Allogeneic NK cells induce the <i>in vitro</i> activation of monocyte-derived and conventional type-2 dendritic cells and trigger an inflammatory response under cancer-associated conditions

Natural killer (NK) cells are innate lymphocytes capable to recognize and kill virus-infected and cancer cells. In the past years, the use of allogeneic NK cells as anti-cancer therapy gained interest due to their ability to induce graft-versus-cancer responses without causing graft-versus-host disease and multiple protocols have been developed to produce high numbers of activated NK cells. While the ability of these cells to mediate tumor kill has been extensively studied, less is known about their capacity to influence the activity of other immune cells that may contribute to a concerted anti-tumor response in the tumor microenvironment (TME). In this study, we analyzed how an allogeneic off-the-shelf cord blood stem cell-derived NK-cell product influenced the activation of dendritic cells (DC). Crosstalk between NK cells and healthy donor monocyte-derived DC (MoDC) resulted in the release of IFNγ and TNF, MoDC activation, and the release of the T-cell-recruiting chemokines CXCL9 and CXCL10. Moreover, in the presence of prostaglandin-E2, NK cell/MoDC crosstalk antagonized the detrimental effect of IL-10 on MoDC maturation leading to higher expression of multiple (co-)stimulatory markers. The NK cells also induced activation of conventional DC2 (cDC2) and CD8 + T cells, and the release of TNF, GM-CSF, and CXCL9/10 in peripheral blood mononuclear cells of patients with metastatic colorectal cancer. The activated phenotype of MoDC/cDC2 and the increased release of pro-inflammatory cytokines and T-cell-recruiting chemokines resulting from NK cell/DC crosstalk should contribute to a more inflamed TME and may thus enhance the efficacy of T-cell-based therapies.</p

Automated Intelligent Monitoring and the Controlling Software System for Solar Panels

The inspection of the solar panels on a periodic basis is important to improve longevity and ensure performance of the solar system. To get the most solar potential of the photovoltaic (PV) system is possible through an intelligent monitoring & controlling system. The monitoring & controlling system has rapidly increased its popularity because of its user-friendly graphical interface for data acquisition, monitoring, controlling and measurements. In order to monitor the performance of the system especially for renewable energy source application such as solar photovoltaic (PV), data-acquisition systems had been used to collect all the data regarding the installed system. In this paper the development of a smart automated monitoring & controlling system for the solar panel is described, the core idea is based on IoT (the Internet of Things). The measurements of data are made using sensors, block management data acquisition modules, and a software system. Then, all the real-time data collection of the electrical output parameters of the PV plant such as voltage, current and generated electricity is displayed and stored in the block management. The proposed system is smart enough to make suggestions if the panel is not working properly, to display errors, to remind about maintenance of the system through email or SMS, and to rotate panels according to a sun position using the Ephemeral table that stored in the system. The advantages of the system are the performance of the solar panel system which can be monitored and analyzed

Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures

Abstract Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with LinCD34+ CD38lowCD45RACD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC

Defining Early Human NK Cell Developmental Stages in Primary and Secondary Lymphoid Tissues

A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56brightCD16+/− and CD56dimCD16+ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body

Enhanced Collateral Growth by Double Transplantation of Gene-Nucleofected Fibroblasts in Ischemic Hindlimb of Rats

BACKGROUND: Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy. METHODS AND RESULTS: Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed. CONCLUSIONS: These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases

Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy

Natural killer cell immunotherapy: from bench to bedside. Natural born killers

Contains fulltext : MMUBN000001_750983515.pdf (publisher's version ) (Closed access)Radboud Universiteit Nijmegen, 15 januari 2013Promotores : Sweep, C.G.J., Witte, T.J.M. de Co-promotores : Dolstra, H., Schaap, N.P.M

Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells.

Contains fulltext : 49545.pdf (publisher's version ) (Open Access)The targeted manipulation of the genetic program of single cells as well as of complete organisms has strongly enhanced our understanding of cellular and developmental processes and should also help to increase our knowledge of primary human stem cells, e.g., hematopoietic stem cells (HSCs), within the next few years. An essential requirement for such genetic approaches is the existence of a reliable and efficient method to introduce genetic elements into living cells. Retro- and lentiviral techniques are efficient in transducing primary human HSCs, but remain labor and time consuming and require special safety conditions, which do not exist in many laboratories. In our study, we have optimized the nucleofection technology, a modified electroporation strategy, to introduce plasmid DNA into freshly isolated human HSC-enriched CD34(+) cells. Using enhanced green fluorescent protein (eGFP)-encoding plasmids, we obtained transfection efficiencies of approximately 80% and a mean survival rate of 50%. Performing functional assays using GFU-GEMM and long-term culture initiating cells (LTC-IC), we demonstrate that apart from a reduction in the survival rate the nucleofection method itself does not recognizably change the short- or long-term cell fate of primitive hematopoietic cells. Therefore, we conclude, the nucleofection method is a reliable and efficient method to manipulate primitive hematopoietic cells genetically