169 research outputs found
In vitro models to study natural killer cell dynamics in the tumor microenvironment
Immunotherapy is revolutionizing cancer therapy. The rapid development of new immunotherapeutic strategies to treat solid tumors is posing new challenges for preclinical research, demanding novel in vitro methods to test treatments. Such methods should meet specific requirements, such as enabling the evaluation of immune cell responses like cytotoxicity or cytokine release, and infiltration into the tumor microenvironment using cancer models representative of the original disease. They should allow high-throughput and high-content analysis, to evaluate the efficacy of treatments and understand immune-evasion processes to facilitate development of new therapeutic targets. Ideally, they should be suitable for personalized immunotherapy testing, providing information for patient stratification. Consequently, the application of in vitro 3-dimensional (3D) cell culture models, such as tumor spheroids and organoids, is rapidly expanding in the immunotherapeutic field, coupled with the development of novel imaging-based techniques and -omic analysis. In this paper, we review the recent advances in the development of in vitro 3D platforms applied to natural killer (NK) cell-based cancer immunotherapy studies, highlighting the benefits and limitations of the current methods, and discuss new concepts and future directions of the field
The art of cellular communication: tunneling nanotubes bridge the divide
The ability of cells to receive, process, and respond to information is essential for a variety of biological processes. This is true for the simplest single cell entity as it is for the highly specialized cells of multicellular organisms. In the latter, most cells do not exist as independent units, but are organized into specialized tissues. Within these functional assemblies, cells communicate with each other in different ways to coordinate physiological processes. Recently, a new type of cell-to-cell communication was discovered, based on de novo formation of membranous nanotubes between cells. These F-actin-rich structures, referred to as tunneling nanotubes (TNT), were shown to mediate membrane continuity between connected cells and facilitate the intercellular transport of various cellular components. The subsequent identification of TNT-like structures in numerous cell types revealed some structural diversity. At the same time it emerged that the direct transfer of cargo between cells is a common functional property, suggesting a general role of TNT-like structures in selective, long-range cell-to-cell communication. Due to the growing number of documented thin and long cell protrusions in tissue implicated in cell-to-cell signaling, it is intriguing to speculate that TNT-like structures also exist in vivo and participate in important physiological processes
Multi-Level Communication of Human Retinal Pigment Epithelial Cells via Tunneling Nanotubes
Background: Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. Methodology and Principal Findings: Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca2+ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. Conclusions and Significance: Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic
Individualization of Hematopoietic Stem Cell Transplantation Using Alpha/Beta T-Cell Depletion
Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with several potentially lethal complications. Higher levels of CD3+ T-cells in the graft have been associated with increased risk of graft-versus-host disease (GVHD), but also beneficial graft-versus-leukemia effect and reduced infections. To tackle post-transplant complications, donor lymphocyte infusions have been used but with an increased risk of GVHD. To reduce this risk, we performed depletion of αβ T-cells and treated 12 patients post-HSCT suffering from infections and/or poor immune reconstitution. The αβ T-cell depleted cell products were characterized by flow cytometry. The median log depletion of αβ T-cells was −4.3 and the median yield of γδ T-cells was 73.5%. The median CD34+ cell dose was 4.4 × 106/kg. All 12 patients were alive 3 months after infusion and after 1 year, two patients had died. No infusion-related side effects were reported and no severe acute GVHD (grade III-IV) developed in any patient post-infusion. Overall, 3 months after infusion 11 out of 12 patients had increased levels of platelets and/or granulocytes. In conclusion, we describe the use of αβ T-cell depleted products as stem cell boosters with encouraging results
Corneal Alternations Induced by Topical Application of Benzalkonium Chloride in Rabbit
