMP-Dependent Protein Kinases, EC 2.7.1.37; Microfilament Proteins; Phosphoproteins; Platelet Aggregation Inhibitors; RNA, 63231-63-0; Thionucleotides; Thrombin, EC 3.4.21.5; vasodilator-stimulated phosphoprotein