Expression of CD95 on mature leukocytes of MRL/lpr mice after transplantation of genetically modified bone marrow stem cells

Bone marrow transplantation (BMT) is commonly used for the treatment of severe haematological and immunological diseases. For instance, the autoimmune lymphoproliferative syndrome (ALPS) caused by a complete expression defect of CD95 (Fas, APO-1) can be cured by allogeneic BMT. However, since this therapy may not generate satisfactory results when only partially compatible donors are available, we were interested in the development of a potential alternative treatment by using lentiviral gene transfer of a normal copy of CD95 cDNA in hematopoietic stem cells. Here, we show that this approach applied to MRL/lpr mice results in the expression of functional CD95 receptors on the surface of lymphocytes, monocytes, and granulocytes. This suggests that correction of CD95 deficiency can be achieved by gene therapy.

The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line

This study investigated the uptake, kinetics and cellular distribution of different surface coated quantum dots (QDs) before relating this to their toxicity. J774.A1 cells were treated with organic, COOH and NH2 (PEG) surface coated QDs (40 nM). Model 20 nm and 200 nm COOH-modified coated polystyrene beads (PBs) were also examined (50 microg ml(-1)). The potential for uptake of QDs was examined by both fixed and live cell confocal microscopy as well as by flow cytometry over 2 h. Both the COOH 20 nm and 200 nm PBs were clearly and rapidly taken up by the J774.A1 cells, with uptake of 20 nm PBs being relatively quicker and more extensive. Similarly, COOH QDs were clearly taken up by the macrophages. Uptake of NH2 (PEG) QDs was not detectable by live cell imaging however, was observed following 3D reconstruction of fixed cells, as well as by flow cytometry. Cells treated with organic QDs, monitored by live cell imaging, showed only a small amount of uptake in a relatively small number of cells. This uptake was insufficient to be detected by flow cytometry. Imaging of fixed cells was not possible due to a loss in cell integrity related to cytotoxicity. A significant reduction (p<0.05) in the fluorescent intensity in a cell-free environment was found with organic QDs, NH2 (PEG) QDs, 20 nm and 200 nm PBs at pH 4.0 (indicative of an endosome) after 2 h, suggesting reduced stability. No evidence of exocytosis was found over 2 h. These findings confirm that surface coating has a significant influence on the mode of NP interaction with cells, as well as the subsequent consequences of that interaction.

The cell surface receptor FGFRL1 forms constitutive dimers that promote cell adhesion

FGFRL1 is a novel member of the fibroblast growth factor (FGF) receptor family. Utilizing the FRET (fluorescence resonance energy transfer) technique, we demonstrate that FGFRL1 forms constitutive homodimers at cell surfaces. The formation of homodimers was verified by co-precipitation of differentially tagged FGFRL1 polypeptides from solution. If overexpressed in cultivated cells, FGFRL1 was found to be enriched at cell-cell contact sites. The extracellular domain of recombinant FGFRL1 promoted cell adhesion, but not cell spreading, when coated on plastic surfaces. Adhesion was mediated by heparan sulfate glycosaminoglycans located at the cell surface. It could specifically be blocked by addition of soluble heparin but not by addition of other glycosaminoglycans. When the amino acid sequence of the putative heparin-binding site was modified by in vitro mutagenesis, the resulting protein exhibited decreased affinity for heparin and reduced activity in the cell-binding assay. Moreover, a synthetic peptide corresponding to the heparin-binding site was able to neutralize the effect of heparin. With its dimeric structure and its adhesion promoting properties, FGFRL1 resembles the nectins, a family of cell adhesion molecules found at cell-cell junctions.

