Reinhold Wintersteiger

The role of nanoparticle size in hemocompatibility

It is expected that nanoparticular matters will be increasingly used for industrial and medical applications. Since it is known that nanoparticles exhibit unique and potential hazardous properties due to their small size, toxicity studies, risk assessment and risk management are of great interest. We focussed on adverse effects on human blood. Processes which warrant special attention are clotting, reactions triggering inflammatory and immune responses and hemolysis. Starting with the determination of size and surface charge in different media we assessed the effect of size and surface charge on induction of coagulation, thrombocyte activation, complement activation, granulocyte activation and hemolysis. We used polystyrene particles as model because they are available in different sizes but constant surface charges. The presence of salts and of protein in the dispersion solution increased particle size and neutralized surface charge. Positively charged particles formed aggregates in buffered solution. Interference of the particles with assays based on fluorescence associated cell sorting was identified. Positive surface charge induced activation of complement. Small size caused thrombocyte and granulocyte activation, and hemolysis. A characterization of particle size and surface charge in the solutions used for the experiments appears important for interpretation of the results. The size dependency of adverse effects in human blood is not linear; negatively charged particles larger than 60 nm hydrodynamic diameter appear to be considerably less hematotoxic than smaller ones.

Macrophages of Human First Trimester Decidua Express Markers Associated to Alternative Activation

Problem:  Depending on the type of their activation, macrophages may promote TH1‐ or TH2‐type of immune responses. To date, not much is known about the activation phenotype of decidua macrophages, which – together with NK cells – constitute the majority of bone marrow derived cells at this location.

Characterization of prostanoid receptors mediating actions of the isoprostanes, 8-iso-PGE2 and 8-iso-PGF2α, in some isolated smooth muscle preparations

We investigated the contracting actions of the isoprostanes (isoPs), 8‐iso‐prostaglandin (PG) F2α and 8‐iso‐PGE2, in comparison to the effects of the thromboxane (TX) A2‐mimetic U 46619 and the traditional prostaglandin PGE2 in the isolated rat aorta, isolated rat gastric fundus and the isolated guinea‐pig ileum. U 46619 and 8‐iso‐PGF2α caused contractions in the rat aorta and rat gastric fundus in a concentration‐dependent manner, whereas these agonists showed no effects in the guinea‐pig ileum. However, 8‐iso‐PGE2 and PGE2 caused contractions in all isolated organs used. The prostanoid TP‐receptor antagonist SQ 29,548 (10 nM) significantly antagonized vasoconstrictions induced by the agonists used in the rat aorta. SQ 29,548 at a final concentration of 3 μM, but not at lower concentrations, significantly inhibited contractions induced by U 46619, 8‐iso‐PGF2α and 8‐iso‐PGE2 in the rat fundus. Responses to PGE2 were unchanged. The prostanoid EP1‐receptor antagonist SC 51089 (3 μM) significantly inhibited contractions induced by 8‐iso‐PGE2 and PGE2 in the rat fundus and in the guinea‐pig ileum. SC 51089 had no effect on responses to any of the agonists tested. Our results show that 8‐iso‐PGE2, in contrast to 8‐iso‐PGF2α, can also cause contractions by activation of the EP1‐receptors in the rat gastric fundus and the guinea‐pig ileum. The findings of the present study do not support the existence of a unique isoP‐receptor in the tissues used.

Differential Phenotypic Properties of Human Peripheral Blood CD56dim+ and CD56bright+ Natural Killer Cell Subpopulations

We investigated the presence of integrins and various other cell surface markers on the surface of freshly isolated CD56dim+ and CD56bright+ NK cells, the latter comprising a mean of 6.5% of total peripheral blood (PB) CD3-CD56+ NK cells. This small subpopulation stained more intensively for CD2, CD11c, CD15s, CD44, CD49e, CD55, CD62L, HLA-DR, and GZS-1, whereas there was no expression of CD49f in contrast to CD56dim+ cells. The myeloid antigen CDw65 was present on 11% of CD56bright+ cells, other myeloid markers were not found. 28% of CD56bright+ cells were positive for CD45R0. These results support the notion that CD56bright+ NK cells, based on their different marker profile, may possess different functional and biodistributional properties and--due to the signal-transducing capabilities of several adhesion molecules--differential patterns of stimulatability compared to the majority of PB-NK cells.

