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Sample records for ensheathing cell conditioned

  1. Effects of different sera conditions on olfactory ensheathing cells in vitro.

    PubMed

    Lu, Meng; Dong, Jun; Lu, Teng; Lv, Hongjun; Yang, Pinglin; Cheng, Zhijian; Li, Jin; Liang, Baobao; Xu, Junkui; Li, Haopeng; He, Xijing

    2015-01-01

    Transplantation of olfactory ensheathing cells (OEC) is a promising therapy in spinal cord injury (SCI) treatment. However, the therapeutic efficacy of this method is unstable due to unknown reasons. Considering the alterations in the culture environment that occur during OEC preparation for transplantation, we hypothesize that these changes may cause variations in the curative effects of this method. In this study, we compared OEC cultured in medium containing different types and concentrations of serum. After purification and passage, the OEC were cultured for 7 days in different media containing 5%, 10%, 15% or 20% fetal bovine serum (FBS) or rat serum (RS), or the cells were cultured in FBS-containing medium first, followed by medium containing RS. In another group, the OEC were first cultured in 10% FBS for 3 days and then cultured with rat spinal cord explants with 10% RS for another 4 days. An MTT assay and P75 neurotrophin receptor immunofluorescence staining were used to examine cell viability and OEC numbers, respectively. The concentration of neurotrophin-3 (NT-3), which is secreted by OEC into the culture supernatant, was detected using the enzyme-linked immunosorbent assay (ELISA). RT-PCR was applied to investigate the NT-3 gene expression in OEC according to different groups. Compared with FBS, RS reduced OEC proliferation in relation to OEC counts (χ2 = 166.279, df = 1, p < 0.01), the optical density (OD) value in the MTT assay (χ2 = 34.730, df = 1, p < 0.01), and NT-3 concentration in the supernatant (χ2 = 242.997, df = 1, p < 0.01). OEC cultured with spinal cord explants secreted less NT-3 than OEC cultured alone (F = 9.611, df = 5.139, p < 0.01). Meanwhile, the order of application of different sera was not influential. There was statistically significant difference in NT-3 gene expression among different groups when the serum concentration was 15% (χ2 = 64.347, df = 1, p < 0.01). In conclusion, different serum conditions may be responsible

  2. The mitochondrial protective mechanism of olfactory ensheathing cells conditioned medium protects against H2O2-induced injury in astrocytes.

    PubMed

    Liu, Jinbo; Qiu, Jing; Xiong, Yuyun; Liu, Zhiyuan; Gao, Jing

    2013-10-25

    Our previous studies showed that olfactory ensheathing cells conditioned medium (OECCM), has a positive effect against apoptosis in ASTs and prevents the morphology changes in the, mitochondria, but the accurate mechanism is still unknown. In this study, we examined the mitochondrial mechanism of OECCM which may protect against H2O2-induced injury in ASTs. It was, found that OECCM could protect ASTs from the injury of 500μmol/L H2O2, decrease the intracellular, ROS and Ca(2+) level, as well inhibit apoptosis and the expression of cleaved caspase-3. Further, investigations showed that OECCM could increase both the mitochondrial membrane potential and the, ATP level, as well enhance the cell respiratory function. In summary, OECCM could protect ASTs from, damages induced by H2O2. Its mechanism may be related to the decrease of ROS generation and the, overloading of Ca(2+), then stabilization of the mitochondrial function. PMID:24036462

  3. Voltage-dependent K+ currents contribute to heterogeneity of olfactory ensheathing cells

    PubMed Central

    Rela, Lorena; Piantanida, Ana Paula; Bordey, Angelique; Greer, Charles A.

    2015-01-01

    The olfactory nerve is permissive for axon growth throughout life. This has been attributed in part to the olfactory ensheathing glial cells that encompass the olfactory sensory neuron fascicles. Olfactory ensheathing cells also promote axon growth in vitro and when transplanted in vivo to sites of injury. The mechanisms involved remain largely unidentified owing in part to the limited knowledge of the physiological properties of ensheathing cells. Glial cells rely for many functions on the properties of the potassium channels expressed; however, those expressed in ensheathing cells are unknown. Here we show that olfactory ensheathing cells express voltage-dependent potassium currents compatible with inward rectifier (Kir) and delayed rectifier (KDR) channels. Together with gap junction coupling, these contribute to the heterogeneity of membrane properties observed in olfactory ensheathing cells. The relevance of K+ currents expressed by ensheathing cells is discussed in relation to plasticity of the olfactory nerve. PMID:25856239

  4. Femtosecond laser surgery of olfactory ensheathing cells protuberance

    NASA Astrophysics Data System (ADS)

    Yang, H. F.; Zhou, M.; di, J. K.; Zhao, E. L.; Gong, A. H.