Biomechanical comparison of different surface modifications for dental implants

Purpose: A satisfactory clinical outcome in dental implant treatment relies on primary stability for immediate load bearing. While the geometric design of an implant contributes to mechanical stability, the nature of the implant surface itself is also critically important. Biomechanical and microcomputerized tomographic evaluation of implant osseointegration was performed to compare alternative structural, chemical and biochemical, and/or pharmaceutical surface treatments applied to an identical established implant design. Materials and Methods: Dental implants with the same geometry but with 6 different surface treatments were tested in vivo in a sheep model (pelvis). Peri-implant bone density and removal torque were compared at 2, 4, and 8 weeks after implantation. Implant surfaces tested were: sandblasted and acid-etched titanium (Ti), sandblasted and etched zirconia, Ti coated with calcium phosphate (CaP), Ti modified via anodic plasma-chemical treatment (APC), bisphosphonate-coated Ti (Ti + Bisphos), and Ti coated with collagen containing chondroitin sulfate (CS). Results: All dental implants were well integrated at the time of sacrifice. There were no significant differences observed in peri-implant bone density between implant groups. After 8 weeks of healing, removal torque values for Ti, Ti + CaP, Ti + Bisphos, and Ti + collagen + CS were significantly higher than those for zirconia and Ti + APC. Conclusions: Whereas the sandblasted/acid-etched Ti implant can still be considered the reference standard surface for dental implants, functional surface modifications such as bisphosphonate or collagen coating seem to enhance early peri-implant bone formation and should be studied further.

An in-situ surface electrochemistry approach toward whole-cell studies: Charge transfer between Geobacter sulfurreducens and electrified metal/electrolyte interfaces through linker molecules

Electrochemical reactivity and structure properties of electrogenic bacteria, Geobacter sulfurreducens (Gs) were studied to explore the heterogeneous electron transfer at the bacteria/electrode interface using electrochemical and in-situ spectroscopic techniques. The redox behavior of Gs adsorbed on a gold electrode, which is modified with a ω-functionalized self-assembled monolayer (SAM) of alkanethiols, depends strongly on the terminal group. The latter interacts directly with outermost cytochromes embedded into the outer membrane of the Gs cells. The redox potential of bacterial cells bound electrostatically to a carboxyl-terminated SAM is close to that observed for bacteria attached to a bare gold electrode, revealing a high electronic coupling at the cell/SAM interface. The redox potentials of bacterial cells adsorbed on amino- and pyridyl-terminated SAMs are significantly different suggesting that the outermost cytochromes changes their conformation upon adsorption on these SAMs. No redox activity of Gs was found with CH3-, N(CH3)3+- and OH-terminated SAMs. Complementary in-situ spectroscopic studies on bacteria/SAMs/Au electrode assemblies were carried out to monitor structure changes of the bacterial cells upon polarization. Spectro-electrochemical techniques revealed the electrochemical turnover of the oxidized and reduced states of outer membrane cytochromes (OMCs) in Gs, providing evidence that the OMCs are responsible for the direct electron transfer to metal electrodes, such as gold or silver, during the electricity production. Furthermore, we observed spectroscopic signatures of the native structure of the OMCs and no conformational change during the oxidation/reduction process of the microorganisms. These findings indicate that the carboxyl-anchoring group provides biocompatible conditions for the outermost cytochromes of the Gs, which facilitate the heterogeneous electron transfer at the microorganism/electrode interface.

Ferrocene-terminated alkanethiol self-assembled monolayers: An electrochemical and in situ surface-enhanced infra-red absorption spectroscopy study

The effect of anions on the redox behavior and structure of 11-ferrocenyl-1-undecanethiol (FcC11) monolayers (SAM) on Au(1 1 1) single crystal and Au(1 1 1-25 nm) thin film electrodes was investigated in 0.1 M solutions of HPF6, HClO4, HBF4, HNO3, and H2SO4 by cyclic voltammetry (CV) and in situ surface-enhanced infrared reflection-absorption spectroscopy (SEIRAS). We demonstrate that the FcC11 redox peaks shift toward positive potentials and broaden with increasing hydrophilicity of the anions. In situ surface-enhanced IR-spectroscopy (SEIRAS) provided direct access for the incorporation of anions into the oxidized adlayer. The coadsorption of anions is accompanied by the penetration of water molecules. The latter effect is particularly pronounced in aqueous HNO3 and H2SO4 electrolytes. The adlayer permeability increases with increasing hydrophilicity of the anions. We also found that even the neutral (reduced) FcC11 SAM is permeable for water molecules. Based on the property of interfacial water to reorient upon charge inversion, we propose a spectroscopic approach for estimating the potential of zero total charge of the FcC11-modified Au(1 1 1) electrodes in aqueous electrolytes.