Reaction patterns of monoclonal antibodies to HLA-G in human tissues and on cell lines: a comparative study

We compared the immunohistochemical reaction patterns of HLA-G-specific antibodies 87G, 4H84, G233, 16G1, and BFL.1 on human placentas under three different preparative conditions and on cryosections of other human tissues. Human and murine cell lines, either naturally expressing or transfected with HLA-G, were analyzed for their reaction patterns by immunocytochemistry and flow cytometry. Antibodies HCA2, TP25.99, W6/32 to classical HLA class I, anti-beta(2)-m and various non-HLA-G expressing cell lines were used as controls. The binding ability of the antibodies depends on the histotechnical procedure used. 4H84 and HCA2 bind to HLA-G despite aldehyde fixation and also paraffin embedding. 87G does not bind HLA-G in studies involving fixation with aldehydes. G233 labels HLA-G in aldehyde fixed but not paraffin embedded tissues. By immunocytochemistry HLA-G2 is merely detected with antibodies 4H84 and HCA2. MAb 16G1 binds to HLA-Gsol transfected cell lines only. The HLA-G specificity of mAb BFL.1 was considered as doubtful because it failed to react with most of the HLA-G transfected cell lines. Binding of 87G to the surface of monocytes or U-937 cells stimulated with IFN-gamma and GM-CSF is an Fc-receptor mediated phenomenon.

Solvent- andpH-dependence of the absorption and fluorescence spectra of harman: Detection of three ground state and four excited state species

ThepH-dependence of the absorption and fluorescence spectra of the alkaloid harman has been investigated. Three species, namely the cation, the neutral molecule and the anion have been found in absorption, whilst four species, namely the cation, the neutral molecule, the anion and the zwitterion were detected by fluorimetry. The zwitterion must be formed by a double proton transfer during lifetime of the excited state. Fluorescence quantum yields are entirely different for the various species, being highest for the cation (ϕf in 1N sulfuric acid 0.89).Unlike quinine sulfate the fluorescence of harman cation is not quenched by chloride ion, which suggests its use as a fluorescence standard superior to quinine.The ground statepKas of harman are 7.37 and 14.6, the excieted statepKas, as calculated from theFörster-Weller-equation, are 12.0 and 8.65. Thus the observed zwitterion fluorescence is predicted from the calculations.ZusammenfassungDiepH-Abhängigkeit des Absorptions- und Fluoreszenzspektren des Alkaloids Harman wurde untersucht. Drei Spezies, nämlich das Kation, das Neutralmolekül und das Anion, wurden in Absorption gefunden, während vier Formen, nämlich das Kation, das Neutralmolekül, das Anion und das Zwiterion, durch Fluoreszenz nachgewiesen wurden. Das Zwitterion wird durch einen doppelten Protonentransfer während der Lebenszeit des angeregten Zustandes gebildet. Die Fluoreszenzquantenausbeuten der einzelnen Formen sind deutlich verschieden und für das Kation am höchsten (ϕf=0,89 in 1N Schwefelsäure). Im Gegensatz zum Chininsulfat wird Harman durch Chloridion nicht gelöscht, was seine Verwendung als Fluoreszenzstandard nahelegt.Die Grundzustands-pK-Werte des Harmans betragen 7,37 und 14,6, diepK-Werte des angeregten Zustandes, wie sie aus derFörster-Weller-Gleichung erhalten werden, betragen 12,0 und 8,65. Die beobachtete Fluoreszenz aus der zwitterionischen Form wird somit auch von den Berechnungen vorausgesagt.