    2009-03-01

    Protuberance cutting and recoverable damage of olfactory ensheathing cells (OECs) using femto-second laser surgery are reported. The damages of OECs protuberance are induced by using femtosecond laser surgery with different laser powers and scanning velocities. Through optimizing the parameters, steady cutting of OEC protuberance is achieved. After femtosecond laser surgery with 150 μ W and 10 μm/s is conducted, we found that the cell recovers its viability, such as growth, spread, and motility 1.5 h after the damage, which prove cell’s recovery and regeneration. Current work offers a novel method for establishing cell damage model and studying on cytokinetics.

  5. Short Report: Olfactory Ensheathing Cells Promote Differentiation of Neural Stem Cells and Robust Neurite Extension

    PubMed Central

    Sethi, Rosh; Sethi, Roshan; Redmond, Andy

    2014-01-01

    Aims The goal of this study was to gain insight into the signaling between olfactory ensheathing cells (OECs) and neural stem cells (NSCs). We sought to understand the impact of OECs on NSC differentiation and neurite extension and to begin to elucidate the factors involved in these interactions to provide new targets for therapeutic interventions. Materials and Methods We utilized lines of OECs that have been extremely well characterized in vitro and in vivo along with well studied NSCs in gels to determine the impact of the coculture in three dimensions. To further elucidate the signaling, we used conditioned media from the OECs as well as fractioned components on NSCs to determine the molecular weight range of the soluble factors that was most responsible for the NSC behavior. Results We found that the coculture of NSCs and OECs led to robust NSC differentiation and extremely long neural processes not usually seen with NSCs in three dimensional gels in vitro. Through culture of NSCs with fractioned OEC media, we determined that molecules larger than 30 kDa have the greatest impact on the NSC behavior. Conclusions Overall, our findings suggest that cocultures of NSCs and OECs may be a novel combination therapy for neural injuries including spinal cord injury (SCI). Furthermore, we have identified a class of molecules which plays a substantial role in the behavior that provides new targets for investigating pharmacological therapies. PMID:24996386

  6. Evidence for a Notch1-mediated transition during olfactory ensheathing cell development.

    PubMed

    Miller, Sophie R; Perera, Surangi N; Benito, Cristina; Stott, Simon R W; Baker, Clare V H

    2016-09-01

    Olfactory ensheathing cells (OECs) are a unique glial population found in both the peripheral and central nervous system: they ensheath bundles of unmyelinated olfactory axons from their peripheral origin in the olfactory epithelium to their central synaptic targets in the glomerular layer of the olfactory bulb. Like all other peripheral glia (Schwann cells, satellite glia, enteric glia), OECs are derived from the embryonic neural crest. However, in contrast to Schwann cells, whose development has been extensively characterised, relatively little is known about their normal development in vivo. In the Schwann cell lineage, the transition from multipotent Schwann cell precursor to immature Schwann cell is promoted by canonical Notch signalling. Here, in situ hybridisation and immunohistochemistry data from chicken, mouse and human embryos are presented that suggest a canonical Notch-mediated transition also occurs during OEC development. PMID:27271278

  7. The influence of ensheathing cells on olfactory receptor cell neurite outgrowth in vitro.

    PubMed

    Kafitz, K W; Greer, C A

    1998-11-30

    We previously reported that laminin substrates increased primary (1 degree) neurite outgrowth from olfactory receptor cells (ORCs) in vitro. To further explore mechanisms underlying the outgrowth of ORC neurites, we have cocultured ORCs with the ensheathing cells (ENSH) from the olfactory nerve. ORCs were plated either: (i) directly on monolayers of ENSH (prepared with minor modifications as reported by Doucette and Devon, or (ii) on coverslips suspended above the ENSH monolayer to investigate diffusible trophic influences of ENSH. In addition, ORCs were cocultured with either olfactory bulb glia (OBG) or hippocampal astrocytes (HG) or grown on either laminin (LN) substrates or poly-L-lysine (PLL) controls. The length of ORC neurites was determined after 48 hr in vitro. Immunocytochemical characterization of the ENSH cultures for p75 nerve growth factor (NGF) receptor and glial fibrillary acidic protein (GFAP) revealed that those cultures contained more than 80% ENSH. In OBG cultures approximately 10% and in HG cultures no cells with ENSH characteristics were found. All cells with ENSH characteristics were also LN-immunoreactive. After 48 hr in culture ORCs had the longest 1 degree neurites when they were cocultured with ENSH. No significant differences in the 1 degree neurite length were found comparing ORCs grown directly on ENSH and ORCs physically separated from ENSH. ORCs cultured on HG and on EHS-LN showed no significant differences in the ORC 1 degree neurite length, but on both substrates the ORC 1 degree neurites were significantly shorter than on ENSH. The length of the ORC secondary neurites did not vary significantly in the different culture conditions. Our results suggest that while LN appears to contribute to ORC neurite extension, additional diffusible factors released from ENSH are likely to be further determinants of neurite outgrowth. Because the OBG and HG cocultures did not influence ORC neurite outgrowth as significantly as did the ENSH, it