Emergence of Canine Distemper Virus Strains With Modified Molecular Signature and Enhanced Neuronal Tropism Leading to High Mortality in Wild Carnivores

An ongoing canine distemper epidemic was first detected in Switzerland in the spring of 2009. Compared to previous local canine distemper outbreaks, it was characterized by unusually high morbidity and mortality, rapid spread over the country, and susceptibility of several wild carnivore species. Here, the authors describe the associated pathologic changes and phylogenetic and biological features of a multiple highly virulent canine distemper virus (CDV) strain detected in and/or isolated from red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), stone (Martes foina) and pine (Martes martes) martens, from a Eurasian lynx (Lynx lynx), and a domestic dog. The main lesions included interstitial to bronchointerstitial pneumonia and meningopolioencephalitis, whereas demyelination-the classic presentation of CDV infection-was observed in few cases only. In the brain lesions, viral inclusions were mainly in the nuclei of the neurons. Some significant differences in brain and lung lesions were observed between foxes and mustelids. Swiss CDV isolates shared together with a Hungarian CDV strain detected in 2004. In vitro analysis of the hemagglutinin protein from one of the Swiss CDV strains revealed functional and structural differences from that of the reference strain A75/17, with the Swiss strain showing increased surface expression and binding efficiency to the signaling lymphocyte activation molecule (SLAM). These features might be part of a novel molecular signature, which might have contributed to an increase in virus pathogenicity, partially explaining the high morbidity and mortality, the rapid spread, and the large host spectrum observed in this outbreak.

CO2 Electroreduction on Cu-Modified Platinum Single Crystal Electrodes in Aprotic Media

Techniques of electrode modification by copper deposits are developed that allow obtaining compact bulk quasi-epitaxial deposits on basal Pt(hkl) single crystal faces. The issues of the deposit roughness and characterization are discussed. Problems of drying and transferring electrodes with copper deposits into other solutions are considered. The obtained deposits are used for CO2 electroreduction in propylene carbonate and acetonitrile solutions of 0.1 M TBAPF6, and the relationship between the electrode surface structure and its electrocatalytic activity in CO2 electroreduction is discussed. We also demonstrate that the restructuring of Cu deposits occurs upon CO2 electroreduction. Complementary reactivity studies are presented for bare Pt(hkl) and Cu(hkl) single crystal electrodes. Cu-modified Pt(hkl) electrodes display the highest activity as compared to bare Pt(hkl) and Cu(hkl). Particularly, the Cu/Pt(110) electrode shows the highest activity among the electrodes under study. Such high activity of Cu/Pt(hkl) electrodes can be explained not only by the increasing actual surface area but also by structural effects, namely by the presence of a large amount of specific defect sites (steps, kinks) on Cu crystallites.

Relating in situ gas measurements to the surface outgassing properties of cometary nuclei

The sensitivity of the gas flow field to changes in different initial conditions has been studied for the case of a highly simplified cometary nucleus model. The nucleus model simulated a homogeneously outgassing sphere with a more active ring around an axis of symmetry. The varied initial conditions were the number density of the homogeneous region, the surface temperature, and the composition of the flow (varying amounts of H2O and CO2) from the active ring. The sensitivity analysis was performed using the Polynomial Chaos Expansion (PCE) method. Direct Simulation Monte Carlo (DSMC) was used for the flow, thereby allowing strong deviations from local thermal equilibrium. The PCE approach can be used to produce a sensitivity analysis with only four runs per modified input parameter and allows one to study and quantify non-linear responses of measurable parameters to linear changes in the input over a wide range. Hence the PCE allows one to obtain a functional relationship between the flow field properties at every point in the inner coma and the input conditions. It is for example shown that the velocity and the temperature of the background gas are not simply linear functions of the initial number density at the source. As probably expected, the main influence on the resulting flow field parameter is the corresponding initial parameter (i.e. the initial number density determines the background number density, the temperature of the surface determines the flow field temperature, etc.). However, the velocity of the flow field is also influenced by the surface temperature while the number density is not sensitive to the surface temperature at all in our model set-up. Another example is the change in the composition of the flow over the active area. Such changes can be seen in the velocity but again not in the number density. Although this study uses only a simple test case, we suggest that the approach, when applied to a real case in 3D, should assist in identifying the sensitivity of gas parameters measured in situ by, for example, the Rosetta spacecraft to the surface boundary conditions and vice versa.

