Lack of toxic effects of F 12511, a novel potent inhibitor of acyl-coenzyme A: cholesterol O-acyltransferase, on human adrenocortical cells in culture.

Inhibition of acyl-coenzyme A: cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) reduces intracellular cholesteryl esters that are substrates for steroidogenesis in adrenal cells. The adrenal side effects of ACAT inhibitors remain a key point for their development as antiatherosclerotic agents. The aim of this study was to characterize the effects of a novel and powerful ACAT inhibitor, F 12511 (S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-phenylacetanilide, on the NCI-H295R cell line, which has functional properties comparable to those of normal human adrenal cells. F 12511 incubated with cultured cells for 4-72 hr strongly inhibited cholesteryl oleate formation. The concentrations required to produce 50% inhibition (IC50) values) ranged from 20 to 50 nM; in the presence of low-density lipoproteins (LDL), this effect was paralleled by a decrease in cholesteryl ester mass and an increase in intracellular free cholesterol. At concentrations 100-fold larger than the IC(50) value for up to 48 hr, F 12511 reduced neither the basal release of cortisol and aldosterone nor the production of cortisol stimulated by forskolin. F 12511 did not modify the mRNA levels of the steroidogenic enzyme genes cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), or cytochrome P450 21-hydroxylase (P450c21) or those of the LDL receptor and high-density lipoprotein scavenger receptor class B, type I (SR-BI) genes, either in the presence or absence of adenosine 3',5'-cyclic monophosphate stimulation for 24 hr. Exposure to F 12511 at up to 3 microM for 24 or 48 hr did not result in significant change in morphological and ultrastructural characteristics; the cytoplasm contained large numbers of mitochondria with intact crystae, and the same typical features of secretory activity were observed in NCI-H295R control cells. Exposure to 3 microM of F 12511 for 96 hr also did not affect cell viability. These data demonstrate that reduction of the substrate for steroidogenesis by the ACAT inhibitor F 12511 impairs neither steroid production nor transcription of genes involved in steroidogenesis and lipoprotein uptake in the pluripotent human adrenal cell line NCI-H295R.

Effects of tumor necrosis factor on receptor-mediated endocytosis and barrier functions of bovine brain capillary endothelial cell monolayers.

Tumor necrosis factor alpha (TNF-alpha) plays a crucial role in the pathogenesis of the central nervous system infections. In an in vitro reconstructed blood-brain barrier model, a significant dysregulation of receptor mediated endocytosis of low density lipoproteins (LDL) and transferrin (Tf) is demonstrated at delayed phase of direct TNF-alpha activation. Concomitant with the increase in LDL uptake, we demonstrate a decrease of Tf-receptor mediated endocytosis. The potential role of TNF action in the differential or opposite routing of macromolecules is also characterized by a stimulation of their transcytosis. These findings may provide a new insight into the inflammatory effect of TNF-alpha on brain capillary endothelial cells.

High-density-lipoprotein subfraction 3 interaction with glycosylphosphatidylinositol-anchored proteins.

To elucidate further the binding of high-density-lipoprotein subfraction 3 (HDL3) to cells, the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-proteins) was studied. Treatment of cultured cells, such as fibroblasts or SK-MES-1 cells, with a phosphatidylinositol-specific phospholipase C (PI-PLC) significantly decreases specific HDL3 binding. Moreover, PI-PLC treatment of cultured cells or cellular plasma membrane fractions results in releasing proteins. These proteins have a soluble form and can also bind HDL3, as revealed by ligand blotting experiments with HDL3. In order to obtain enriched GPI-proteins, we used a detergent-free purification method to prepare a caveolar membrane fraction. In the caveolar fraction, we obtained, by ligand blotting experiments, the enrichment of two HDL3-binding proteins with molecular masses of 120 and 80 kDa. These proteins were also revealed in a plasma membrane preparation with two other proteins, with molecular masses of 150 and 104 kDa, and were sensitive to PI-PLC treatment. Electron microscopy also showed the binding of Au-labelled HDL3 inside the caveolar membrane invaginations. In SK-MES-1 cells, HDL3 are internalized into a particular structure, resulting in the accumulation and concentration of such specific membrane domains. To sum up, a demonstration has been made of the implication of GPI-proteins as well as caveolae in the binding of HDL3 to cells.