  8. An electromagnetic cell-stretching device for mechanotransduction studies of olfactory ensheathing cells.

    PubMed

    Harshad, Kamble; Jun, Myeongjun; Park, Sungsu; Barton, Matthew J; Vadivelu, Raja K; St John, James; Nguyen, Nam-Trung

    2016-06-01

    Olfactory ensheathing cells (OECs) are primary candidates for cell transplantation therapy to repair spinal cord injury (SCI). However, the post transplantation survival of these cells remains a major hurdle for a success using this therapy. Mechanical stimuli may contribute to the maintenance of these cells and thus, mechanotransduction studies of OECs may serve as a key benefit to identify strategies for improvement in cell transplantation. We developed an electromagnetic cell stretching device based on a single sided uniaxial stretching approach to apply tensile strain to OECs in culture. This paper reports the design, simulation and characterisation of the stretching device with preliminary experimental observations of OECs in vitro. The strain field of the deformable membrane was investigated both experimentally and numerically. Heterogeneity of the device provided an ideal platform for establishing strain requirement for the OEC culture. The cell stretching system developed may serve as a tool in exploring the mechanobiology of OECs for future SCI transplantation research. PMID:27194027

  9. Axon ensheathment and metabolic supply by glial cells in Drosophila.

    PubMed

    Schirmeier, Stefanie; Matzat, Till; Klämbt, Christian

    2016-06-15

    Neuronal function requires constant working conditions and a well-balanced supply of ions and metabolites. The metabolic homeostasis in the nervous system crucially depends on the presence of glial cells, which nurture and isolate neuronal cells. Here we review recent findings on how these tasks are performed by glial cells in the genetically amenable model organism Drosophila melanogaster. Despite the small size of its nervous system, which would allow diffusion of metabolites, a surprising division of labor between glial cells and neurons is evident. Glial cells are glycolytically active and transfer lactate and alanine to neurons. Neurons in turn do not require glycolysis but can use the glially provided compounds for their energy homeostasis. Besides feeding neurons, glial cells also insulate neuronal axons in a way similar to Remak fibers in the mammalian nervous system. The molecular mechanisms orchestrating this insulation require neuregulin signaling and resemble the mechanisms controlling glial differentiation in mammals surprisingly well. We hypothesize that metabolic cross talk and insulation of neurons by glial cells emerged early during evolution as two closely interlinked features in the nervous system. This article is part of a Special Issue entitled SI: Myelin Evolution. PMID:26367447

  10. A lab-on-a-chip device for investigating the fusion process of olfactory ensheathing cell spheroids.

    PubMed

    Munaz, Ahmed; Vadivelu, Raja K; John, James A St; Nguyen, Nam-Trung

    2016-08-01

    Understanding the process of fusion of olfactory ensheathing cell spheroids will lead to improvement of cell transplantation therapies to repair spinal cord injuries. The successful fusion of transplanted spheroids will enable alternative transplantation strategies to be developed for in vivo applications. This paper describes the use of a microfluidic device to trap and fuse olfactory ensheathing cell spheroids. The velocity, the pressure distribution in the device were simulated numerically to predict the trapping location. The simulation predicted the optimum flow rates for trapping the spheroids in the later experiments. Simulated particle trajectories were verified experimentally with tracing of fluorescent micro particles. The fusion process of the spheroids was investigated over a period of 48 hours. The microfluidic platform presented here can be used for testing potential drugs that can promote the fusion process and improve the transplantation therapy. PMID:27387270

  11. Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles

    PubMed Central

    Vadivelu, Raja K.; Ooi, Chin H.; Yao, Rebecca-Qing; Tello Velasquez, Johana; Pastrana, Erika; Diaz-Nido, Javier; Lim, Filip; Ekberg, Jenny A. K.; Nguyen, Nam-Trung; St John, James A.

