Oxygen tension modulates the effects of TNFα in compressed chondrocytes.

OBJECTIVE AND DESIGN: Oxygen tension and biomechanical signals are factors that regulate inflammatory mechanisms in chondrocytes. We examined whether low oxygen tension influenced the cells response to TNFα and dynamic compression. MATERIALS AND METHODS: Chondrocyte/agarose constructs were treated with varying concentrations of TNFα (0.1-100 ng/ml) and cultured at 5 and 21 % oxygen tension for 48 h. In separate experiments, constructs were subjected to dynamic compression (15 %) and treated with TNFα (10 ng/ml) and/or L-NIO (1 mM) at 5 and 21 % oxygen tension using an ex vivo bioreactor for 48 h. Markers for catabolic activity (NO, PGE2) and tissue remodelling (GAG, MMPs) were quantified by biochemical assay. ADAMTS-5 and MMP-13 expression were examined by real-time qPCR. 2-way ANOVA and a post hoc Bonferroni-corrected t test were used to analyse data. RESULTS: TNFα dose-dependently increased NO, PGE2 and MMP activity (all p < 0.001) and induced MMP-13 (p < 0.05) and ADAMTS-5 gene expression (pp < 0.01) with values greater at 5 % oxygen tension than 21 %. The induction of catabolic mediators by TNFα was reduced by dynamic compression and/or L-NIO (all p < 0.001), with a greater inhibition observed at 5% than 21 %. The stimulation of GAG synthesis by dynamic compression was greater at 21 % than 5 % oxygen tension and this response was reduced with TNFα or reversed with L-NIO. CONCLUSIONS: The present findings revealed that TNFα increased production of NO, PGE2 and MMP activity at 5 % oxygen tension. The effects induced by TNFα were reduced by dynamic compression and/or the NOS inhibitor, linking both types of stimuli to reparative activities. Future therapeutics should develop oxygen-sensitive antagonists which are directed to interfering with the TNFα-induced pathways.

Development of a two-stage gene selection method that incorporates a novel hybrid approach using the cuckoo optimization algorithm and harmony search for cancer classification.

For each cancer type, only a few genes are informative. Due to the so-called 'curse of dimensionality' problem, the gene selection task remains a challenge. To overcome this problem, we propose a two-stage gene selection method called MRMR-COA-HS. In the first stage, the minimum redundancy and maximum relevance (MRMR) feature selection is used to select a subset of relevant genes. The selected genes are then fed into a wrapper setup that combines a new algorithm, COA-HS, using the support vector machine as a classifier. The method was applied to four microarray datasets, and the performance was assessed by the leave one out cross-validation method. Comparative performance assessment of the proposed method with other evolutionary algorithms suggested that the proposed algorithm significantly outperforms other methods in selecting a fewer number of genes while maintaining the highest classification accuracy. The functions of the selected genes were further investigated, and it was confirmed that the selected genes are biologically relevant to each cancer type.

Chondrocyte dedifferentiation increases cell stiffness by strengthening membrane-actin adhesion.

OBJECTIVE: Chondrocyte dedifferentiation is known to influence cell mechanics leading to alterations in cell function. This study examined the influence of chondrocyte dedifferentiation in monolayer on cell viscoelastic properties and associated changes in actin organisation, bleb formation and membrane-actin cortex interaction. METHOD: Micropipette aspiration was used to estimate the viscoelastic properties of freshly isolated articular chondrocytes and the same cells after passage in monolayer. Studies quantified the cell membrane-actin cortex adhesion by measuring the critical pressure required for membrane detachment and bleb formation. We then examined the expression of ezrin, radixin and moesin (ERM) proteins which are involved in linking the membrane and actin cortex and combined this with theoretical modelling of bleb dynamics. RESULTS: Dedifferentiated chondrocytes at passage 1 (P1) were found to be stiffer compared to freshly isolated chondrocytes (P0), with equilibrium modulus values of 0.40 and 0.16 kPa respectively. The critical pressure increased from 0.59 kPa at P0 to 0.74 kPa at P1. Dedifferentiated cells at P1 exhibited increased cortical F-actin organisation and increased expression of total and phosphorylated ERM proteins compared to cells at P0. Theoretical modelling confirmed the importance of membrane-actin cortex adhesion in regulating bleb formation and effective cellular elastic modulus. CONCLUSION: This study demonstrates that chondrocyte dedifferentiation in monolayer strengthens membrane-actin cortex adhesion associated with increased F-actin organisation and up-regulation of ERM protein expression. Thus dedifferentiated cells have reduced susceptibility to bleb formation which increases cell modulus and may also regulate other fundamental aspects of cell function such as mechanotransduction and migration.