1,25-Dihydroxyvitamin D3 regulates gamma 1 transpeptidase activity in rat brain.

gamma-Glutamyl transpeptidase (gamma-GT), primarily described as a kidney enzyme, is also expressed in several cell types of the central nervous system (CNS). It is involved in the glutathione cycle and in cysteine transport. Here we report that the specific activity of this enzyme is transiently increased in the rat brain, following a treatment with 1,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D. In vitro experiments showed that this positive regulatory effect does not affect endothelial cells of the brain microvessels, but does affect pericytes and parenchymal astrocytes. Changes in the specific activity of gamma-GT were not correlated with any important modification of brain amino acid concentrations. Since gamma-GT is though to participate in the scavenging of reactive oxygen species, these data suggest that 1,25-D3 could be an effector controlling detoxification processes in the brain.

Intracarotid tumor necrosis factor-alpha administration increases the blood-brain barrier permeability in cerebral cortex of the newborn pig: quantitative aspects of double-labelling studies and confocal laser scanning analysis.

Tumor necrosis factor-alpha (TNF-alpha) plays a crucial role in the pathogenesis of the central nervous system infections. The aim of the present study was to analyze quantitatively the changes in the blood-brain barrier (BBB) permeability after the intracarotid injection of TNF-alpha. Recombinant human TNF-alpha was injected into the left internal carotid artery of anesthetized newborn pigs (n = 48) in the doses of 0, 1000, 10 000 and 100 000 IU, respectively. Before, as well as 1, 2, 4, 8, and 16 h after the challenge, the extravasation of a small (sodium fluorescein (SF), mw 376), and a large (Evan's blue-albumin (EBA), mw 67 000) tracer was determined concomitantly by spectrophotometry in the cerebral cortex of the animals. There was a time- and dose-dependent increase in BBB permeability both for SF and EBA; however, significant (P < 0.05) BBB opening for albumin only developed 2 h after the challenge. In the morphological study the same excitable tracers, identical experimental protocol and groups were used. Cryostat sections of brain tissue were viewed for optical sectioning with a confocal laser scanning microscope equipped with an argon/krypton ion laser. A diffuse BBB opening for SF and a moderate perivascular extravasation for EBA were found in the cortices of TNF-alpha-treated animals. We conclude that significant increases in intravascular TNF-alpha-concentration during neonatal infections may result in vasogenic brain edema formation.

Hypoxia increases the susceptibility to oxidant stress and the permeability of the blood-brain barrier endothelial cell monolayer.

Using a cell culture model of the blood-brain barrier (BBB), we investigated the brain capillary endothelial cell (EC) response to hypoxia. The activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase and the GSH level of brain capillary ECs alone or in coculture with astrocytes, as well as those of pericytes, were compared with those obtained with freshly isolated microvessels. These results demonstrated that brain capillary ECs cocultured with astrocytes and used in the presence of a coculture-conditioned medium provided a relevant in vitro model for studying the effect of hypoxia-reoxygenation at the BBB level. The effect of hypoxia on antioxidant enzymes, GSH, and ATP levels was studied, as well as the modification of the permeability to small weight molecules. A decrease in all enzymes and the GSH level could explain an increase in the susceptibility of the brain capillary ECs to further oxidant injury. Second, profound rearrangements of F-actin filaments of the ECs and a decrease in the ATP level could be associated with an increase in the permeability of the monolayer. Furthermore, an apoptotic process was detected by in situ end labeling of DNA. These results indicate that hypoxia distorts the function of ECs and that these cells in culture provide a valuable tool for exploring mechanisms after hypoxia-reoxygenation.

Exposure of tumor necrosis factor-alpha to luminal membrane of bovine brain capillary endothelial cells cocultured with astrocytes induces a delayed increase of permeability and cytoplasmic stress fiber formation of actin.

Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, has long been known to be involved in the pathogenesis of central nervous system infections and of certain neurodegenerative diseases. However, the possible role of the blood-brain barrier (BBB), the active interface between the blood circulation and brain tissue, remained unknown during these pathological conditions. In our in vitro reconstructed BBB model, 1-hr exposure of recombinant human TNF-alpha (in concentrations of 50, 250, and 500 U/ml, respectively) to the luminal membrane of bovine brain capillary endothelial cells (BBCEC) did not change significantly the transendothelial flux of either sucrose (m.w. 342 Da), or inulin (m.w. 5 kDa) up to 4 hr (early phase), except for a slight decrease (P < 0.05) in sucrose permeation at 2-4 hr with the highest dose of TNF-alpha. On the other hand, at 16 hr after the 1-hr challenge with TNF-alpha (delayed phase) at all 3 concentrations, significant increase was induced in the permeability of BBCEC monolayers for both markers. These changes of permeability were accompanied by a selective reorganization of F-actin filaments into stress fibers, while the intracellular distribution of vimentin remained similar to the control. These results suggest that BBCEC can respond directly to TNF-alpha by a delayed increase of permeability and reorganization of actin filaments.

In vivo cooperation of two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, leads to transformation and immortalization of chicken myelomonocytic cells.

To investigate a possible in vivo cooperation between the p61/63myc and P135gag-myb-ets proteins, we used a previously constructed retrovirus, named MHE226, which contains the fused v-myb and v-ets oncogenes of the E26 retrovirus and the v-myc oncogene of MH2. For that purpose, chicken neuroretina cells producing MHE226 and pseudotyped with the Rous associated virus-1 (RAV-1) helper virus were injected in 1-day-old chickens. In control experiments, we also injected chicken neuroretina cells producing E26 (RAV-1), RAV-1 alone, or constructs lacking one of the oncogenes of MHE226. The average life span of MHE226-infected chickens is half that of E26-infected chickens. MHE226-infected chickens harbor tumors scattered in many organs, but compared with E26, MHE226 induced a weak leukemia. Study of integration sites suggests that the majority of the tumors results from clonal or oligoclonal events. Cell cultures were derived from the tumors of MHE226-infected chickens and grown in standard medium without addition of exogenous chicken myelomonocytic growth factor. These cells still divide at high rate after more than 100 passages and can thus be considered immortalized. By using several criteria, these cells were characterized as precursors of the myelomonocytic lineages.

Evidence for eosinophil activation in eosinophilic cystitis.

Eosinophilic cystitis (EC) is a rare condition. Recent studies have shown that activated eosinophils release cytotoxic cationic proteins which can induce tissue damage. Moreover, in vitro studies have shown that interleukin-5 (IL-5) is a cytokine able to attract and activate eosinophils. The goal of this study was to detect a possible activation of eosinophils in EC using electron microscopy, in situ hybridization with an IL-5 RNA probe and immunochemistry with a specific anti-IL-5 antibody. Using these combined methods in a typical case of EC, we found numerous activated eosinophils synthesizing and secreting IL-5 protein. IL-5 could enhance the activation of eosinophils and their cytotoxic potential in bladder tissues. This mechanism might explain the chronicity of the lesions in EC.

Synthesis of interleukin-5 by activated eosinophils in patients with eosinophilic heart diseases.

Eosinophilic endomyocardial disease represents a major evolutive risk in chronic eosinophilia-associated disorders. Eosinophil granule proteins appear to be involved in cardiac injury, but the mechanisms leading to eosinophil infiltration and degranulation are not clear. Interleukin-5 (IL-5) has been recently shown to be produced by eosinophils and might play a role in both chemoattraction and degranulation of eosinophils. In four cases of eosinophilic diseases with severe cardiac failure, we evaluated the proportion of eosinophil phenotypes and the serum levels of eosinophil cationic protein (ECP) and soluble IL-2 receptor (sIL-2R), markers of disease activity in the hypereosinophilic syndromes. All four patients showed a markedly increased proportion of hypodense eosinophils with elevated serum ECP and sIL-2R levels. In all four patients, extracellular deposition of eosinophil granule proteins and features of eosinophil activation were observed in cardiac tissues. The synthesis of IL-5 by eosinophils was detected in myocardial sections and blood cells by in situ hybridization and by immunostaining with a monoclonal antibody against human IL-5. Sixty percent to 90% of tissue eosinophils expressed IL-5 mRNA and IL-5 protein. These data suggest that IL-5 can be produced by eosinophils at the sites of myocardial tissue damage and might participate in local eosinophil activation.