    2015-01-01

    We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures. PMID:26462469

  12. Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles

    NASA Astrophysics Data System (ADS)

    Vadivelu, Raja K.; Ooi, Chin H.; Yao, Rebecca-Qing; Tello Velasquez, Johana; Pastrana, Erika; Diaz-Nido, Javier; Lim, Filip; Ekberg, Jenny A. K.; Nguyen, Nam-Trung; St John, James A.

    2015-10-01

    We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures.

  13. Linckosides enhance proliferation and induce morphological changes in human olfactory ensheathing cells.

    PubMed

    Tello Velasquez, Johana; Yao, Rebecca-Qing; Lim, Filip; Han, Chunguang; Ojika, Makoto; Ekberg, Jenny A K; Quinn, Ronald J; John, James A St

    2016-09-01

    Linckosides are members of the steroid glycoside family isolated from the starfish Linckia laevigata. These natural compounds have notable neuritogenic activity and synergistic effects on NGF-induced neuronal differentiation of PC12 cells. Neurogenic factors or molecules that are able to mimic their activities are known to be involved in the survival, proliferation and migration of neurons and glial cells; however how glial cells respond to specific neurogenic molecules such as linckosides has not been investigated. This study aimed to examine the effect of three different linckosides (linckoside A, B and granulatoside A) on the morphological properties, proliferation and migration of human olfactory ensheathing cells (hOECs). The proliferation rate after all the treatments was higher than control as detected by MTS assay. Additionally, hOECs displayed dramatic morphological changes characterized by a higher number of processes after linckoside treatment. Interestingly changes in microtubule organization and expression levels of some early neuronal markers (GAP43 and βIII-tubulin) were also observed. An increase in the phosphorylation of ERK 1/2 after addition of the compounds suggests that this pathway may be involved in the linckoside-mediated effects particularly those related to morphological changes. These results are the first description of the stimulating effects of linckosides on hOECs and raise the potential for this natural compound or its derivatives to be used to regulate and enhance the therapeutic properties of OECs, particularly for cell transplantation therapies. PMID:27343824

  14. Nose-to-brain delivery: evaluation of polymeric nanoparticles on olfactory ensheathing cells uptake.

    PubMed

    Musumeci, Teresa; Pellitteri, Rosalia; Spatuzza, Michela; Puglisi, Giovanni

    2014-02-01

    The nasal route has received a great deal of attention as a convenient and reliable method for the brain target on administration of drugs. When drugs are loaded into nanoparticles (NPs) the interaction with mucosa transports directly into the brain, skipping the blood-brain barrier and achieving rapid cerebrospinal fluid levels. Poly-lactic acid (PLA), poly-lactic-co-glycolic acid (PLGA), and chitosan (CS) were chosen to prepare NPs. After optimization of CS nanocarriers, our goal was to evaluate the different type of NPs uptake into olfactory ensheathing cells (OECs). We then correlated obtained biological data to zeta potential measurements of cells treated with NPs. Rodhamine-loaded NPs were used to study the uptake of OECs carried out by confocal microscopy at different times (1, 2, and 4 h). Our results showed that uptake of rodhamine-NPs by OECs was time dependent and it was influenced by the carrier charge. Confocal imaging of OECs demonstrated that NPPLGA showed a higher increase in uptake compared with NPPLA and NPCS after 1 h and it increased at 2-4 h. Zeta potential values of treated cells were more amplified with respect to untreated cells. The highest values were showed by unloaded NPPLGA, confirming microscopy data. PMID:24395679

  15. Streptococcus pneumoniae infection regulates expression of neurotrophic factors in the olfactory bulb and cultured olfactory ensheathing cells.

    PubMed

    Ruiz-Mendoza, S; Macedo-Ramos, H; Santos, F A; Quadros-de-Souza, L C; Paiva, M M; Pinto, T C A; Teixeira, L M; Baetas-da-Cruz, W

