Small-Molecule Immunosuppressive Drugs and Therapeutic Immunoglobulins Differentially Inhibit NK Cell Effector Functions in vitro

Small-molecule immunosuppressive drugs (ISD) prevent graft rejection mainly by inhibiting T lymphocytes. Therapeutic immunoglobulins (IVIg) are used for substitution, antibody-mediated rejection (AbMR) and HLA-sensitized recipients by targeting distinct cell types. Since the effect of ISD and IVIg on natural killer (NK) cells remains somewhat controversial in the current literature, the aim of this comparative study was to investigate healthy donor's human NK cell functions after exposure to ISD and IVIg, and to comprehensively review the current literature. NK cells were incubated overnight with IL2/IL12 and different doses and combinations of ISD and IVIg. Proliferation was evaluated by 3[H]-thymidine incorporation; phenotype, degranulation and interferon gamma (IFNγ) production by flow cytometry and ELISA; direct NK cytotoxicity by standard 51[Cr]-release and non-radioactive DELFIA assays using K562 as stimulator and target cells; porcine endothelial cells coated with human anti-pig antibodies were used as targets in antibody-dependent cellular cytotoxicity (ADCC) assays. We found that CD69, CD25, CD54, and NKG2D were downregulated by ISD. Proliferation was inhibited by methylprednisolone (MePRD), mycophenolic acid (MPA), and everolimus (EVE). MePRD and MPA reduced degranulation, MPA only of CD56bright NK cells. MePRD and IVIg inhibited direct cytotoxicity and ADCC. Combinations of ISD demonstrated cumulative inhibitory effects. IFNγ production was inhibited by MePRD and ISD combinations, but not by IVIg. In conclusion, IVIg, ISD and combinations thereof differentially inhibit NK cell functions. The most potent drug with an effect on all NK functions was MePRD. The fact that MePRD and IVIg significantly block NK cytotoxicity, especially ADCC, has major implications for AbMR as well as therapeutic strategies targeting cancer and immune cells with monoclonal antibodies

Investigation of the influence of exogenous tissue factor on endothelial cell proliferation, apoptosis and survival

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The influence of exogenous tissue factor on the regulators of proliferation and apoptosis in endothelial cells

Background: The exposure of tissue factor (TF) at the site of injury or trauma is a rapid process that leads to the initiation of blood coagulation as well as homeostatic processes giving rise to vascular repair. Aims and Methods: By exposing human endothelial cells to combinations of exogenous TF and factor VIIa (FVIIa) in serum-free medium, the influence of TF concentrations on cellular proliferation and apoptosis was investigated. Results: Lower concentrations of TF resulted in increased cellular proliferation as well as upregulation of cyclin D1, downregulation of p21 and p27 and induction of tube formation in vitro. Conversely, incubation with higher concentrations of TF resulted in the activation of caspase-3, expression of p53 and Bax, translocation of p53 into the nucleus and induction of DNA fragmentation. Incubation of the cells with TF/FVIIa led to a lower proliferation rate with additional upregulation in p27. Conclusions: TF seems to have a bifunctional role in determining the fate of endothelial cells, depending on the concentration and the interactions of this protein. The release of TF in the locality of the injured tissue makes this protein an ideal factor for ascertaining the level of injury and determining the fate of the cells. Copyright © 2007 S. Karger AG

T-bet and Eomesodermin in NK Cell Development, Maturation, and Function

Recent reports give insights into the role of the T-box transcription factors, T-bet and Eomesodermin (Eomes), in NK cell biology. In this mini-review, we recapitulate the initial reports that delineate T-bet and Eomes as master regulators of NK cell development, maturation, and function. We discuss how T-bet and Eomes expression is regulated during NK cell development and peripheral maturation. Furthermore, we summarize the current literature on the role of T-bet and Eomes in the transcriptional regulation of NK cell function and review possible effects of T-box transcription factor anomalies during aging, infection, cancer, and after hematopoietic stem cell transplantation. We discuss how the current data argue in favor of a model of T-bet and Eomes synergy in transcriptional regulation of NK cell function and identify T-box transcription factors as potential targets for therapeutic interventions

Low molecular weight dextran sulfate binds to human myoblasts and improves their survival after transplantation in mice