Membrane-bound interleukin-21 and CD137 ligand induce functional human natural killer cells from peripheral blood mononuclear cells through STAT-3 activation.

Natural killer (NK) cell adoptive transfer is a promising approach for cancer immunotherapy; however, its development has been hindered by the lack of efficient methods to produce large numbers of functional NK cells. In this study, we engineered the leukaemia cell line K562 to express CD137 ligand (CD137L) and membrane-bound interleukin (mbIL)-21 on the cell surface, and used these cells to expand NK cells from the peripheral blood mononuclear cells. We found that purity of the NK cells (CD3(-) CD56(+) /CD16(+) ) increased from less than 30% to above 95% after a 3-week expansion and proliferation of the cells was sustained for more than 8 weeks. The surface expression of NK cell activating and inhibitory receptors, except for NKp80, was clearly increased with the expansion, and NK cell-mediated killing activity was also enhanced significantly. However, these changes in both phenotype and function were clearly reversed by JSI-124, a specific signal transducer and activator of transcription-3 (STAT-3) inhibitor. Taken together, data showed that the combination of mbIL-21 and CD137L could efficiently induce the formation of functional human NK cells from peripheral blood mononuclear cells, and STAT-3 inhibition could impair this induction. Therefore, STAT-3 activation may benefit human NK cell proliferation and cytotoxicity, and provide valuable clinical applications in NK cell immunotherapy against viral infectious diseases and cancers.

Involvement of limbic structures in patients with isolated rapid eye movement sleep behavior disorder.

This study aimed to investigate the alterations in limbic structure volumes and limbic covariance network in patients with isolated rapid eye movement (REM) sleep behavior disorder (iRBD) and to compare them with healthy controls. We retrospectively enrolled 35 patients with iRBD and 35 healthy controls who underwent three-dimensional T1-weighted brain MRI. Volumetric analysis of subcortical limbic structures, including the hippocampus, amygdala, thalamus, mammillary body, hypothalamus, basal forebrain, septal nuclei, fornix, and nucleus accumbens, was performed. Furthermore, the limbic covariance network was examined using graph theory based on the limbic structure volumes. Some of the limbic structure volumes differed significantly. The right amygdala and hypothalamus volumes were lower in the patients with iRBD than in the healthy controls (0.101% vs. 0.114%, p = 0.016, and 0.027% vs. 0.030%, p = 0.045, respectively). However, there were no significant differences in the limbic covariance network between the groups. This study demonstrated that the volumes of the right amygdala and hypothalamus are lower in patients with iRBD, even without cognitive impairments, than in healthy controls. However, there were no significant differences in the limbic covariance network between the groups. The involvements of the limbic structures could be related to the conversion to neurodegenerative diseases in patients with iRBD.

Cell mechanics, structure, and function are regulated by the stiffness of the three-dimensional microenvironment.

This study adopts a combined computational and experimental approach to determine the mechanical, structural, and metabolic properties of isolated chondrocytes cultured within three-dimensional hydrogels. A series of linear elastic and hyperelastic finite-element models demonstrated that chondrocytes cultured for 24 h in gels for which the relaxation modulus is <5 kPa exhibit a cellular Young's modulus of ∼5 kPa. This is notably greater than that reported for isolated chondrocytes in suspension. The increase in cell modulus occurs over a 24-h period and is associated with an increase in the organization of the cortical actin cytoskeleton, which is known to regulate cell mechanics. However, there was a reduction in chromatin condensation, suggesting that changes in the nucleus mechanics may not be involved. Comparison of cells in 1% and 3% agarose showed that cells in the stiffer gels rapidly develop a higher Young's modulus of ∼20 kPa, sixfold greater than that observed in the softer gels. This was associated with higher levels of actin organization and chromatin condensation, but only after 24 h in culture. Further studies revealed that cells in stiffer gels synthesize less extracellular matrix over a 28-day culture period. Hence, this study demonstrates that the properties of the three-dimensional microenvironment regulate the mechanical, structural, and metabolic properties of living cells.

