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1.  Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity 
ACS Nano  2014;8(3):2252-2260.
Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy.
PMCID: PMC4004316  PMID: 24564881
magnetic nanoparticles; cancer immunotherapy; T cell; adoptive immunotherapy; receptor clustering; membrane organization
2.  Nanoscale Artificial Antigen Presenting Cells for T Cell Immunotherapy 
Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50–100 nm in diameter) and avidin-coated quantum dot nanocrystals, (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo.
PMCID: PMC4114774  PMID: 23891987
Nanoparticle; Artificial Antigen Presenting Cell; Immunotherapy; T cell
3.  Work–family conflict, emotional exhaustion and performance-based self-esteem: reciprocal relationships 
The three constructs of work–family conflict, emotional exhaustion and performance-based self-esteem are all related to tremendous negative consequences for the individual, the organization as well as for society. Even though there are studies that connect two of those constructs, the prospective relations between all three of them have not been studied yet.
We explored the prospective relations between the three constructs in a large Swedish data set representative of the Swedish workforce. Gender differences in the relations were investigated. Longitudinal data with a 2-year time lag were gathered from 3,387 working men and women who responded to the 2006 and 2008 waves of the Swedish Longitudinal Occupational Survey of Health. Four different cross-lagged models were analysed.
In the best fitting model, higher levels of work–family conflict at time 1 were associated with an increased level of performance-based self-esteem at time 2, but not with emotional exhaustion, after controlling for having children, gender, education and age. Also, relationships between emotional exhaustion at time 1 and work–family conflict and performance-based self-esteem at time 2 could be established. Furthermore, relationships between performance-based self-esteem time 1 and work–family conflict and emotional exhaustion time 2 were found. Multiple-group analysis did not show any differences in the relations of the tested constructs over time for either men or women.
We conclude that the three constructs are interrelated and best understood through a reciprocal model. No differences were found between men and women.
PMCID: PMC4286621  PMID: 24664455
Emotional exhaustion; Performance-based self-esteem; Self-esteem; Structural equation modelling; Work–family conflict
4.  Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set 
BMC Biotechnology  2013;13:43.
Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency.
109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring.
Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.
PMCID: PMC3691671  PMID: 23688045
Transgenic pig; Knockout pig; Somatic cell nuclear transfer; Multi-factorial analysis
5.  Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig 
BMC Biotechnology  2012;12:84.
Somatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs) as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination.
PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs) and porcine ear fibroblasts (PEFs) could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT.
The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.
PMCID: PMC3537537  PMID: 23140586
Pig; Primary kidney cells; Fibroblasts; Nuclear transfer; Genetic engineering
6.  Detection and Isolation of Viable Mouse IL-17-Secreting T Cells 
The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37°C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37°C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.
PMCID: PMC2865976  PMID: 19229172
7.  Evolution of surface-based deformable image registration for adaptive radiotherapy of non-small cell lung cancer (NSCLC) 
To evaluate the performance of surface-based deformable image registration (DR) for adaptive radiotherapy of non-small cell lung cancer (NSCLC).
Based on 13 patients with locally advanced NSCLC, CT images acquired at treatment planning, midway and the end of the radio- (n = 1) or radiochemotherapy (n = 12) course were used for evaluation of DR. All CT images were manually [gross tumor volume (GTV)] and automatically [organs-at-risk (OAR) lung, spinal cord, vertebral spine, trachea, aorta, outline] segmented. Contours were transformed into 3D meshes using the Pinnacle treatment planning system and corresponding mesh points defined control points for DR with interpolation within the structures. Using these deformation maps, follow-up CT images were transformed into the planning images and compared with the original planning CT images.