    2016-03-11

    Streptococcus pneumoniae is the causative agent of numerous diseases including severe invasive infections such as bacteremia and meningitis. It has been previously shown that strains of S. pneumoniae that are unable to survive in the bloodstream may colonize the CNS. However, information on cellular components and pathways involved in the neurotropism of these strains is still scarce. The olfactory system is a specialized tissue in which olfactory receptor neurons (ORNs) are interfacing with the external environment through several microvilli. Olfactory ensheathing cells (OECs) which also form the glial limiting membrane at the surface of the olfactory bulb (OB) are the only cells that ensheathe the ORNs axons. Since previous data from our group showed that OECs may harbor S. pneumoniae, we decided to test whether infection of the OB or OEC cultures modulates the expression levels of neurotrophic factor's mRNA and its putative effects on the activation and viability of microglia. We observed that neurotrophin-3 (NT-3) and glial cell-line-derived neurotrophic factor (GDNF) expression was significantly higher in the OB from uninfected mice than in infected mice. A similar result was observed when we infected OEC cultures. Brain-derived neurotrophic factor (BNDF) expression was significantly lower in the OB from infected mice than in uninfected mice. In contrast, in vitro infection of OECs resulted in a significant increase of BDNF mRNA expression. An upregulation of high-mobility group box 1 (HMGB1) expression was observed in both OB and OEC cultures infected with S. pneumoniae. Moreover, we found that conditioned medium from infected OEC cultures induced the expression of the pro-apoptotic protein cleaved-caspase-3 and an apparently continuous nuclear factor-kappa B (NF-κB) p65 activation in the N13 microglia. Altogether, our data suggest the possible existence of an OEC-pathogen molecular interface, through which the OECs could interfere on the activation and

  16. Olfactory ensheathing cells form the microenvironment of migrating GnRH-1 neurons during mouse development.

    PubMed

    Geller, Sarah; Kolasa, Elise; Tillet, Yves; Duittoz, Anne; Vaudin, Pascal

    2013-04-01

    During development, GnRH-1 neurons differentiate extracerebraly from the nasal placode and migrate from the vomeronasal organ to the forebrain along vomeronasal and terminal nerves. Numerous studies have described the influence of different molecules on the migration of GnRH-1 neurons, however, the role of microenvironment cells remains poorly understood. This study used GFAP-GFP transgenic mice to detect glial cells at early developmental stages. Using nasal explant cultures, the comigration of glial cells with GnRH-1 neurons was clearly demonstrated. This in vitro approach showed that glial cells began migrating from the explants before GnRH-1 neurons. They remained ahead of the GnRH-1 migratory front and stopped migrating after the GnRH-1 neurons. The association of these glial cells with the axons combined with gene expression analysis of GFAP-GFP sorted cells enabled them to be identified as olfactory ensheathing cells (OEC). Immunohistochemical analysis revealed the presence of multiple glial cell-type markers showing several OEC subpopulations surrounding GnRH-1 neurons. Moreover, these OEC expressed genes whose products are involved in the migration of GnRH-1 neurons, such as Nelf and Semaphorin 4. In situ data confirmed that the majority of the GnRH-1 neurons were associated with glial cells along the vomeronasal axons in nasal septum and terminal nerves in the nasal forebrain junction as early as E12.5. Overall, these data demonstrate an OEC microenvironment for migrating GnRH-1 neurons during mouse development. The fact that this glial cell type precedes GnRH-1 neurons and encodes for molecules involved in their nasal migration suggests that it participates in the GnRH-1 system ontogenesis. PMID:23404564

  17. Synergetic effects of ciliary neurotrophic factor and olfactory ensheathing cells on optic nerve reparation (complete translation)

    PubMed Central

    Yin, Dan-ping; Chen, Qing-ying; Liu, Lin

    2016-01-01

    At present, there is no effective treatment for the repair of the optic nerve after injury, or improvement of its microenvironment for regeneration. Intravitreally injected ciliary neurotrophic factor (CNTF) and olfactory ensheathing cells (OECs) promote the long-distance regrowth of severed optic nerve fibers after intracranial injury. Here, we examined the efficacy of these techniques alone and in combination, in a rat model of optic nerve injury. We injected condensed OEC suspension at the site of injury, or CNTF into the vitreous body, or both simultaneously. Retrograde tracing techniques showed that 4 weeks postoperatively, the number of surviving retinal ganglion cells and their axonal density in the optic nerve were greater in rats subjected to OEC injection only than in those receiving CNTF injection only. Furthermore, combined OEC + CNTF injection achieved better results than either monotherapy. These findings confirm that OECs are better than CNTF at protecting injured neurons in the eye, but that combined OEC and CNTF therapy is notably more effective than either treatment alone. PMID:27482233

  18. Olfactory ensheathing cell-neurite alignment enhances neurite outgrowth in scar-like cultures

    PubMed Central

    Khankan, Rana R.; Wanner, Ina B.; Phelps, Patricia E.

