Infrared- and Raman-spectroscopy measurements of a transition in the crystal structure and a closing of the energy gap of BiTeI under pressure.

BiTeI is a giant Rashba spin splitting system, in which a noncentrosymmetric topological phase has recently been suggested to appear under high pressure. We investigated the optical properties of this compound, reflectivity and transmission, under pressures up to 15 GPa. The gap feature in the optical conductivity vanishes above p∼9 GPa and does not reappear up to at least 15 GPa. The plasma edge, associated with intrinsically doped charge carriers, is smeared out through a phase transition at 9 GPa. Using high-pressure Raman spectroscopy, we follow the vibrational modes of BiTeI, providing additional clear evidence that the transition at 9 GPa involves a change of crystal structure. This change of crystal structure possibly inhibits the high-pressure topological phase from occurring.

Antiinflammatory effects of reconstituted high-density lipoprotein during human endotoxemia.

High-density lipoprotein (HDL) has been found to neutralize LPS activity in vitro and in animals in vivo. We sought to determine the effects of reconstituted HDL (rHDL) on LPS responsiveness in humans in a double-blind, randomized, placebo-controlled, cross-over study. rHDL, given as a 4-h infusion at 40 mg/kg starting 3.5 h before endotoxin challenge (4 ng/kg), reduced flu-like symptoms during endotoxemia, but did not influence the febrile response. rHDL potently reduced the endotoxin-induced release of TNF, IL-6, and IL-8, while only modestly attenuating the secretion of proinflammatory cytokine inhibitors IL-1ra, soluble TNF receptors and IL-10. In addition, rHDL attenuated LPS-induced changes in leukocyte counts and the enhanced expression of CD11b/CD18 on granulocytes. Importantly, rHDL infusion per se, before LPS administration, was associated with a downregulation of CD14, the main LPS receptor, on monocytes. This effect was biologically relevant, since monocytes isolated from rHDL-treated whole blood showed reduced expression of CD14 and diminished TNF production upon stimulation with LPS. These results suggest that rHDL may inhibit LPS effects in humans in vivo not only by binding and neutralizing LPS but also by reducing CD14 expression on monocytes.

Studies onthe chemical nature of lysine-binding sites and on their localization in human plasminogen.

The isolated 'kringle' structures 1 and 4 of human plasminogen lost lysine affinity upon photo-oxidation of histidine, but mostly retained it in the presence of 6-aminohexanoic acid. Lysine affinity was lost and could be partially restored after blocking of histidine with diethylpyrocarbonate and deblocking, or after esterification of COOH-groups and saponification. Only His-31 and most likely Asp-54 qualify as participants in a lysine binding site when the primary structures of the 'kringles' are considered.

Human lymphocyte membrane proteins treated with neuraminidase.

Human peripheral blood lymphocytes were surface-iodinated, treated with neuraminidase from Vibrio cholerae and lysed with non-ionic detergent. In addition, surface membrane fractions were isolated from surface-iodinated cells in the absence of detergents and treated with neuraminidase after membrane isolation. The effect of neuraminidase treatment on the membrane proteins was studied by two-dimensional gel electrophoresis. One surface-labelled protein of 45 000 molecular weight which is characterized by its association with the detergent-resistant matrix of the cells and by its specific enrichment in an isolated membrane fraction, was found to be particularly sensitive to neuraminidase treatment both of intact cells and isolated membranes. A prominent labelled protein of apparent molecular weight of 60 000 is observed in the soluble fraction after neuraminidase treatment of intact cells. The analogous protein is detected when isolated membrane fractions are treated with neuraminidase.

Two detergent-insoluble proteins of the human lymphocyte membrane are enriched in an isolated membrane fraction.