The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor.

Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on the measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen-presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with an SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells.

Anisotropic strain transfer through the aortic valve and its relevance to the cellular mechanical environment.

Aortic valve interstitial cells are responsible for maintaining the valve in response to their local mechanical environment. However, the complex organization of the extracellular matrix means cell strains cannot be directly derived from gross strains, and knowledge of tissue structure-function correlations is fundamental towards understanding mechanotransduction. This study investigates strain transfer through the valve, hypothesizing that organization of the valve matrix leads to non-homogenous local strains. Radial and circumferential samples were cut from aortic valve leaflets and subjected to quasi-static mechanical characterization. Further samples were imaged using confocal microscopy, to determine local strains in the matrix. Mechanical data demonstrated that the valve was significantly stronger and stiffer when loaded circumferentially, comparable with previous studies. Micromechanical studies demonstrated that strain transfer through the matrix is anisotropic and indirect, with local strains consistently smaller than applied strains in both orientations. Under radial loading, strains were transferred linearly to cells. However, under circumferential loading, strains were only one-third of applied values, with a less direct relationship between applied and local strains. This may result from matrix reorganization, and be important for preventing cellular damage during normal valve function. These findings should be taken into account when investigating interstitial cell behaviours, such as cell metabolism and mechanotransduction.

Monolayer expansion induces an oxidative metabolism and ROS in chondrocytes.

This study tests the hypothesis that articular chondrocytes shift from a characteristically glycolytic to an oxidative energy metabolism during population expansion in monolayer. Bovine articular chondrocytes were cultured in monolayer under standard incubator conditions for up to 14 days. Cellular proliferation, oxygen consumption, lactate production, protein content, ROS generation and mitochondrial morphology were examined. Lactate release increased approximately 5-fold within 1 week, but this was limited to approximately 2-fold increase when normalized to cellular protein content. By contrast, per cell oxidative phosphorylation increased 98-fold in 1 week. The increase in oxidative phosphorylation was evident within 24h, preceding cell proliferation and was associated with augmented reactive oxygen species generation. The autologous chondrocyte implantation procedure requires 14-21 days for population expansion. The alterations in metabolic phenotype we report within 7 days in vitro are thus pertinent to autologous chondrocyte implantation with significant implications for the chondrocyte functionality.

The effects of direct current stimulation on isolated chondrocytes seeded in 3D agarose constructs.

Endogenous electrical activity has been detected in articular cartilage. It has previously been suggested that the associated electrical currents and potentials are important to the mechanotransduction processes in cartilage. The present study investigates the effects of direct current on cell proliferation and matrix synthesis, using the well established 3D chondrocyte--agarose model system. Bovine chondrocytes isolated from metacarpalphalangeal joints were seeded in agarose constructs and exposed to a current density of 4 mA/cm2 for 6 h, a magnitude and period which was shown to maintain cell viability. The influence of the optimized electric stimulus was assessed by protein incorporation and mRNA measurements, using radiolabels and real-time QPCR, respectively. Results indicated no systematic influences of electrical current on protein synthesis, cell proliferation and mRNA expression levels. These data suggest that both the mode of stimulation and the model system are critical for the in vitro modulation of chondrocyte metabolism.

Dynamic compression influences interleukin-1beta-induced nitric oxide and prostaglandin E2 release by articular chondrocytes via alterations in iNOS and COX-2 expression.