    2015-01-01

    The regenerative capacity of the adult CNS neurons after injury is strongly inhibited by the spinal cord lesion site environment that is composed primarily of the reactive astroglial scar and invading meningeal fibroblasts. Olfactory ensheathing cell (OEC) transplantation facilitates neuronal survival and functional recovery after a complete spinal cord transection, yet the mechanisms by which this recovery occurs remain unclear. We used a unique multicellular scar-like culture model to test if OECs promote neurite outgrowth in growth inhibitory areas. Astrocytes were mechanically injured and challenged by meningeal fibroblasts to produce key inhibitory elements of a spinal cord lesion. Neurite outgrowth of postnatal cerebral cortical neurons was assessed on three substrates: quiescent astrocyte control cultures, reactive astrocyte scar-like cultures, and scar-like cultures with OECs. Initial results showed that OECs enhanced total neurite outgrowth of cortical neurons in a scar-like environment by 60%. We then asked if the neurite growth-promoting properties of OECs depended on direct alignment between neuronal and OEC processes. Neurites that aligned with OECs were nearly three times longer when they grew on inhibitory meningeal fibroblast areas and twice as long on reactive astrocyte zones compared to neurites not associated with OECs. Our results show that OECs can independently enhance neurite elongation and that direct OEC-neurite cell contact can provide a permissive substrate that overcomes the inhibitory nature of the reactive astrocyte scar border and the fibroblast-rich spinal cord lesion core. PMID:25863021

  19. Generation of magnetized olfactory ensheathing cells for regenerative studies in the central and peripheral nervous tissue.

    PubMed

    Riggio, Cristina; Nocentini, Sara; Catalayud, Maria Pilar; Goya, Gerardo Fabian; Cuschieri, Alfred; Raffa, Vittoria; del Río, José Antonio

    2013-01-01

    As olfactory receptor axons grow from the peripheral to the central nervous system (CNS) aided by olfactory ensheathing cells (OECs), the transplantation of OECs has been suggested as a plausible therapy for spinal cord lesions. The problem with this hypothesis is that OECs do not represent a single homogeneous entity, but, instead, a functionally heterogeneous population that exhibits a variety of responses, including adhesion and repulsion during cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. In this paper, we report a system based on modified OECs carrying magnetic nanoparticles as a proof of concept experiment enabling specific studies aimed at exploring the potential of OECs in the treatment of spinal cord injuries. Our studies have confirmed that magnetized OECs (i) survive well without exhibiting stress-associated cellular responses; (ii) in vitro, their migration can be modulated by magnetic fields; and (iii) their transplantation in organotypic slices of spinal cord and peripheral nerve showed positive integration in the model. Altogether, these findings indicate the therapeutic potential of magnetized OECs for CNS injuries. PMID:23708092

  20. Canine olfactory ensheathing cells from the olfactory mucosa can be engineered to produce active chondroitinase ABC.

    PubMed

    Carwardine, Darren; Wong, Liang-Fong; Fawcett, James W; Muir, Elizabeth M; Granger, Nicolas

    2016-08-15

    A multitude of factors must be overcome following spinal cord injury (SCI) in order to achieve clinical improvement in patients. It is thought that by combining promising therapies these diverse factors could be combatted with the aim of producing an overall improvement in function. Chondroitin sulphate proteoglycans (CSPGs) present in the glial scar that forms following SCI present a significant block to axon regeneration. Digestion of CSPGs by chondroitinase ABC (ChABC) leads to axon regeneration, neuronal plasticity and functional improvement in preclinical models of SCI. However, the enzyme activity decays at body temperature within 24-72h, limiting the translational potential of ChABC as a therapy. Olfactory ensheathing cells (OECs) have shown huge promise as a cell transplant therapy in SCI. Their beneficial effects have been demonstrated in multiple small animal SCI models as well as in naturally occurring SCI in canine patients. In the present study, we have genetically modified canine OECs from the mucosa to constitutively produce enzymatically active ChABC. We have developed a lentiviral vector that can deliver a mammalian modified version of the ChABC gene to mammalian cells, including OECs. Enzyme production was quantified using the Morgan-Elson assay that detects the breakdown products of CSPG digestion in cell supernatants. We confirmed our findings by immunolabelling cell supernatant samples using Western blotting. OECs normal cell function was unaffected by genetic modification as demonstrated by normal microscopic morphology and the presence of the low affinity neurotrophin receptor (p75(NGF)) following viral transduction. We have developed the means to allow production of active ChABC in combination with a promising cell transplant therapy for SCI repair. PMID:27423610

