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Flow cytometry has become a widely-used and powerful tool for the characterization of cells according to their expression of specific proteins. However, sensitivity of this method is still limited since conventionally labeled antibodies can be conjugated with at maximum 1-10 dye molecules. This fact resulted in the need to develop new techniques in order to identify molecules which are expressed in very low but functionally relevant amounts. In the past, we have successfully used a liposome-based high-sensitivity immunofluorescence technique to measure the expression of low abundant membrane bound glucocorticoid receptors (mGR) on different cell types. The use of this technique allows the detection of as few as 50-100 antigen molecules per cell which is due to a 100-fold to 1000-fold increase in fluorescence signal intensity compared with conventional methods. The higher sensitivity is achieved since thousands of dye molecules can be enclosed in liposomes. Another modern high-sensitivity immunofluorescence staining method is the purchasable Fluorescence Amplification by Sequential Employment of Reagents (FASER) procedure. Here, we aimed at comparing sensitivity and specificity of these two techniques for the detection of the mGR. Our data demonstrate the FASER technique to be more sensitive and also more specific for the detection of mGR as compared to the liposome technique. However, both methods have advantages and disadvantages which are discussed in detail. [ABSTRACT FROM AUTHOR]

The accumulation of oxidatively damaged proteins is a well-known hallmark of aging and several neurodegenerative diseases including Alzheimer's, Parkinson's and Huntigton's diseases. These highly oxidized protein aggregates are in general not degradable by the main intracellular proteolytic machinery, the proteasomal system. One possible strategy to reduce the accumulation of such oxidized protein aggregates is the prevention of the formation of oxidized protein derivatives or to reduce the protein oxidation to a degree that can be handled by the proteasome. To do so an antioxidative strategy might be successful. Therefore, we undertook the present study to test whether antioxidants are able to prevent the protein oxidation and to influence the proteasomal degradation of moderate oxidized proteins. As a model protein we choose ferritin. H2O2 induced a concentration dependent increase of protein oxidation accompanied by an increased proteolytic susceptibility. This increase of proteolytic susceptibility is limited to moderate hydrogen peroxide concentrations, whereas higher concentrations are accompanied by protein aggregate formation. Protective effects of the vitamin E derivative Trolox, the pyridoindole derivative Stobadine and of the standardized extracts of flavonoids from bark of Pinus Pinaster Pycnogenol® and from leaves of Ginkgo biloba (EGb 761) were studied on moderate damaged ferritin. [ABSTRACT FROM AUTHOR]

Pigs with mutated calcium release channels are stress-susceptible and are known for increased postmortem metabolism which results in inferior meat quality. The lipid composition, lipid peroxidation, and oxidation of proteins of skeletal muscle homogenates from the longissimus muscle of pigs were investigated. Muscle tissue samples were obtained from malignant hyperthermia-resistant (MHR) and hyperthermia-susceptible (MHS) pigs in a postmortem time course. Differences in the n-3 polyunsaturated fatty acid composition with higher values in the susceptible pigs were detected (MHR versus MHS: 141±5 versus 116±4 μg/g wet wt.; P<0.05). This was accompanied by a dramatic 40% postmortem vitamin E loss in MHS muscles (22 h MHR versus 22 h MHS: 1.66±0.11 versus 1.06±0.16 μg/g wet wt.; P<0.05). Muscle tissue from MHS pigs oxidized at a significantly higher rate in comparison with tissue from MHR animals. This difference was also observed for spontaneous malondialdehyde formation as well as for oxidant-induced peroxidation rate (0.75 h MHR versus 0.75 h MHS: 0.81±0.09 versus 1.33±0.09 nmol/g wet wt.; P<0.05). No difference was observed in protein oxidation level in MHS and MHR animals. It is concluded that the enhanced lipid oxidation rate could contribute to the dramatic changes in meat quality from MHS pigs. [Copyright &y& Elsevier]