Toxoplasma gondii: differential location of antigens secreted from encysted bradyzoites.

Since we have previously demonstrated the protective role against infection played by Toxoplasma excreted-secreted antigens (Darcy et al. Parasite Immunology, 10, 553-567, 1988), the aim of the present work was an attempt to precisely define the location of GRA1, GRA2, and GRA5 in both the tachyzoite and the bradyzoite stages from distinct strains, in order to explore the mechanisms of secretion by Toxoplasma gondii. Three monoclonal antibodies (Charif et al. 1990) and colloidal immunogold labeling were used to localize the 27-, 28.5-, and 21-kDa target antigens to the matrix of the dense granules of tachyzoites and bradyzoites. They were, moreover, detected in the parasitophorous vacuole and in the cyst ground substance after host cell invasion. Our data suggest that a selective sorting mechanism for dense-granule contents exists at least in encysted bradyzoites. GRA2 was found preferentially associated with the ground substance of the cyst wall and the tubular elements of the network of the modified host cell phagosome, whereas GRA5 was located on the delimiting membrane of both the cyst wall and the parasitophorous vacuole. These observations reveal the selective targeting of dense-granule molecules, which could have different functions and fates when exocytosed into the parasite-containing vacuole.

Immunological properties of apoB-containing lipoprotein particles in human atherosclerotic arteries.

In this study, we have documented immunochemical properties of apolipoprotein (apo) B-containing particles (LpB) extracted from human atherosclerotic lesions obtained during vascular reconstructive surgery of patients. These properties were compared to those of particles purified from corresponding atherosclerotic plasma and healthy control plasma. LpB immunoreactivities were tested in solid phase competitive binding radioimmunoassays using five anti-apoB monoclonal antibodies (MAb) for which epitopes have been previously located on the protein. The regions encompassed amino acids 405 to 539 (MAb B1), 1854 to 1879 (MAb B4), 3506 (MAb BA11), and 4355 (MAb BL3). The fifth antibody (MAb BL5) recognizes a conformationally expressed epitope. LpB from lesions presented a significantly decreased immunoreactivity as compared to LpB from respective plasma except for the epitope recognized by MAb BA11 located precisely in the low density lipoprotein (LDL) receptor binding site. The accessibility of the four sequential epitopes was similar on LpB from atherosclerotic and healthy plasma while it was decreased for the conformational one in LpB from atherosclerotic samples. These altered immunoreactivities were not related to changes in chemical composition of LpB as this was quite comparable in all preparations. With regard to electronegativity, apoB fragmentation, immunological accessibility, and size distribution of the particles, changes seem to increase in the following order from healthy plasma, atherosclerotic plasma, and the corresponding lesions. The results confirm some structural characteristics of oxidatively modified particles from human atherosclerotic lesions and to a lesser degree from respective plasma, but more specifically demonstrate a global conformational change in LpB from lesions, this change being perhaps initiated in the plasma.

Activated eosinophils in adult coeliac disease: evidence for a local release of major basic protein.

The eosinophil population is increased in the jejunal mucosa of patients with coeliac disease. Eosinophils may participate in the mucosal damage by releasing their granule components that have cytotoxic properties such as eosinophil cationic protein (ECP) and major basic protein (MBP). This study aimed to assess the presence of ECP and MBP in the jejunal mucosa of 10 adult patients with active coeliac disease who presented with villous flattening. Endoscopic jejunal biopsy specimens were obtained from macroscopically flattened jejunal mucosa and were processed for ultrastructural study and immunogold labelling using anti-MBP, anti-ECP, and anti-IgA antibodies. Numerous eosinophils were found in the upper part of the lamina propria and showed two types of morphological change: some were lytic and others exhibited ultrastructural signs of activation, containing altered granules with fading of the central core. IgA plasma cells were intermingled with eosinophils and had dense deposits on the external side of their cytoplasmic membrane. MBP was detected in central cores of granules but also diffusely in their matrix and in tight association with dense extracellular deposits. Conversely, ECP was detected only in the matrix of eosinophil granules. This study showed that numerous eosinophils are in an activated state in the mucosa of patients with active coeliac disease and release cytotoxic proteins such as MBP, which could contribute to the mucosal damage. The observation that eosinophils and IgA plasmocytes were closely associated in the mucosa supports a role for IgA in eosinophil recruitment and activation in coeliac disease.