Fluorescence derivatization of tertiary amines with 2-naphthyl chloroformate

Abstract 2-Naphthyl chloroformate (NCF) was found to be a suitable fluorescence reagent for the derivatization of drugs containing a tertiary amino group. The tertiary amines undergo dealkylation when heated with NCF, forming fluorescent carbamates. The reaction has been applied to the pre-column derivatization of some antihistamines.

Enantioselective sequential-injection chemiluminescence immunoassays for 3,3′,5-triiodothyronine (T3) and thyroxine (T4)

This study deals with the development of enantioselective flow-through immunosensors for triiodothyronine (T 3) and tetraiodothyronine (thyroxine, T4) on the basis of a competitive assay using enantioselective antibodies. The instrumental set-up is based on a simple sequential-injection system equipped with a chemiluminescence detector and an immunoreactor, which consists of a flow-cell packed with immobilized haptens. As haptens, 4-amino- l-phenylalanine (4-amino-l-Phe), 4-amino-d-Phe or l-T3 were used. Antibodies directed against 4-amino-l -o rd-Phe or l-T3 were labeled with an acridinium ester. Three different approaches for immobilizing the haptens were investigated including simple adsorption on polystyrene, chemical binding to an activated methacrylate polymer and binding via the biotin–streptavidin binding (BSB) system. The latter approach showed the best results regarding repeatability and sensivity. Using biotinylated l-T3 immobilized onto a streptavidin-derivatized trisacryl support and labeled anti- l-T3 antibodies, a detection limit of 15.5 fmol/ml for l-T3 was obtained. One assay cycle including regeneration takes only about 5 min. This approach was applied to detect l-T3 in plasma samples without any sample pre-treatment. The average recovery from spiked plasma sample was about 93% with a R.S.D. below 5%.

Influence of isoprostanes on vasoconstrictor effects of noradrenaline and angiotensin II

The isoprostanes, 8-iso-prostaglandin F2alpha and 8-iso-prostaglandin E2, which are released in vivo by free radical-catalyzed peroxidation of arachidonic acid, are potent vasoconstrictors. Increased formation of 8-iso-prostaglandin F2alpha has been detected in human cardiovascular diseases, in which enhanced plasma levels of noradrenaline and angiotensin II have harmful vasoconstrictor effects. Therefore, we investigated the influence of perfusions with the thromboxane A2 mimetic, U 46619, and with the isoprostanes, 8-iso-prostaglandin F2alpha, 8-iso-prostaglandin E2, 8-iso-prostaglandin E1 and 8-iso-prostaglandin F3alpha, on the vasoconstrictor effects of noradrenaline and angiotensin II in the isolated perfused rabbit ear. Our results demonstrate that perfusions with U 46619, 8-iso-prostaglandin E2 and 8-iso-prostaglandin F2alpha, at a subthreshold concentration (30 nM), amplified the vasoconstrictions induced by noradrenaline or angiotensin II significantly. In addition, the results show that U 46619, 8-iso-prostaglandin F2alpha, 8-iso-prostaglandin E2 and 8-iso-prostaglandin E1, which were applied as a bolus, induced much more pronounced vasoconstrictions than prostaglandin F2alpha, prostaglandin E2 and prostaglandin F3alpha. Prostaglandin E1 and 8-iso-prostaglandin F3alpha, showed no effects. In conclusion, it can be assumed that the powerful vasoconstrictions induced by 8-iso-prostaglandin E2 and 8-iso-prostaglandin F2alpha and their potentiating effects on vasoconstrictions induced by noradrenaline or angiotensin II might be of pathophysiological relevance in cardiovascular diseases.

High-performance liquid chromatography of naphthylurethanes with fluorescence detection

The method used previously for the fluorodensitometric determination of compounds with an alcoholic hydroxyl group was examined for its applicability in high-performance liquid chromatography. The conversion of alcoholic substances into urethanes was performed with naphthyl isocyanate. Chloroform—benzene—ethanol and n-heptane—diethyl ether, were used as eluents for the separation of urethanes of various polarity with silica gel columns. Reversed-phase material is also suitable. The detection limits ascertained by means of fluorescence detection are in the picomole range.