Benzalkonium chloride (BAC) is the most common preservative in ophthalmic preparations. Here, we investigated the corneal alternations in rabbits following exposure to BAC. Twenty-four adult male New Zealand albino rabbits were randomly divided into three groups. BAC at 0.01%, 0.05%, or 0.1% was applied twice daily to one eye each of rabbits for 4 days. The contralateral untreated eyes were used as control. Aqueous tear production and fluorescein staining scores of BAC-treated eyes were compared with those of controls. The structure of the central cornea was examined by in vivo confocal microscopy. Expression of mucin-5 subtype AC (MUC5AC) in conjunctiva was detected by immunostainig on cryosections. Corneal barrier function was assessed in terms of permeability to carboxy fluorescein (CF). The distribution and expression of ZO-1, a known marker of tight junction, and reorganization of the perijunctional actomyosin ring (PAMR) were examined by immunofluorescence analysis. Although there were no significant differences between control and BAC-treated eyes in Schirmer scores, corneal fluorescein scores and the number of conjunctival MUC5AC staining cells, in vivo confocal microscopy revealed significant epithelial and stromal defects in all BAC-treated corneas. Moreover, BAC at 0.1% resulted in significant increases in central corneal thickness and endothelial CF permeability, compared with those in control eyes, and endothelial cell damage with dislocation of ZO-1 and disruption of PAMR. Topical application of BAC can quickly impair the whole cornea without occurrence of dry eye. A high concentration of BAC breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR and remodeling of apical junctional complex in vivo
Effects of chirality on the intracellular localization of binuclear ruthenium(II) polypyridyl complexes
Interest in binuclear ruthenium(II) polypyridyl complexes as luminescent cellular imaging agents and for biomedical applications is increasing rapidly. We have investigated the cellular localization, uptake, and biomolecular interactions of the pure enantiomers of two structural isomers of [μ-bipb(phen)4Ru2]4+ (bipb is bis(imidazo[4,5-f]-1,10-phenanthrolin-2-yl)benzene and phen is 1,10-phenanthroline) using confocal laser scanning microscopy, emission spectroscopy, and linear dichroism. Both complexes display distinct enantiomeric differences in the staining pattern of fixed cells, which are concluded to arise from chiral discrimination in the binding to intracellular components. Uptake of complexes in live cells is efficient and nontoxic at 5 μM, and occurs through an energy-dependent mechanism. No differences in uptake are observed between the structural isomers or the enantiomers, suggesting that the interactions triggering uptake are rather insensitive to structural variations. Altogether, these findings show that the complexes investigated are promising for future applications as cellular imaging probes. In addition, linear dichroism shows that the complexes exhibit DNA-condensing properties, making them interesting as potential gene delivery vectors
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Photochemically active DNA-intercalating ruthenium and related complexes – insights by combining crystallography and transient spectroscopy
Recent research on the study of the interaction of ruthenium polypyridyl compounds and defined sequence nucleic acids is reviewed. Particular emphasis is paid to complexes [Ru(LL)2(Int)]2+ containing potentially intercalating ligands (Int) such as dipyridophenazine (dppz), which are known to display light-switching or photo-oxidising behaviour, depending on the nature of the ancillary ligands. X-ray crystallography has made a key contribution to our understanding, and the first complete survey of structural results is presented. These include sequence, enantiomeric, substituent and structural specificities. The use of ultrafast transient spectrocopic methods to probe the ultrafast processes for complexes such as [Ru(TAP)2(dppz)]2+ and [Ru(phen)2(dppz)]2+ when bound to mixed sequence oligonucleotides are reviewed with particular attention being paid to the complementary advantages of transient (visible) absorption and time-resolved (mid) infra-red techniques to probe spectral changes in the metal complex and in the nucleic acid. The observed photophysical properties are considered in light of the structural information obtained from X-ray crystallography. In solution, metal complexes can be expected to bind at more than one DNA step, so that a perfect correlation of the photophysical properties and factors such as the orientation or penetration of the ligand into the intercalation pocket should not be expected. This difficulty can be obviated by carrying out TRIR studies in the crystals. Dppz complexes also undergo insertion, especially with mismatched sequences. Future areas for study such as those involving non-canonical forms of DNA, such as G-quadruplexes or i-motifs are also briefly considered
Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays
New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level
EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF); Scientific Opinion on Flavouring Group Evaluation 10, Revision 2 (FGE.10Rev2): Aliphatic primary and secondary saturated and unsaturated alcohols, aldehydes, acetals, carboxylic acids and esters containing an additional oxygenated functional group and lactones from chemical groups 9, 13 and 30
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