Osseointegration of biochemically modified implants in an osteoporosis rodent model

The present study examined the impact of implant surface modifications on osseointegration in an osteoporotic rodent model. Sandblasted, acid-etched titanium implants were either used directly (control) or were further modified by surface conditioning with NaOH or by coating with one of the following active agents: collagen/chondroitin sulphate, simvastatin, or zoledronic acid. Control and modified implants were inserted into the proximal tibia of aged ovariectomised (OVX) osteoporotic rats (n = 32/group). In addition, aged oestrogen competent animals received either control or NaOH conditioned implants. Animals were sacrificed 2 and 4 weeks post-implantation. The excised tibiae were utilised for biomechanical and morphometric readouts (n = 8/group/readout). Biomechanical testing revealed at both time points dramatically reduced osseointegration in the tibia of oestrogen deprived osteoporotic animals compared to intact controls irrespective of NaOH exposure. Consistently, histomorphometric and microCT analyses demonstrated diminished bone-implant contact (BIC), peri-implant bone area (BA), bone volume/tissue volume (BV/TV) and bone-mineral density (BMD) in OVX animals. Surface coating with collagen/chondroitin sulphate had no detectable impact on osseointegration. Interestingly, statin coating resulted in a transient increase in BIC 2 weeks post-implantation; which, however, did not correspond to improvement of biomechanical readouts. Local exposure to zoledronic acid increased BIC, BA, BV/TV and BMD at 4 weeks. Yet this translated only into a non-significant improvement of biomechanical properties. In conclusion, this study presents a rodent model mimicking severely osteoporotic bone. Contrary to the other bioactive agents, locally released zoledronic acid had a positive impact on osseointegration albeit to a lesser extent than reported in less challenging models.

Surgical exposures and options for instrumentation in acetabular fracture fixation: Pararectus approach versus the modified Stoppa.

BACKGROUND As an alternative to the modified Stoppa approach, the Pararectus approach is used clinically for treatment of acetabular fractures involving the anterior column. The current study assessed the surgical exposure and the options for instrumentation using both of these approaches. METHODS Surgical dissections were conducted on five human cadavers (all male, mean age 88 years (82-97)) using the modified Stoppa and the Pararectus approach, with the same skin incision length (10cm). Distal boundaries of the exposed bony surfaces were marked using a chisel. After removal of all soft-tissues, distances from the boundaries in the false and true pelvis were measured with reference to the pelvic brim. The exposed bone was coloured and calibrated digital images of each inner hemipelvis were taken. The amount of exposed surface using both approaches was assessed and represented as a percentage of the total bony surface of each hemipelvis. For instrumentation, a suprapectineal quadrilateral buttress plate was used. Screw lengths were documented, and three-dimensional CT reconstructions were performed to assess screw trajectories qualitatively. Wilcoxon's signed rank test for paired groups was used (level of significance: p<0.05). RESULTS After utilization of the Pararectus approach, the distances from the farthest boundaries of exposed bone towards the pelvic brim were significantly higher in the false but not the true pelvis, compared to the modified Stoppa approach. The percentage (mean±SD) of exposed bone accessible after utilizing the Pararectus approach was 42±8%, compared to 29±6% using the modified Stoppa (p=0.011). In cadavers exposed by the Pararectus approach, screws placed for posterior fixation and as a posterior column screw were longer by factor 1.8 and 2.1, respectively (p<0.05), and screws could be placed more posteromedial towards the posterior inferior iliac spine or in line with the posterior column directed towards the ischial tuberosity. CONCLUSION Compared to the modified Stoppa, the Pararectus approach facilitates a greater surgical access in the false pelvis, provides versatility for fracture fixation in the posterior pelvic ring and allows for the option to extend the approach without a new incision.