Off-target and tumor-specific accumulation of monocytes, macrophages and myeloid-derived suppressor cells after systemic injection

Solid tumors frequently coexist with a degree of local chronic inflammation. Recruited myeloid cells can therefore be considered as interesting vehicles for tumor-targeted delivery of therapeutic agents. Using in vivo imaging, the short-term accumulation of systemically injected monocytes, macrophages and myeloid-derived suppressor cells (MDSCs) was compared in mice bearing fat pad mammary carcinomas. Monocytes and macrophages demonstrated almost identical in vivo and ex vivo distribution patterns with maximal tumor-associated accumulation seen 48 hours after injection that remained stable over the 4-day follow-up period. However, a substantial accumulation of both cell types was also seen in the liver, spleen and lungs albeit decreasing over time in all three locations. The MDSCs exhibited a similar distribution pattern as the monocytes and macrophages, but demonstrated a better relative on-target fraction over time. Overall, our findings highlight off-target cell accumulation as a major obstacle in the use of myeloid cells as vehicles for therapeutic tumor-targeted agents and indicate that their short-term on-target accumulation is mainly of nonspecific nature

Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells

Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca2+ chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na+/H+ exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions. © 2011 The Authors. Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd

Improving the repeatability and efficacy of intradermal electroporated self-replicating mRNA

Local administration of naked self-replicating mRNA (sr-mRNA) in the skin or muscle using electroporation is effective but hampered by low repeatability. In this manuscript, we demonstrated that intradermal electroporation of sr-mRNA in combination with a protein-based RNase inhibitor increased the expression efficiency, success rate, and repeatability of the data. The RNase inhibitor should be added just before administration because storage of the inhibitor together with the sr-mRNA at -80 degrees C resulted in a partial loss of the beneficial effect. Furthermore, the location of intradermal electroporation also had a major effect on the expression of the sr-mRNA, with the highest and longest expression observed at the tail base of the mice. In contrast with previous work, we did not observe a beneficial effect of calcium ions on the efficacy of naked sr-mRNA after intradermal injection. Finally, another important finding was that the traditional representation of in vivo bioluminescence data as means in logarithmic graphs can mask highly variable data. A more truthful representation can be obtained by showing the individual data points or by displaying median values in combination with interquartile ranges. In conclusion, intradermal sr-mRNA electroporation can be improved by adding an RNase inhibitor and injecting at the tail base

Mononuclear but not polymorphonuclear phagocyte depletion increases circulation times and improves mammary tumor-homing efficiency of donor bone marrow-derived monocytes

Tumor associated macrophages are an essential part of the tumor microenvironment. Consequently, bone marrow-derived monocytes (BMDMs) are continuously recruited to tumors and are therefore seen as ideal delivery vehicles with tumor-targeting properties. By using immune cell depleting agents and macroscopic in vivo fluorescence imaging, we demonstrated that removal of endogenous monocytes and macrophages (but not neutrophils) leads to an increased tumor accumulation of exogenously administered BMDMs. By means of intravital microscopy (IVM), we confirmed our macroscopic findings on a cellular level and visualized in real time the migration of the donor BMDMs in the tumors of living animals. Moreover, IVM also revealed that clodronate-mediated depletion drastically increases the circulation time of the exogenously administered BMDMs. In summary, these new insights illustrate that impairment of the mononuclear phagocyte system increases the circulation time and tumor accumulation of donor BMDMs

Migration of Star Clusters and Nuclear Rings

Star clusters that form in nuclear rings appear to be at slightly larger radii than the gas. We argue that the star clusters move out from the gas in which they are formed because of satellite-disk tidal interactions. In calculating the dynamics of this star cluster and gas ring system, we include the effects of dynamical friction of the background stars in the host galaxy on the star cluster, and inflowing gas along the bar onto the nuclear ring at the two contact points. We show that the final separation is of the order of the Hill radius of the nuclear ring, which is typically 20-30% of its radius. Massive star clusters can reach half of this separation very quickly and produce a factor of a few enhancement in the gas surface density. If this leads to star formation in addition to the (ongoing) formation of star clusters near the contact points, a possible (initial) azimuthal age gradient may become diluted or even disappear. Finally, if the star cluster are massive and/or numerous enough, we expect the nuclear ring to migrate inward, away from the (possibly) associated (inner) Lindblad resonance. We discuss how these predictions may be tested observationally.Comment: 12 pages, 6 figures, referee comments incorporated; published in ApJ, 2009, 697, 61

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins

What determines the complex kinetics of stomatal conductance under blueless PAR in Festuca arundinacea? Subsequent effects on leaf transpiration