Productive human immunodeficiency virus-1 infection of megakaryocytic cells is enhanced by tumor necrosis factor-alpha.

Recent findings have indicated that megakaryocytes may be susceptible to human immunodeficiency virus (HIV) infection, suggesting a potential role for megakaryocytes as viral reservoirs in HIV-infected patients. We report that the megakaryocytic cell line Dami could be productively infected with the HTLV III-B strain of HIV-1, in 26 different experiments (results of 16 experiments are reported); productive infection lasted up to 30 weeks. Despite a lack of detectable surface expression of the CD4 molecule and very low levels of CD4 mRNA, between 40% and 60% of megakaryocytic cells produced viral proteins after contact with HIV-1. Neither cytopathogenic effects nor syncytial formation was observed. Production of high levels of functional viral particles was indicated by analysis of p24 protein levels, reverse transcriptase activity, ultrastructural studies, and the capacity of supernatants from infected Dami cells to infect the Molt-4 T-lymphocytic cell line. HIV-1 RNA and protein levels in infected Dami cells were enhanced by treatment with tumor necrosis factor-alpha (TNF-alpha), and decreased by treatment with interferon-alpha (IFN-alpha) and IFN-gamma. Transient transfection of the megakaryocytic cells with various constructs of the HIV-1 promoter (LTR) linked to the luciferase reporter gene suggested that the effect of TNF-alpha was related, as in monocytic and T-cell lines, to transactivation of the enhancer region of the HIV-1 LTR. These findings indicate that signals provided by the immune system may modulate HIV-1 expression in cells of the megakaryocytic lineage.

Human endothelial cells transfected by SV40 T antigens: characterization and potential use as a source of normal endothelial factors.

A putative role for the vascular endothelium as target for autoantibodies has been suggested in several autoimmune disorders and connective-tissue diseases. However, there are some difficulties linked to the use of cultured endothelial cells (EC) that limit considerably the extensive studies on the nature of endothelial target antigens involved. To overcome this problem, human EC, derived from umbilical veins, were transfected with recombinant plasmid pSV1 which contained the early genes of simian virus SV40. These transfected cells, called EC-pSV1, are able to grow without EC growth supplement and demonstrate a population doubling time of about 50 h. Among the EC properties, EC-pSV1 retain intracellular content of angiotensin-converting enzyme activity, exhibit constitutive production of interleukin 6 and of a growth-promoting activity on early passage EV, express intercellular adhesion molecule 1 (ICAM-1) and its up-regulation by tumor necrosis factor alpha, but have lost the expression of factor VIII-related antigen. Moreover, EC-pSV1 express a 55-kDa antigen found on EC and human platelets, and presumably acting as an antibody target in some cases of non-allergic asthma. However, at the 50-55th generation, morphological changes and altered growth behavior were visible. This work demonstrates that transfection of EC with SV40 T antigens may be of interest, particularly in areas of research including the study of EC targets involved in different human diseases.

Immunopathological study of eosinophils in eosinophilic granuloma of bone: evidence for release of three cationic proteins and subsequent uptake in macrophages.

Eosinophils from two patients with eosinophilic granuloma of bone (EGB) were studied by combined immunohistochemical and immuno-ultrastructural methods with antibodies directed against three eosinophil granule proteins: major basic protein, eosinophil cationic protein, and eosinophil peroxidase. Immunohistostaining showed the presence and distribution of large numbers of eosinophils in the granuloma. Immuno-ultrastructural methods showed alterations of eosinophil fine structure associated with some steps in the release of granule proteins. No granule extrusion was seen, but rather cationic proteins diffused within cytoplasmic tubulo-vesicular structures. Furthermore, the three granule proteins were found within phagolysosomes of surrounding macrophages, suggesting an interaction between eosinophils and phagocytic cells at the destructive stage of EGB.