Human lymphocytes isolated from peripheral blood on Ficoll/Paque density gradients were surface-labelled by 125I/lactoperoxidase or 3H/reductive alkylation and lysed in buffer solutions containing non-ionic or amphoteric detergents (octylphenylpolyoxyethylenes, octylglucoside, cholylamidopropyldimethylammoniopropane sulfonate) under a variety of conditions. The cell lysate was fractionated by sedimentation or by density gradient centrifugation. The large majority of the labelled proteins is solubilized by the detergents. Two proteins of 45 000 and 30 000 molecular weight are the main detergent-insoluble, surface-labelled components. They can be fractionated from detergent lysates of cells in relatively pure form from the other membrane proteins and from nuclear material on density gradients. The same two proteins are specifically enriched in a membrane fraction isolated from a detergent-free cell homogenate by density gradient centrifugation. Cytoskeletal and other intracellular proteins remain associated with these two proteins when fractionated by either of these two independent methods.

Elevation of plasma high-density lipoprotein concentration reduces interleukin-1-induced expression of E-selectin in an in vivo model of acute inflammation.

BACKGROUND: Although there is strong evidence that plasma HDL levels correlate inversely with the incidence of coronary artery disease, the precise mechanism(s) for the protective effect of HDLs remains unclear. We recently showed that HDLs inhibit endothelial cell expression of cytokine-induced leukocyte adhesion molecules in vitro. Our study therefore sought to test the hypothesis that elevating the level of circulating HDLs would inhibit endothelial cell activation in vivo. METHODS AND RESULTS: We used a porcine model of inflammation previously established in our laboratory, in which the level of vascular endothelial cell expression of E-selectin in interleukin (IL)-1alpha-induced skin lesions was measured by the uptake of a radiolabeled anti-E-selectin antibody (1.2B6). Porcine plasma HDL levels were elevated by use of a bolus injection of reconstituted discoidal HDL (recHDL). These particles resemble nascent HDL particles in shape and contain apolipoprotein A-I as the sole protein and soybean phosphatidylcholine as the sole phospholipid. We found that recHDLs inhibited the expression of IL-1alpha-induced E-selectin by porcine aortic endothelial cells in vitro, confirming that the inhibitory effect is conserved with synthetic HDLs and demonstrating that the phenomenon is not restricted to human endothelial cells. In vivo, elevating the circulating level of HDLs approximately 2-fold led to significant inhibition of basal and IL-1alpha-induced E-selectin expression by porcine microvascular endothelial cells. CONCLUSIONS: These observations demonstrate the potential anti-inflammatory action of HDLs and provide support for the further investigation of the mechanisms underlying the inhibitory effects of HDLs on endothelial cell activation.

Monomeric and dimeric IgG1 as probes for assessing high-affinity and low-affinity receptors for IgG on human monocyte-derived macrophages and on activated macrophages.

From a panel of IgG1 myeloma proteins, only one was found to interact with human monocyte FcR in a manner similar to that of polyclonal IgG. This protein was used in binding studies involving human macrophage Fc receptors. A monomeric fraction depleted of dimeric and polymeric IgG1 was crosslinked with bis-diazonium benzidine, and a fraction highly enriched in cross linked IgG1 dimers was radiolabeled. Labeled monomeric and dimeric IgG were allowed to interact with monocytes that had matured to macrophages in vitro. The association with macrophages at 4 degrees C, in the presence of cytochalasin B, reached a plateau after 6 hr. The dissociation induced by excess unlabeled IgG followed similar kinetics as the association, but 20-30% of the bound IgG could not be dissociated. Under equilibrium conditions, evidence for a single FcR population binding monomeric IgG was obtained, the Kd being in the range of 12-42 nM. In contrast, the binding of dimeric IgG was more consistent with a model assuming two populations of binding sites when appropriate curve-fitting calculations were applied. The high-affinity FcR population had a Kd in the range of 0.8-3.5 nM, whereas the Kd of the low-affinity FcR population was in the range of 28-85 nM. When macrophages had been pre-treated with recombinant interferon-gamma, the expression of high-affinity sites was increased by a factor of 1.5-3, but the number of low-affinity sites was not augmented. Cytofluorographic analyses confirmed the increased expression of high-affinity FcR, binding fluoresceinating murine IgG2a. The expression of CD16, a low-affinity FcR expressed on neutrophils, NK cells and macrophages, as well as the expression of the complement receptor type III was little influenced by the rIFN-gamma pretreatment.