Light quality and, in particular, its content of blue light is involved in plant functioning and morphogenesis. Blue light variation frequently occurs within a stand as shaded zones are characterized by a simultaneous decrease of PAR and blue light levels which both affect plant functioning, for example, gas exchange. However, little is known about the effects of low blue light itself on gas exchange. The aims of the present study were (i) to characterize stomatal behaviour in Festuca arundinacea leaves through leaf gas exchange measurements in response to a sudden reduction in blue light, and (ii) to test the putative role of Ci on blue light gas exchange responses. An infrared gas analyser (IRGA) was used with light transmission filters to study stomatal conductance (gs), transpiration (Tr), assimilation (A), and intercellular concentration of CO2 (Ci) responses to blueless PAR (1.80 μmol m−2 s−1). The results were compared with those obtained under a neutral filter supplying a similar photosynthetic efficiency to the blueless PAR filter. It was shown that the reduction of blue light triggered a drastic and instantaneous decrease of gs by 43.2% and of Tr by 40.0%, but a gradual stomatal reopening began 20 min after the start of the low blue light treatment, thus leading to new steady-states. This new stomatal equilibrium was supposed to be related to Ci. The results were confirmed in more developed plants although they exhibited delayed and less marked responses. It is concluded that stomatal responses to blue light could play a key role in photomorphogenetic mechanisms through their effect on transpiration

Comparison and evaluation of experimental mediastinitis models: precolonized foreign body implants and bacterial suspension inoculation seems promising

BACKGROUND: Post-sternotomy mediastinitis (PSM) is a devastating surgical complication affecting 1–3% of patients that undergo cardiac surgery. Staphylococcus aureus is one of the most commonly encountered bacterial pathogen cultured from mediastinal samples obtained from patients with PSM. A component of the membrane of the gram positive bacteria, lipoteichoic acid, stimulates the blood monocytes and macrophages to secrete cytokines, radicals and nitrogen species leading to oxido-inflammatory damage. This seems to be responsible for the high mortality rate in PSM. For the evaluation of the pathogenesis of infection or for the investigation of alternative treatment models in infection, no standard model of mediastinitis seems to be available. In this study, we evaluated four mediastinitis models in rats. METHODS: The rats were divided into four groups to form different infection models. Group A: A suspension of 1 × 10(7 )colony-forming units Staphylococcus aureus in 0,5 mL was inoculated from the right second intercostal space into the mediastinum. Group B: A hole was created in the right second intercostal space and a piece of stainless-steel implant with a length of 0.5 cm was inserted into the mediastinum and a suspension of 1 × 10(7 )cfu bacteria in 0,5 mL was administered via the tail vein. Group C: Precolonized stainless-steel implant was inserted into the mediastinum. Group D: Precolonized stainless-steel implant was inserted into the mediastinum and the bacteria suspension was also injected into the mediastinum. On the 10(th )day, rats were sacrificed and the extension of infection in the mediastenae was evaluated by quantitative cultures. Myeloperoxidase activity (MPO) and malondialdehyde (MDA) levels were determined in the sera to evaluate the neutrophil activation and assess the inflammatory oxidation. RESULTS: The degree of infection in group C and D were 83.3% and 100% respectively (P < 0.001). MDA levels were significantly higher in these two groups than the others (P < 0.001). CONCLUSION: Infected implants and high bacterial concentration administration were the two important components that played a significant role in the outcome of a successful infection in mediastinum in a rat model

A review of elliptical and disc galaxy structure, and modern scaling laws

A century ago, in 1911 and 1913, Plummer and then Reynolds introduced their models to describe the radial distribution of stars in `nebulae'. This article reviews the progress since then, providing both an historical perspective and a contemporary review of the stellar structure of bulges, discs and elliptical galaxies. The quantification of galaxy nuclei, such as central mass deficits and excess nuclear light, plus the structure of dark matter halos and cD galaxy envelopes, are discussed. Issues pertaining to spiral galaxies including dust, bulge-to-disc ratios, bulgeless galaxies, bars and the identification of pseudobulges are also reviewed. An array of modern scaling relations involving sizes, luminosities, surface brightnesses and stellar concentrations are presented, many of which are shown to be curved. These 'redshift zero' relations not only quantify the behavior and nature of galaxies in the Universe today, but are the modern benchmark for evolutionary studies of galaxies, whether based on observations, N-body-simulations or semi-analytical modelling. For example, it is shown that some of the recently discovered compact elliptical galaxies at 1.5 < z < 2.5 may be the bulges of modern disc galaxies.Comment: Condensed version (due to Contract) of an invited review article to appear in "Planets, Stars and Stellar Systems"(www.springer.com/astronomy/book/978-90-481-8818-5). 500+ references incl. many somewhat forgotten, pioneer papers. Original submission to Springer: 07-June-201