A progressive tumor shrinkage was observed with median GTV volumes of 170 cm3 (range 42 cm3 - 353 cm3), 124 cm3 (19 cm3 - 325 cm3) and 100 cm3 (10 cm3 - 270 cm3) at treatment planning, mid-way and at the end of treatment. Without DR, correlation coefficients (CC) were 0.76 ± 0.11 and 0.74 ± 0.10 for comparison of the planning CT and the CT images acquired mid-way and at the end of treatment, respectively; DR significantly improved the CC to 0.88 ± 0.03 and 0.86 ± 0.05 (p = 0.001), respectively. With manual landmark registration as reference, DR reduced uncertainties on the GTV surface from 11.8 mm ± 5.1 mm to 2.9 mm ± 1.2 mm. Regarding the carina and intrapulmonary vessel bifurcations, DR reduced uncertainties by about 40% with residual errors of 4 mm to 6 mm on average. Severe deformation artefacts were observed in patients with resolving atelectasis and pleural effusion, in one patient, where the tumor was located around large bronchi and separate segmentation of the GTV and OARs was not possible, and in one patient, where no clear shrinkage but more a decay of the tumor was observed.
The surface-based DR performed accurately for the majority of the patients with locally advanced NSCLC. However, morphological response patterns were identified, where results of the surface-based DR are uncertain.
PMCID: PMC2804595  PMID: 20025753
8.  Investigation of the usability of conebeam CT data sets for dose calculation 
To investigate the feasibility and accuracy of dose calculation in cone beam CT (CBCT) data sets.
Kilovoltage CBCT images were acquired with the Elekta XVI system, CT studies generated with a conventional multi-slice CT scanner (Siemens Somatom Sensation Open) served as reference images. Material specific volumes of interest (VOI) were defined for commercial CT Phantoms (CATPhan® and Gammex RMI®) and CT values were evaluated in CT and CBCT images. For CBCT imaging, the influence of image acquisition parameters such as tube voltage, with or without filter (F1 or F0) and collimation on the CT values was investigated. CBCT images of 33 patients (pelvis n = 11, thorax n = 11, head n = 11) were compared with corresponding planning CT studies. Dose distributions for three different treatment plans were calculated in CT and CBCT images and differences were evaluated. Four different correction strategies to match CT values (HU) and density (D) in CBCT images were analysed: standard CT HU-D table without adjustment for CBCT; phantom based HU-D tables; patient group based HU-D tables (pelvis, thorax, head); and patient specific HU-D tables.
CT values in the CBCT images of the CATPhan® were highly variable depending on the image acquisition parameters: a mean difference of 564 HU ± 377 HU was calculated between CT values determined from the planning CT and CBCT images. Hence, two protocols were selected for CBCT imaging in the further part of the study and HU-D tables were always specific for these protocols (pelvis and thorax with M20F1 filter, 120 kV; head S10F0 no filter, 100 kV). For dose calculation in real patient CBCT images, the largest differences between CT and CBCT were observed for the standard CT HU-D table: differences were 8.0% ± 5.7%, 10.9% ± 6.8% and 14.5% ± 10.4% respectively for pelvis, thorax and head patients using clinical treatment plans. The use of patient and group based HU-D tables resulted in small dose differences between planning CT and CBCT: 0.9% ± 0.9%, 1.8% ± 1.6%, 1.5% ± 2.5% for pelvis, thorax and head patients, respectively. The application of the phantom based HU-D table was acceptable for the head patients but larger deviations were determined for the pelvis and thorax patient populations.
The generation of three HU-D tables specific for the anatomical regions pelvis, thorax and head and specific for the corresponding CBCT image acquisition parameters resulted in accurate dose calculation in CBCT images. Once these HU-D tables are created, direct dose calculation on CBCT datasets is possible without the need of a reference CT images for pixel value calibration.
PMCID: PMC2648965  PMID: 19087250
9.  Influence of increased target dose inhomogeneity on margins for breathing motion compensation in conformal stereotactic body radiotherapy 
Breathing motion should be considered for stereotactic body radiotherapy (SBRT) of lung tumors. Four-dimensional computer tomography (4D-CT) offers detailed information of tumor motion. The aim of this work is to evaluate the influence of inhomogeneous dose distributions in the presence of breathing induced target motion and to calculate margins for motion compensation.
Based on 4D-CT examinations, the probability density function of pulmonary tumors was generated for ten patients. The time-accumulated dose to the tumor was calculated using one-dimensional (1D) convolution simulations of a 'static' dose distribution and target probability density function (PDF). In analogy to stereotactic body radiotherapy (SBRT), different degrees of dose inhomogeneity were allowed in the target volume: minimum doses of 100% were prescribed to the edge of the target and maximum doses varied between 102% (P102) and 150% (P150). The dose loss due to breathing motion was quantified and margins were added until this loss was completely compensated.