Improved permeabilization procedure for flow cytometric detection of internal antigens. Analysis of interleukin-2 production.

A cell membrane permeabilizing treatment is described which involves the use of lysolecithin at low concentration in acidic acetate buffer and paraformaldehyde fixation. It preserved well-separated scatter cytograms of small and large lymphocytes. The accuracy of the immunochemical detection of internal antigens by flow cytofluorography was demonstrated by the linear relationship between the percentage of fluorescent cells detected and the proportion of intracellular antigen-containing cells in mixtures with antigen-negative cell lines. Cell cycle analysis by dual nuclear staining with propidium iodide and FITC-conjugated Ki-67 antibody recognising in vitro stimulated human T lymphocytes verified that the proliferating lymphocytes retained their increased light scatter properties after permeabilization. Enumeration of interleukin-2 (IL-2) producing cells by their cytoplasmic immunofluorescence showed that enlarged lymphocytes were the main IL-2 producing cells. This improved permeabilization procedure, by gating small and enlarged lymphocytes separately, makes it possible to determine by two color fluorescence the immunophenotype of activated T cells committed to interleukin production.

Fast double antibody radioimmunoassay of human granulocyte myeloperoxidase and its application to plasma.

The haem enzyme myeloperoxidase (MPO) (EC 1.11.1.7) with a spectral A430/A280 ratio greater than 0.7 and a specific activity of 125 U/mg was purified from isolated human neutrophils. To obtain a radioimmunoassay (RIA) for this enzyme, a specific antiserum against human neutrophil MPO was raised in rabbits and used at an initial dilution of 1/10,000. MPO labelled with 125iodine by a technique of self-labelling in the presence of H2O2, had a specific activity of 24 mCi/mg. After incubation at room temperature (2 h) and separation by double antibody precipitation in the presence of polyethylene glycol, the sensitivity of the RIA was 21 ng/ml. The RIA showed good precision and accuracy with intra- and interassay coefficients of variation of less than 7% for MPO concentrations ranging from 100 to 800 ng/ml, and satisfactory recoveries of known amounts of exogenous MPO in plasma. For the measurement of MPO in blood, the best sampling technique was to collect blood into EDTA. Rapid centrifugation (within 20 min) was necessary for blood collected into heparin. Mean MPO values in normal individuals were 340 +/- 98 ng/ml in EDTA plasma (n = 152) and 332 +/- 82 ng/ml in heparinized plasma (n = 34). When MPO was measured 12-6 h after injury in critically ill patients high values (above 1000 ng/ml) were found in 6/15 patients with multiple injuries. In patients with sepsis (n = 22), MPO values were always above 1000 ng/ml.

Antipeptide antibody against the human low-density-lipoprotein receptor. Characterization and cross-reactivity with bovine lymphocytes.

A dodecapeptide corresponding to the external N-terminal sequence of the human low-density-lipoprotein (LDL) receptor was synthesized. Antibodies raised in rabbits against the peptide were purified and were shown to react specifically with the peptide and with human LDL receptor of fibroblasts, HeLa cells and lymphocytes using binding studies and immunoblotting. By indirect immunogold analysis, antibodies bound to the LDL receptor of human lymphocytes could be revealed as clusters. Anti-receptor peptide immunoglobulins specifically bound to the human HeLa cell's LDL receptor with a lower affinity than LDL (Kd x 3). The anti-receptor peptide immunoglobulins and 125I-labelled-LDL competed with each other for the LDL-receptor sites. Antibodies failed to react with lymphocytes of subjects with the homozygous form of familial hypercholesterolaemia. Cross-reactivity with the dodecapeptide of the bovine LDL receptor was limited, but this cross-reactivity was confirmed by the binding of anti-receptor peptide immunoglobulins to the LDL receptor from bovine lymphocytes.