Uta Reibetanz

The Leipzig high-energy ion nanoprobe: A report on first results

Abstract The high-energy ion nanoprobe LIPSION at the University of Leipzig has been operational since October 1998. Its magnetic quadrupole lens system, arranged as a separated Russian quadruplet, has been developed by the Microanalytical Research Centre (MARC), Melbourne. The ultrastable single-ended 3.5 MV SINGLETRON™ accelerator (High Voltage Engineering Europa) supplies H+ and He+ ion beams with a beam brightness in the range of 10–20 A rad −2 m −2 eV −1 [D.J.W. Mous, R.G. Haitsma, T. Butz, R.-H. Flagmeyer, D. Lehmann, J. Vogt, Nucl. Instr. and Meth. B 130 (1997) 31]. Due to this high brightness, the excellent optical properties of the focusing system of the nanoprobe and the suppression of mechanical vibrations, lateral resolutions of 100 nm for the low current mode (STIM) and 340 nm at a current of 10 pA (PIXE, RBS, SEI modes) were achieved. Further improvements are expected.

Colloidal DNA Carriers for Direct Localization in Cell Compartments by pH Sensoring

Multifunctional colloidal microparticles allow the integration of various active agents as well as reporter molecules into one system without interfering combining delivery and sensing functions. In this study, calcium carbonate particles were functionalized with fluorescein isothiocyanate-labeled poly(allylamine hydrochloride) (FITC-PAH) allowing particle localization in cell compartments of different pH. Plasmid DNA (pEGFP-C1 and pDsRed1-N1) as a reporter agent for drug release in the cytoplasm and rhodamine-B-isothiocyanate-labeled protamine (RITC-PRM) were integrated into biocompatible and biodegradable PRM/DXS multilayers. The uptake and processing of the particles by HEK293T/17 cells were investigated via flow cytometry and confocal laser scanning microscopy. The presented data show a clear correlation between the fluorescence intensity of the FITC-labeled core, that is, the particle localization after cellular uptake, and the expression of fluorescent proteins by the cells without further cell staining. In conclusion, this particle design allows the simultaneous study of particle location and processing to monitor the transport and release of active agents and should thus be an invaluable tool for the study and design of nano- and microcarrier systems.

Non-Vital Polymorphonuclear Leukocytes Express Myeloperoxidase on their Surface

The heme-containing enzyme myeloperoxidase (MPO) becomes expressed to the cell surface of non-vital polymorphonuclear leukocytes (PMNs) as evidenced by flow cytometry analysis and confocal fluorescence microscopy. While only a very small percentage of freshly isolated cells was able to bind the MPO antibody, PMN suspensions cultured for 36 h or longer time periods contained an increasing number of cells able to interact with these antibodies. Two distinct patterns of fluorescence for the MPO antibodies were observed. Antibodies were localised either in surface patches or distributed over the whole cell body. The latter type dominated in cell samples cultured for more than three days, while the first type was predominantly found in samples cultured for lower time periods. We observed also two peaks for fluorescence distribution by flow cytometry after addition of MPO antibodies to PMNs. Myeloperoxidase was localised at phosphatidylserine epitopes at the surface of non-vital PMNs as evidenced by coincubation with fluorescent MPO antibodies and FITC-labelled annexin V. Myeloperoxidase bound to the outer surface of PMNs uses hydrogen peroxide as a substrate as shown by appearance of an intense chemiluminescence using the impermeable luminescent protein Pholasin. Thus, myeloperoxidase becomes expressed to the surface of non-vital polymorphonuclear leukocytes colocalised with phosphatidylserine that may indicate a role of myeloperoxidase in apoptosis of PMNs.