Interleukin-1beta (IL-1beta) induces the release of nitric oxide (.NO) and prostaglandin E2 (PGE2) by chondrocytes and this effect can be reversed with the application of dynamic compression. Previous studies have indicated that integrins may play a role. In addition, IL-1beta upregulates the expression of iNOS and COX-2 mRNA via upstream activation of p38 MAPK. The current study examines the involvement of these pathways in mediating .NO and PGE2 release in IL-1beta stimulated bovine chondrocytes subjected to dynamic compression. Bovine chondrocytes were seeded in agarose constructs and cultured with 0 or 10 ng.ml(-1) IL-1beta with or without the application of 15% dynamic compressive strain at 1 Hz. Selected inhibitors were used to interrogate the role of alpha5beta1 integrin signalling and p38 MAPK activation in mediating the release of .NO and PGE2 in response to both IL-1beta and dynamic compression. The relative expression levels of iNOS and COX-2 were assessed using real-time quantitative PCR. Nitrite, a stable end product of .NO, was measured using the Griess assay and PGE2 release was measured using an enzyme immunoassay. IL-1beta enhanced .NO and PGE2 release and this effect was reversed by the application of dynamic compression. Co-incubation with an integrin binding peptide (GRGDSP) abolished the compression-induced effect. Real-time quantitative PCR analysis revealed that IL-1beta enhanced iNOS and COX-2 mRNA levels, with the maximum expression at 6 or 12 hours. Dynamic compression reduced this effect via a p38 MAPK sensitive pathway. These results suggest that dynamic compression acts to abrogate of .NO and PGE2 release by directly influencing the expression levels of iNOS and COX-2.

Mechanical loading modulates intracellular calcium signaling in human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a promising cell source for tissue-engineered connective tissue repair strategies. Additionally, increasing evidence confirms the role of the mechanical environment in maintaining tissue homeostasis, with calcium signaling implicated as a mediator in mechanotransduction pathways. Spontaneous intracellular calcium signaling was observed in a subset of MSCs embedded within 4% alginate hydrogel constructs, measured by a Ca2+ indicator fluo-4 in conjunction with confocal laser-scanning microscopy. By the use of pair-wise analysis, it was shown that distinct populations of MSCs up-regulated and down-regulated the frequency of calcium transients with the application of a 20% static uniaxial compressive strain of 20 min duration, delivered after a 20 min unstrained period. Calcium transients in control specimens were monitored throughout two unstrained 20 min periods. These values were statistically significant (p<0.05) by chi 2 test of independence. This dual-response indicator highlights the heterogeneous nature of MSC populations, which may have important implications for their successful use in cell therapies.

Appropriate Amount of Regular Exercise Is Associated with a Reduced Mortality Risk.

PURPOSE: This study aimed at investigating whether there is a continuous dose-response relationship between the amount of physical activity (PA) and longevity benefit. METHODS: We evaluated the records of 23,257,723 Koreans age ≥20 yr who had undergone one biennial medical evaluation by the National Health Insurance Corporation. Participants with ≥20 min of vigorous or ≥30 min of moderate PA or walking were stratified into four groups: 0 d·wk; 1-3 d·wk; 4 to 5 d·wk; and 6-7 d·wk. After calculating total metabolic equivalent task-hours per week (MET·h·wk), we created eight categories of MET-hours per week (0, 0.1-4.9, 5.0-9.9, 10.0-14.9, 15.0-19.9, 20.0-24.9, 25.0-29.9, and ≥30.0). Multivariate Cox proportional hazard analyses were performed. RESULTS: A reverse J-shaped risk curve was observed, with the lowest mortality risk in the participants exercising 4 to 5 d·wk (reference). Participants who did not exercise at all and those who exercised with a PA frequency of 1 to 3 d·wk or 6 to 7 d·wk showed a significantly increased mortality risk compared with the reference group. When we repeated the Cox analysis among the 8 MET·h·wk categories with the participants reporting 20.0 to 24.9 MET·h·wk of PA as the reference group, we found that those with physical inactivity and 25.0-29.9 or ≥30.0 MET·h·wk of PA showed a higher mortality risk than the reference group. These relationships were persistently observed after adjustment for confounders. CONCLUSIONS: An appropriate amount of regular exercise in each specific type of PA was associated with the lowest risk of mortality. The inactive participants showed an increased mortality risk, and daily PA did not show any additional benefit in the mortality risk.