Isolation and purification of the human thymocyte antigens T6 and M241.

T6 and M241 antigens are products of the Class I major histocompatibility complex. The T6 and M241 antigens can be detected on human cortical thymocytes and on dendritic cells in the skin by monoclonal antibodies. Here we report a method of purification of the T6 and M241 antigens. Amino acid sequence data of purified antigens indicate that the heavy chains are blocked at their N-termini, whereas the partial N-terminal amino acid sequence of the light chains is identical to that of the human beta 2-microglobulin. In order to obtain sequence data from the heavy chains a method is described for isolation of purified cyanogen bromide fragments by electrophoretic methods.

Reconstituted high density lipoprotein (rHDL) modulates platelet activity in vitro and ex vivo.

A reconstituted high density lipoprotein (rHDL) prepared for clinical use was tested for its influence on platelet activity modulated by various stimuli. In a first series of in vitro experiments, rHDL was added to blood in a concentration series, and platelet rich plasma (PRP) was isolated. Platelets were stimulated with arachidonic acid, collagen, epinephrine or ADP, and platelet aggregation was assessed. rHDL mediated a dose dependent inhibition of the platelet activity. With purified platelets rHDL inhibited the release reaction induced by collagen, but not by thrombin, as measured by CD62P (P-Selectin) expression on the plasma membrane. Ex vivo experiments were performed with PRP from volunteers, previously infused with 25 mg rHDL/kg body weight and 40 mg rHDL/kg body weight, respectively. Platelet activity in PRP was assessed before, and up to 30 h after the end of the rHDL infusion. A transient inhibition of the platelet aggregation induced by arachidonic acid and collagen was observed which was more pronounced in the group receiving 40 mg rHDL/kg body weight. In both groups of experiments, in vitro and ex vivo, the inhibition of the platelet activity was also dependent on the stimulus used.

Differential effects of reconstituted high-density lipoprotein on coagulation, fibrinolysis and platelet activation during human endotoxemia.

High-density lipoproteins (HDL) can bind and neutralize lipopolysaccharides (LPS) in vitro and in vivo. HDL can also affect fibrinolytic activity and can directly influence platelet function by reducing platelet aggregation. In this study, the effects of reconstituted HDL (rHDL) on LPS-induced coagulation, fibrinolysis and platelet activation in humans were investigated. In a double-blind, randomized, placebo-controlled, cross-over study, eight healthy male volunteers were injected with LPS (4 ng/kg) on two occasions, once in conjunction with rHDL (40 mg/kg, given as a 4 h infusion starting 3.5 h prior to LPS injection), and once in conjunction with placebo. rHDL significantly reduced LPS-induced activation of coagulation (plasma levels of prothrombin fragment F1 + 2) and fibrinolysis (plasma levels of tissue type plasminogen activator antigen, t-PA). No effect was observed on LPS-induced inhibition of the fibrinolytic pathway (PAI-1) or on the transient thrombocytopenia elicited by LPS. Furthermore, rHDL treatment significantly enhanced the inhibition of collagen-stimulated inhibition of platelet aggregation during endotoxemia, but had no such effect on arachidonate-stimulated platelet aggregation. rHDL treatment per se also reduced collagen-induced platelet aggregation. These results indicate that rHDL modifies the procoagulant state associated with endotoxemia.

Biochemical comparison of the T6 antigen and HLA-A,B antigens.

The human thymic differentiation antigen T6, which was found to be associated with beta 2-microglobulin, was compared to the HLA-A,B antigens. Using a heteroantiserum prepared against denatured heavy chains of HLA-A,B antigens, no cross-reactivity with denatured T6 could be detected. The molecular weight of the protein backbone of T6 was found to be 34,000 as compared to 40,000 for the HLA-A,B antigens. Also, not only was the percentage of carbohydrate of T6 (25-35%) different from the HLA-A,B antigens (10%), but lectin binding studies showed that their sugar composition may differ. The two forms of T6, which previously had been found on MOLT-4 cells, appeared to have different levels of glycosylation, but apparently had the same protein backbone. T6, like HLA, has a hydrophobic domain, since it could be labeled with [125I]iodonaphthylazide. We conclude from these studies that T6 may be a class I MHC antigen which is different from the classical HLA-A,B antigens.