Myeloperoxidase binds to non-vital spermatozoa on phosphatidylserine epitopes

The heme protein myeloperoxidase is released from stimulated polymorphonuclear leukocytes, a cell species found in increasing amounts in the male and female genital tract of patients with genital tract inflammations. Myeloperoxidase binds only to a fraction of freshly prepared human spermatozoa. The number of spermatozoa able to bind myeloperoxidase raised considerably in samples containing pre-damaged cells or in acrosome-reacted samples. In addition, myeloperoxidase released from zymosan-stimulated polymorphonuclear leukocytes was also able to bind to pre-damaged spermatozoa. The ability of spermatozoa to bind myeloperoxidase coincided with the binding of annexin V to externalized phosphatidylserine epitopes indicating the loss of plasma membrane integrity and with the incorporation of ethidium homodimer I. Myeloperoxidase did not interact with intact spermatozoa. Annexin V and myeloperoxidase bind to the same binding sites as verified by double fluorescence techniques, flowcytometry analyses as well as competition experiments. We demonstrated also that myeloperoxidase is eluted together with pure phosphatidylserine liposomes or liposomes composed of phosphatidylserine and phosphatidylcholine in gel filtration, but not with pure phosphatidylcholine liposomes. In conclusion, myeloperoxidase interacts with apoptotic spermatozoa via binding to externalized phosphatidylserine indicating a yet unknown role of this protein in recognition and removal of apoptotic cells during inflammation.

Activity increase in respiratory chain complexes by rubella virus with marginal induction of oxidative stress.

ABSTRACT Mitochondria are important for the viral life cycle, mainly by providing the energy required for viral replication and assembly. A highly complex interaction with mitochondria is exerted by rubella virus (RV), which includes an increase in the mitochondrial membrane potential as a general marker for mitochondrial activity. We aimed in this study to provide a more comprehensive picture of the activity of mitochondrial respiratory chain complexes I to IV. Their activities were compared among three different cell lines. A strong and significant increase in the activity of mitochondrial respiratory enzyme succinate:ubiquinone oxidoreductase (complex II) and a moderate increase of ubiquinol:cytochrome c oxidoreductase (complex III) were detected in all cell lines. In contrast, the activity of mitochondrial respiratory enzyme cytochrome c oxidase (complex IV) was significantly decreased. The effects on mitochondrial functions appear to be RV specific, as they were absent in control infections with measles virus. Additionally, these alterations of the respiratory chain activity were not associated with an elevated transcription of oxidative stress proteins, and reactive oxygen species (ROS) were induced only marginally. Moreover, protein and/or mRNA levels of markers for mitochondrial biogenesis and structure were elevated, such as nuclear respiratory factors (NRFs) and mitofusin 2 (Mfn2). Together, these results establish a novel view on the regulation of mitochondrial functions by viruses.

Inhibition of Human Neutrophil Elastase by α1-Antitrypsin Functionalized Colloidal Microcarriers

Layer-by-layer (LbL)-coated microcarriers offer a good opportunity as transport systems for active agents into specific cells and tissues. The assembling of oppositely charged polyelectrolytes enables a modular construction of the carriers and therefore an optimized integration and application of drug molecules. Here, we report the multilayer incorporation and transport of α(1)-antitrypsin (AT) by colloidal microcarriers. AT is an anti-inflammatory agent and shows inhibitory effects toward its pro-inflammatory antagonist, human neutrophil elastase (HNE). The highly proteolytic enzyme HNE is released by polymorphonuclear leukocytes (PMNs) during inflammatory processes and can cause host tissue destruction and pain. The high potential of this study is based on a simultaneous intra- and extracellular application of AT-functionalized LbL carriers. Carrier application in PMNs results in significant HNE inhibition within 21 h. Microcarriers phagocytosed by PMNs were time dependently decomposed inside phagolysosomes, which enables the step-by-step release of AT. Here, AT inactivates HNE before being released, which avoids a further HNE concentration increase in the extracellular space and, subsequently, reduces the risk of further tissue destruction. Additionally, AT surface-functionalized microcarriers allow the inhibition of already released HNE in the extracellular space. Finally, this study demonstrates the successful application of LbL carriers for a concurrent extra- and intracellular HNE inhibition aiming the rebalancing of protease and antiprotease concentrations and the subsequent termination of chronic inflammations.