Bioenergetic reprogramming of articular chondrocytes by exposure to exogenous and endogenous reactive oxygen species and its role in the anabolic response to low oxygen.

Monolayer culture is integral to many cell-based cartilage repair strategies, but chondrocytes lose regenerative potential with increasing duration in vitro. This coincides with elevated reactive oxygen species (ROS) levels and a bioenergetic transformation characterized by increasing mitochondrial function. This study investigates ROS as stimuli for bioenergetic reprogramming and the effect of antioxidants on the propensity of chondrocytes to regenerate a cartilaginous matrix. Articular chondrocytes were cultured in monolayer under a 2% O2 atmosphere. Oxidative stress was increased using 50 µm H2 O2 or a 20% O2 culture atmosphere, or decreased using the antioxidant N-acetyl-cysteine (NAC). Mitochondrial function was characterized using 200 nm Mitotracker green and an oxygen biosensor. After two population doublings ± NAC, chondrocytes were encapsulated in alginate beads (1 × 107 cells/ml) for an additional 10 days before DMB assay of glycosaminoglycan content. The beads were cultured under both 20% O2 and the more physiological 5% O2 condition. Chondrocytes expanded in 20% O2 exhibited elevated mitochondrial mass and functional capacity, which was partially mimicked by the exogenous ROS, H2 O2 . Oligomycin treatment revealed that the increased oxygen consumption was coupled to oxidative phosphorylation. NAC limited these markers of bioenergetic reprogramming during culture-expansion with no significant effect on subsequent GAG production under 20% O2 . However, NAC treatment in monolayer abolished the hypoxic induction of GAG in alginate beads. This supports the hypothesis of a causal relationship between exposure to ROS and acquired mitochondrial function in chondrocytes. Additionally, mitochondrial function may be required for the hypoxic induction of GAG synthesis by chondrocytes. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose.

In numerous cell types, the cytoskeleton has been widely implicated in mechanotransduction pathways involving stretch-activated ion channels, integrins and deformation of intracellular organelles. Studies have also demonstrated that the cytoskeleton can undergo remodelling in response to mechanical stimuli such as tensile strain or fluid flow. In articular chondrocytes, the mechanotransduction pathways are complex, inter-related and as yet, poorly understood. Furthermore, little is known of how the chondrocyte cytoskeleton responds to physiological mechanical loading. This study utilises the well-characterised chondrocyte-agarose model and an established confocal image-analysis technique to demonstrate that both static and cyclic, compressive strain and hydrostatic pressure all induce remodelling of actin microfilaments. This remodelling was characterised by a change from a uniform to a more punctate distribution of cortical actin around the cell periphery. For some loading regimes, this remodelling was reversed over a subsequent 1h unloaded period. This reversible remodelling of actin cytoskeleton may therefore represent a mechanism through which the chondrocyte alters its mechanical properties and mechanosensitivity in response to physiological mechanical loading.

Genetics of skin appendage neoplasms and related syndromes.

In the past decade the molecular basis of many inherited syndromes has been unravelled. This article reviews the clinical and genetic aspects of inherited syndromes that are characterised by skin appendage neoplasms, including Cowden syndrome, Birt-Hogg-Dube syndrome, naevoid basal cell carcinoma syndrome, generalised basaloid follicular hamartoma syndrome, Bazex syndrome, Brooke-Spiegler syndrome, familial cylindromatosis, multiple familial trichoepitheliomas, and Muir-Torre syndrome.

Integrin-mediated mechanotransduction in IL-1 beta stimulated chondrocytes.