With the time-weighted mean tumor position as the isocentre, a close correlation with a quadratic relationship between the standard deviation of the PDF and the margin size was observed. Increased dose inhomogeneity in the target volume required smaller margins for motion compensation: margins of 2.5 mm, 2.4 mm and 1.3 mm were sufficient for compensation of 11.5 mm motion range and standard deviation of 3.9 mm in P105, P125 and P150, respectively. This effect of smaller margins for increased dose inhomogeneity was observed for all patients. Optimal sparing of the organ-at-risk surrounding the target was achieved for dose prescriptions P105 to P118. The internal target volume concept over-compensated breathing motion with higher than planned doses to the target and increased doses to the surrounding normal tissue.
Treatment planning with inhomogeneous dose distributions in the target volume required smaller margins for compensation of breathing induced target motion with the consequence of lower doses to the surrounding organs-at-risk.
PMCID: PMC2637830  PMID: 19055768
10.  Does Intensity Modulated Radiation Therapy (IMRT) prevent additional toxicity of treating the pelvic lymph nodes compared to treatment of the prostate only? 
To evaluate the risk of rectal, bladder and small bowel toxicity in intensity modulated radiation therapy (IMRT) of the prostate only compared to additional irradiation of the pelvic lymphatic region.
For ten patients with localized prostate cancer, IMRT plans with a simultaneous integrated boost (SIB) were generated for treatment of the prostate only (plan-PO) and for additional treatment of the pelvic lymph nodes (plan-WP). In plan-PO, doses of 60 Gy and 74 Gy (33 fractions) were prescribed to the seminal vesicles and to the prostate, respectively. Three plans-WP were generated with prescription doses of 46 Gy, 50.4 Gy and 54 Gy to the pelvic target volume; doses to the prostate and seminal vesicles were identical to plan-PO. The risk of rectal, bladder and small bowel toxicity was estimated based on NTCP calculations.
Doses to the prostate were not significantly different between plan-PO and plan-WP and doses to the pelvic lymph nodes were as planned. Plan-WP resulted in increased doses to the rectum in the low-dose region ≤ 30 Gy, only, no difference was observed in the mid and high-dose region. Normal tissue complication probability (NTCP) for late rectal toxicity ranged between 5% and 8% with no significant difference between plan-PO and plan-WP. NTCP for late bladder toxicity was less than 1% for both plan-PO and plan-WP. The risk of small bowel toxicity was moderately increased for plan-WP.
This retrospective planning study predicted similar risks of rectal, bladder and small bowel toxicity for IMRT treatment of the prostate only and for additional treatment of the pelvic lymph nodes.
PMCID: PMC2253547  PMID: 18190681
11.  Instruction for Cytokine Expression in T Helper Lymphocytes in Relation to Proliferation and Cell Cycle Progression 
The Journal of Experimental Medicine  1999;190(10):1439-1450.
T helper (Th) lymphocytes, when reactivated, recall expression of those cytokines they had been instructed to express in earlier activations, even in the absence of specific cytokine-inducing factors. In cells that memorize their expression, the cytokine genes are modified by chromatin rearrangement and demethylation, suggesting that they have been somatically imprinted. Here we show, by using inhibitors blocking the cell cycle in various stages, that for the instruction of a Th cell to express interleukin (IL)-4 or IL-10 upon restimulation, entry of the cell into the S phase of the first cell cycle after initial activation is required. Separation of the IL-4 receptor (IL-4R) and T cell antigen receptor (TCR) signals in time, demonstrates that this instruction is dependent on concomitant signaling from both receptors. In Th cells, inhibited to progress into the first S phase after activation, the IL-4R and TCR signals can be memorized for at least 1 d, priming the T cell to become instructed for expression of IL-4 upon restimulation, when entering the S phase after release of the cell cycle block. The requirement of the initial S phase of T cell activation, for instruction of Th cells to express IL-4 or IL-10 upon restimulation points to the decisive role of epigenetic modification of cytokine genes as a molecular correlate of the memory to express particular cytokines.
PMCID: PMC2195692  PMID: 10562319
cytokine memory; T cell differentiation; interleukin 4; interleukin 10; CFSE

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