Immunologically restricted patients such as those with autoimmune diseases or malignancies often suffer from delayed or insufficient fracture healing. In human fracture hematomas and the surrounding bone marrow obtained from immunologically restricted patients, we analyzed the initial inflammatory phase on cellular and humoral level via flow cytometry and multiplex suspension array. Compared with controls, we demonstrated higher numbers of immune cells like monocytes/macrophages, natural killer T (NKT) cells, and activated T helper cells within the fracture hematomas and/or the surrounding bone marrow. Also, several pro-inflammatory cytokines such as Interleukin (IL)-6 and Tumor necrosis factor α (TNFα), chemokines (e.g., Eotaxin and RANTES), pro-angiogenic factors (e.g., IL-8 and Macrophage migration inhibitory factor: MIF), and regulatory cytokines (e.g., IL-10) were found at higher levels within the fracture hematomas and/or the surrounding bone marrow of immunologically restricted patients when compared to controls. We conclude here that the inflammatory activity on cellular and humoral levels at fracture sites of immunologically restricted patients considerably exceeds that of control patients. The initial inflammatory phase profoundly differs between these patient groups and is probably one of the reasons for prolonged or insufficient fracture healing often occurring within immunologically restricted patients. [ABSTRACT FROM AUTHOR]

Purpose: Two of the most common joint diseases are rheumatoid arthritis (RA) and osteoarthritis (OA). Cartilage degradation and erosions are important pathogenetic mechanisms in both joint diseases and have presently gained increasing interest. The aim of the present study was to investigate the effects of the synovial fluid environment of OA patients in comparison with synovial fluids of RA patients on human chondrocytes in vitro. Methods: Primary human chondrocytes were incubated in synovial fluids gained from patients with OA or RA. The detection of vital cell numbers was determined by histology and by using the Casy Cell Counter System. Cytokine and chemokine secretion was determined by a multiplex suspension array. Results: Microscopic analysis showed altered cell morphology and cell shrinkage following incubation with synovial fluid of RA patients. Detection of vital cells showed a highly significant decrease of vital chondrocyte when treated with RA synovial fluids in comparison with OA synovial fluids. An active secretion of cytokines such as vascular endothelial growth factor (VEGF) of chondrocytes treated with OA synovial fluids was observed. Conclusions: Significantly increased levels of various cytokines in synovial fluids of RA, and surprisingly of OA, patients were shown. Activation of pro-inflammatory cytokines of human chondrocytes by synovial fluids of OA patient supports a pro-inflammatory process in the pathogenesis of OA. [ABSTRACT FROM AUTHOR]

Iron oxide particles are a promising marker in molecular magnetic resonance imaging. They are used to label distinct cell populations either in vitro or in vivo. We investigated for the first time whether small citrate-coated very small superparamagnetic iron oxide particles (VSOPs) can lead to an increase in cellular oxidative stress. We incubated rat macrophages (RAW) in vitro with iron oxide particles. We observed a massive uptake of VSOPs measured both with atomic absorption spectroscopy and with NMR, which could be visualized by confocal laser scanning microscopy. After incubation, cells were lysed and the levels of malonyldialdehyde (MDA) and protein carbonyls were determined. We found a significant increase in both MDA and protein carbonyl levels after incubation with the particles. Surprisingly, 24 h after incubation, a significant indication of oxidative stress could no longer be observed. The increase in oxidative stress seems to be transient and closely linked to the incubation procedure. The iron chelator desferal and the intracellular spin trap PBN caused a significant reduction in oxidative stress to almost control levels. This indicates that the augmentation of oxidative stress is closely linked to the free iron during incubation. Proliferation assays showed that incorporation of VSOPs did not lead to long-term cytotoxic effects even though the iron oxide particles remained in the cell. Magnetic labeling of cells with VSOPs seems to cause transient oxidative conditions not affecting cellular viability and seems to be a usable approach for molecular magnetic resonance imaging. [Copyright &y& Elsevier]