  1. Microencapsulation improves inhibitory effects of transplanted olfactory ensheathing cells on pain after sciatic nerve injury

    PubMed Central

    Zhao, Hao; Yang, Bao-lin; Liu, Zeng-xu; Yu, Qing; Zhang, Wen-jun; Yuan, Keng; Zeng, Hui-hong; Zhu, Gao-chun; Liu, De-ming; Li, Qing

    2015-01-01

    Olfactory bulb tissue transplantation inhibits P2X2/3 receptor-mediated neuropathic pain. However, the olfactory bulb has a complex cellular composition, and the mechanism underlying the action of purified transplanted olfactory ensheathing cells (OECs) remains unclear. In the present study, we microencapsulated OECs in alginic acid, and transplanted free and microencapsulated OECs into the region surrounding the injured sciatic nerve in rat models of chronic constriction injury. We assessed mechanical nociception in the rat models 7 and 14 days after surgery by measuring paw withdrawal threshold, and examined P2X2/3 receptor expression in L4–5 dorsal root ganglia using immunohistochemistry. Rats that received free and microencapsulated OEC transplants showed greater withdrawal thresholds than untreated model rats, and weaker P2X2/3 receptor immunoreactivity in dorsal root ganglia. At 14 days, paw withdrawal threshold was much higher in the microencapsulated OEC-treated animals. Our results confirm that microencapsulated OEC transplantation suppresses P2X2/3 receptor expression in L4–5 dorsal root ganglia in rat models of neuropathic pain and reduces allodynia, and also suggest that transplantation of microencapsulated OECs is more effective than transplantation of free OECs for the treatment of neuropathic pain. PMID:26487865

  2. Biofunctionalization of conductive hydrogel coatings to support olfactory ensheathing cells at implantable electrode interfaces.

    PubMed

    Hassarati, Rachelle T; Marcal, Helder; John, L; Foster, R; Green, Rylie A

    2016-05-01

    Mechanical discrepancies between conventional platinum (Pt) electrodes and neural tissue often result in scar tissue encapsulation of implanted neural recording and stimulating devices. Olfactory ensheathing cells (OECs) are a supportive glial cell in the olfactory nervous system which can transition through glial scar tissue while supporting the outgrowth of neural processes. It has been proposed that this function can be used to reconnect implanted electrodes with the target neural pathways. Conductive hydrogel (CH) electrode coatings have been proposed as a substrate for supporting OEC survival and proliferation at the device interface. To determine an ideal CH to support OECs, this study explored eight CH variants, with differing biochemical composition, in comparison to a conventional Pt electrodes. All CH variants were based on a biosynthetic hydrogel, consisting of poly(vinyl alcohol) and heparin, through which the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) was electropolymerized. The biochemical composition was varied through incorporation of gelatin and sericin, which were expected to provide cell adherence functionality, supporting attachment, and cell spreading. Combinations of these biomolecules varied from 1 to 3 wt %. The physical, electrical, and biological impact of these molecules on electrode performance was assessed. Cyclic voltammetry and electrochemical impedance spectroscopy demonstrated that the addition of these biological molecules had little significant effect on the coating's ability to safely transfer charge. Cell attachment studies, however, determined that the incorporation of 1 wt % gelatin in the hydrogel was sufficient to significantly increase the attachment of OECs compared to the nonfunctionalized CH. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 104B: 712-722, 2016. PMID:26248597

  3. Olfactory Ensheathing Cells Express α7 Integrin to Mediate Their Migration on Laminin

    PubMed Central

    Ingram, Norianne T.; Khankan, Rana R.; Phelps, Patricia E.

    2016-01-01

    The unique glia located in the olfactory system, called olfactory ensheathing cells (OECs), are implicated as an attractive choice for transplantation therapy following spinal cord injury because of their pro-regenerative characteristics. Adult OECs are thought to improve functional recovery and regeneration after injury by secreting neurotrophic factors and making cell-to-cell contacts with regenerating processes, but the mechanisms are not well understood. We show first that α7 integrin, a laminin receptor, is highly expressed at the protein level by OECs throughout the olfactory system, i.e., in the olfactory mucosa, olfactory nerve, and olfactory nerve layer of the olfactory bulb. Then we asked if OECs use the α7 integrin receptor directly to promote neurite outgrowth on permissive and neutral substrates, in vitro. We co-cultured α7+/+ and α7lacZ/lacZ postnatal cerebral cortical neurons with α7+/+ or α7lacZ/lacZ OECs and found that genotype did not effect the ability of OECs to enhance neurite outgrowth by direct contact. Loss of α7 integrin did however significantly decrease the motility of adult OECs in transwell experiments. Twice as many α7+/+ OECs migrated through laminin-coated transwells compared to α7+/+ OECs on poly-L-lysine (PLL). This is in contrast to α7lacZ/lacZ OECs, which showed no migratory preference for laminin substrate over PLL. These results demonstrate that OECs express α7 integrin, and that laminin and its α7 integrin receptor contribute to adult OEC migration in vitro and perhaps also in vivo. PMID:27078717