Human cutaneous dendritic cells express two glycoproteins T6 and M241 which are biochemically identical to those found on cortical thymocytes.

Monoclonal antibodies anti-T6 and anti-M241 define unique cell populations within different lineages: cortical thymocytes and dendritic cells in the skin. T6 positive cutaneous dendritic cells are located predominantly in the epidermis and belong to the Langerhans/indeterminate lineage, whereas, most of the M241 positive cells are located in the perivascular regions of the dermis. Biochemical analysis of thymocytes and cutaneous dendritic cells was performed in order to determine whether the reactivity of these antibodies with these cell types is due to sharing of antigenic determinants by two unrelated proteins, or whether similar proteins are present on cells of different lineages. Our results indicate that T6 antigens are borne by the same glycoprotein (49K) on cortical thymocytes and Langerhans/indeterminate cells. Similarly, M241 antigens isolated from thymocytes and cutaneous dendritic cells are found on the same glycoprotein (43K).

Inhibition by immunoglobulins of Staphylococcus aureus adherence to fibronectin-coated foreign surfaces.

Recent data suggest that fibronectin may favor Staphylococcus aureus infection by promoting attachment to either injured tissues or implanted foreign bodies. Using a previously described in vitro assay, we show that promotion of S. aureus adherence by surface-bound fibronectin, adsorbed on polymethylmethacrylate (PMMA) coverslips, is antagonized by antistaphylococcal antibodies present in immunoglobulin G (IgG) purified from human plasma. Among the different organisms tested, the protein A-deficient strain Wood 46 of S. aureus was the most strongly inhibited by purified IgG or whole serum dose-dependently. Bacterial adherence was not influenced by preincubating fibronectin-coated PMMA with either purified IgG or whole serum. However, inhibition of bacterial adherence was directly related to the extent of IgG binding to S. aureus Wood 46. When F(ab')2 fragments of purified IgG were tested in the adherence assay, they could also reduce the interaction between S. aureus Wood 46 and fibronectin-coated PMMA. Two other staphylococcal strains were also tested in the adherence inhibition assay: Whereas the protein A-rich strain Cowan I of S. aureus was moderately inhibited by purified IgG or whole serum, S. epidermidis KH 11 was not at all inhibited by IgG which bound poorly to the bacterial cells. This study has demonstrated that bacterial coating by humoral factors, and specifically IgG, may influence significantly subsequent adherence of S. aureus to surface-bound fibronectin.

Anti-D immunoglobulin in Rh(D) negative volunteers: clearance of Rh(D) positive red cells and kinetics of serum anti-D levels.

Properties of a new anti-D immunoglobulin were assessed in Rh(D) negative healthy male adults. Six volunteers received intravenous, and five volunteers intramuscular injections of 200 micrograms anti-D, 48 hours after pre-treatment with 5 mL of Rh(D) positive erythrocytes. Immediately after intravenous administration of anti-D, a rapid decrease of the Rh(D) positive erythroyctes was noted. After intramuscular injection of anti-D, there was a lag phase of 6 hours until the erythrocytes decreased, and the elimination rate was slower. Twenty-four hours after injection of anti-D, the Rh(D) positive erythrocytes were at the detection limit or no longer detectable in all volunteers. After intravenous administration, anti-D serum levels decreased from 45 ng/mL at 2 hours to 29 ng/mL at 24 hours, whereas after intramuscular administration, anti-D became detectable at 4 hours and increased to 11 ng/mL at 24 hours. During subsequent months, anti-D serum levels decreased at similar rates in both groups. After six months, anti-D was not detectable in any of the volunteers. Thus, the new anti-D immunoglobulin induced elimination of the Rh(D) positive erythrocytes and suggested that Rh(D) immunization of the volunteers was prevented.