Mechanical loading and interleukin-1 beta (IL-1 beta) influence the release of nitric oxide (*NO) and prostaglandin E2 (PGE2) from articular chondrocytes via distinct signalling mechanisms. The exact nature of the interplay between the respective signalling pathways remains unclear. Recent studies have shown that integrins act as mechanoreceptors and may transduce extracellular stimuli into intracellular signals, thereby influencing cellular response. The current study demonstrates that the application of dynamic compression induced an inhibition of *NO and an upregulation of cell proliferation and proteoglycan synthesis in the presence and absence of IL-1 beta. PGE2 release was not affected by dynamic compression in the absence of IL-1 beta but was inhibited in the presence of the cytokine. The integrin binding peptide, GRGDSP, abolished or reversed the compression-induced alterations in all four parameters assessed in the presence and absence of IL-1 beta. The non-binding control peptide, GRADSP, had no effect. These data clearly demonstrate that the metabolic response of the chondrocytes to dynamic compression in the presence and absence of IL-1 beta, are integrin mediated.

Chondrocyte deformation induces mitochondrial distortion and heterogeneous intracellular strain fields.

Chondrocyte mechanotransduction is poorly understood but may involve cell deformation and associated distortion of intracellular structures and organelles. This study quantifies the intracellular displacement and strain fields associated with chondrocyte deformation and in particular the distortion of the mitochondria network, which may have a role in mechanotransduction. Isolated articular chondrocytes were compressed in agarose constructs and simultaneously visualised using confocal microscopy. An optimised digital image correlation technique was developed to calculate the local intracellular displacement and strain fields using confocal images of fluorescently labelled mitochondria. The mitochondria formed a dynamic fibrous network or reticulum, which co-localised with microtubules and vimentin intermediate filaments. Cell deformation induced distortion of the mitochondria, which collapsed in the axis of compression with a resulting loss of volume. Compression generated heterogeneous intracellular strain fields indicating mechanical heterogeneity within the cytoplasm. The study provides evidence supporting the potential involvement of mitochondrial deformation in chondrocyte mechanotransduction, possibly involving strain-mediated release of reactive oxygen species. Furthermore the heterogeneous strain fields, which appear to be influenced by intracellular structure and organisation, may generate significant heterogeneity in mechanotransduction behaviour for cells subjected to identical levels of deformation.

Concentration and M/G ratio influence the physiochemical and mechanical properties of alginate constructs for tissue engineering.

The diffusion and mechanical properties of calcium alginate gels were determined using constructs of different alginate concentrations and guluronic acid contents. It was found that the diffusion of small molecules such as sulphate, glucose and thymidine was not impeded by any of the alginates tested at concentrations of 1% and 3% (w/v). By contrast, the diffusion of large molecules, including insulin like growth factor-1 (IGF-1), human growth hormone and bovine serum albumin was impeded by alginate. This effect was enhanced with increasing alginate concentration, but was less evident for alginates with increased guluronic acid content. These findings have significant implications in tissue engineering where cells such as chondrocytes depend on the supply of factors such as IGF-1 to remain viable. An increase in both alginate concentration and guluronic acid content also increased the compressive properties, as determined by both tangent and equilibrium modulus, of alginate constructs. Although the 3% alginate constructs exhibited enhanced stiffness compared to some reported cartilage substitute biomaterials, such as PGA, their absolute values were still appreciably less stiff than articular cartilage.

Cellular engineering and therapy in combination with cord blood allografting in pediatric recipients.

Cord blood (CB) transplantation is an alternate source of human hematopoietic progenitor cells for allogeneic stem cell transplantation in children and adolescents with both malignant and nonmalignant diseases. Current limitations included delay in hematopoietic reconstitution, increased incidence of primary graft failure and slow cellular immunoreconstitution. These limitations lead to a significant increase in primary graft failure, infectious complications and increased transplant-related mortality. There is a number of experimental approaches currently under investigation including cellular engineering to circumvent these limitations. In this review, we summarize the recent findings of utilizing ex vivo CB expansion with Notch1 ligand Delta 1, mesenchymal progenitor cells, the use of human placenta-derived stem cells and CB-derived natural killer cells. Early and preliminary results suggest some of these experimental cellular strategies may in part ameliorate the incidence of primary graft failure, delays in hematopoietic reconstitution and/or slowness in cellular immune reconstitution following unrelated CB transplantation.