Fluorescent bead arrays by means of layer-by-layer polyelectrolyte adsorption

Colloids with graduated fluorescence intensities were fabricated by means of layer-wise adsorption of fluorescein isothiocyanate-labelled poly(allyl amine hydrochloride) (FITC-PAH) together with poly(styrene sulfonate) (PSS) on silica particles. The graduated fluorescence was adjusted by variation of the fluorescent layer number and mixing labelled PAH with unlabelled PAH in one layer. The graduation of fluorescence intensities was adjusted in a geometric progression. It was shown that a proper label content is crucial if self-quenching phenomena are involved. The approach of mixing FITC-PAH with unlabelled polyelectrolyte during adsorption was unsatisfactory since competition in adsorption occurs. The system shows excellent stability at least over a period of two years.

The architecture of cartilage: Elemental maps and scanning transmission ion microscopy/tomography

Articular cartilage is not just a jelly-like cover of the bone within the joints but a highly sophisticated architecture of hydrated macromolecules, collagen fibrils and cartilage cells. Influences on the physiological balance due to age-related or pathological changes can lead to malfunction and subsequently to degradation of the cartilage. Many activities in cartilage research are dealing with the architecture of joint cartilage but have limited access to elemental distributions. Nuclear microscopy is able to yield spatially resolved elemental concentrations, provides density information and can visualise the arrangement of the collagen fibres. The distribution of the cartilage matrix can be deduced from the elemental and density maps. The findings showed a varying content of collagen and proteoglycan between zones of different cell maturation. Zones of higher collagen content are characterised by aligned collagen fibres that can form tubular structures. Recently we focused on STIM tomography to investigate the three dimensional arrangement of the collagen structures.

Visualisation of collagen fibrils in joint cartilage using STIM

Abstract The scanning transmission ion microscopy (STIM) method was used to investigate the collagen network structure of the articular cartilage from a pigs knee in comparison with high resolution nuclear magnetic resonance imaging (microscopic NMR-tomography) and polarised light microscopy (PLM). Single collagen fibrils down to 200 nm in diameter were visualised. It was proved that the cartilage collagen network consists partly of zones of oriented fibrils as suggested by NMR measurements. Radially oriented fibrils were found in the zone near the calcified zone (hypertrophic zone) of both tibia and femur, and in the tibial radial zone. Tangentially oriented fibrils were found in the femoral and tibial superficial zone and in a second zone of the femoral cartilage. Polarisation light microscopy reveals broader zones of orientation than it was found with STIM.

Influence of Layer-by-Layer (LbL) Assembled CaCO3-Carriers on Macrophage Signaling Cascades

Numerous drawbacks in the current medical treatment of chronic inflammations still require the design of sensitive and gentle methods without side effects. Layer-by-layer (LbL) coated microcarriers loaded with a cocktail of anti-inflammatory substances are supposed to be a new challenge for the medical treatment of immunoreactive cells such as macrophages and polymorphonuclear leukocytes (PMN). Nevertheless, microcarrier application requires biocompatibility of the system itself. Therefore, the aim of this study was to investigate microcarrier CaCO(3) systems LbL coated with biopolymers and a lipid bilayer, respectively, regarding the maintenance of the release of pro-inflammatory cytokines as TNFα and IL1β at normal levels. Only marginal increases after microcarrier interaction were allowed. The required microcarrier optimization results in the maximum use of a carrier/cell ratio of 1:1 for biopolymer-coated carriers and a carrier/cell ratio up to 5:1 for lipid-bilayer-coated carriers during the coincubation with macrophage-like cells. Low formation of reactive oxygen species (ROS) could not be maintained by either reduced carrier/cell ratios or by a surface lipid bilayer.