Myoblast transplantation represents a promising therapeutic strategy in the treatment of several genetic muscular disorders including Duchenne muscular dystrophy. Nevertheless, such an approach is impaired by the rapid death, limited migration, and rejection of transplanted myoblasts by the host. Low molecular weight dextran sulfate (DXS), a sulfated polysaccharide, has been reported to act as a cytoprotectant for various cell types. Therefore, we investigated whether DXS could act as a "myoblastprotectant" either in vitro or in vivo after transplantation in immunodeficient mice. In vitro, DXS bound human myoblasts in a dose-dependent manner and significantly inhibited staurosporine-mediated apoptosis and necrosis. DXS pretreatment also protected human myoblasts from natural killer cell-mediated cytotoxicity. When human myoblasts engineered to express the renilla luciferase transgene were transplanted in immunodeficient mice, bioluminescence imaging analysis revealed that the proportion of surviving myoblasts 1 and 3 days after transplantation was two times higher when cells were preincubated with DXS compared to control (77.9 ± 10.1% vs. 39.4 ± 4.9%, p = 0.0009 and 38.1 ± 8.5% vs. 15.1 ± 3.4%, p = 0.01, respectively). Taken together, we provide evidence that DXS acts as a myoblast protectant in vitro and is able in vivo to prevent the early death of transplanted myoblasts

Evaluation of clinical IMRT treatment planning using the gATE Monte Carlo simulation platform for absolute and relative dose calculations

International audiencePURPOSE:The objective of this study was to evaluate and validate the use of the Geant4 application for emission tomography (GATE) Monte Carlo simulation platform for clinical intensity modulated radiotherapy (IMRT) dosimetry studies. METHODS: The first step consisted of modeling a 6 MV photon beam linear accelerator (LINAC), with its corresponding validation carried out using percent depth dose evaluation, transverse profiles, tissue phantom ratio, and output factor on water phantom. The IMRT evaluation was performed by comparing simulation and measurements in terms of absolute and relative doses using IMRT dedicated quality assurance phantoms considering seven different patient datasets. RESULTS: Concerning the LINAC simulated model validation tissue phantom ratios at 20 and 10 cm in water TPR(10) (20) obtained from GATE and measurements were 0.672 ± 0.063 and 0.675, respectively. In terms of percent depth dose and transverse profiles, error ranges were, respectively: 1.472% ± 0.285% and 4.827% ± 1.323% for field size of 4 × 4, 5 × 5, 10 × 10, 15 × 15, 20 × 20, 25 × 25, 30 × 30, and 40 × 40 cm(2). Most errors were observed at the edge of radiation fields because of higher dose gradient in these areas. Output factors showed good agreement between simulation and measurements with a maximum error of 1.22%. Finally, for IMRT simulations considering seven patient datasets, GATE provided good results with a relative error of 0.43% ± 0.25% on absolute dose between simulated and measured beams (measurements at the isocenter, volume 0.125 cm(3)). Planar dose comparisons were also performed using gamma-index analysis. For the whole set of beams considered the mean gamma-index value was 0.497 ± 0.152 and 90.8% ± 3.6% of the evaluated dose points satisfied the 5% ∕ 4 mm criterion. CONCLUSIONS: These results show that GATE allows reliable simulation of complex beams in radiotherapy after an accurate LINAC modeling is validated. A simple cross-calibration procedure proposed in this work allows obtaining absolute dose values even in complex fields

Impact of the accuracy of automatic tumour functional volume delineation on radiotherapy treatment planning.

International audienceOver the past few years several automatic and semi-automatic PET segmentation methods for target volume definition in radiotherapy have been proposed. The objective of this study is to compare different methods in terms of dosimetry. For such a comparison, a gold standard is needed. For this purpose, realistic GATE-simulated PET images were used. Three lung cases and three H&N cases were designed with various shapes, contrasts and heterogeneities. Four different segmentation approaches were compared: fixed and adaptive thresholds, a fuzzy C-mean and the fuzzy locally adaptive Bayesian method. For each of these target volumes, an IMRT treatment plan was defined. The different algorithms and resulting plans were compared in terms of segmentation errors and ground-truth volume coverage using different metrics (V(95), D(95), homogeneity index and conformity index). The major differences between the threshold-based methods and automatic methods occurred in the most heterogeneous cases. Within the two groups, the major differences occurred for low contrast cases. For homogeneous cases, equivalent ground-truth volume coverage was observed for all methods but for more heterogeneous cases, significantly lower coverage was observed for threshold-based methods. Our study demonstrates that significant dosimetry errors can be avoided by using more advanced image-segmentation methods