  4. Olfactory Ensheathing Cells Express α7 Integrin to Mediate Their Migration on Laminin.

    PubMed

    Ingram, Norianne T; Khankan, Rana R; Phelps, Patricia E

    2016-01-01

    The unique glia located in the olfactory system, called olfactory ensheathing cells (OECs), are implicated as an attractive choice for transplantation therapy following spinal cord injury because of their pro-regenerative characteristics. Adult OECs are thought to improve functional recovery and regeneration after injury by secreting neurotrophic factors and making cell-to-cell contacts with regenerating processes, but the mechanisms are not well understood. We show first that α7 integrin, a laminin receptor, is highly expressed at the protein level by OECs throughout the olfactory system, i.e., in the olfactory mucosa, olfactory nerve, and olfactory nerve layer of the olfactory bulb. Then we asked if OECs use the α7 integrin receptor directly to promote neurite outgrowth on permissive and neutral substrates, in vitro. We co-cultured α7+/+ and α7lacZ/lacZ postnatal cerebral cortical neurons with α7+/+ or α7lacZ/lacZ OECs and found that genotype did not effect the ability of OECs to enhance neurite outgrowth by direct contact. Loss of α7 integrin did however significantly decrease the motility of adult OECs in transwell experiments. Twice as many α7+/+ OECs migrated through laminin-coated transwells compared to α7+/+ OECs on poly-L-lysine (PLL). This is in contrast to α7lacZ/lacZ OECs, which showed no migratory preference for laminin substrate over PLL. These results demonstrate that OECs express α7 integrin, and that laminin and its α7 integrin receptor contribute to adult OEC migration in vitro and perhaps also in vivo. PMID:27078717

  5. Potential of Olfactory Ensheathing Cells from Different Sources for Spinal Cord Repair

    PubMed Central

    Mayeur, Anne; Duclos, Célia; Honoré, Axel; Gauberti, Maxime; Drouot, Laurent; do Rego, Jean-Claude; Bon-Mardion, Nicolas; Jean, Laetitia; Vérin, Eric; Emery, Evelyne; Lemarchant, Sighild; Vivien, Denis; Boyer, Olivier; Marie, Jean-Paul; Guérout, Nicolas

    2013-01-01

    Spinal cord injury (SCI) induces a permanent disability in patients. To this day no curative treatment can be proposed to restore lost functions. Therefore, extensive experimental studies have been conducted to induce recovery after SCI. One of the most promising therapies is based on the use of olfactory ensheathing cells (OECs). OECs can be obtained from either the olfactory bulbs (OB-OECs) or from olfactory mucosa (OM-OECs), involving a less invasive approach for autotransplantation. However the vast majority of experimental transplantations have been focusing on OB-OECs although the OM represents a more accessible source of OECs. Importantly, the ability of OM-OECs in comparison to OB-OECs to induce spinal cord recovery in the same lesion paradigm has never been described. We here present data using a multiparametric approach, based on electrophysiological, behavioral, histological and magnetic resonance imaging experiments on the repair potential of OB-OECs and OM-OECs from either primary or purified cultures after a severe model of SCI. Our data demonstrate that transplantation of OECs obtained from OB or OM induces electrophysiological and functional recovery, reduces astrocyte reactivity and glial scar formation and improves axonal regrowth. We also show that the purification step is essential for OM-OECs while not required for OB-OECs. Altogether, our study strongly indicates that transplantation of OECs from OM represents the best benefit/risk ratio according to the safety of access of OM and the results induced by transplantations of OM-OECs. Indeed, purified OM-OECs in addition to induce recovery can integrate and survive up to 60 days into the spinal cord. Therefore, our results provide strong support for these cells as a viable therapy for SCI. PMID:23638158

  6. Borderless regulates glial extension and axon ensheathment.

    PubMed

    Cameron, Scott; Chen, Yixu; Rao, Yong

    2016-06-15

    Ensheathment of axons by glial processes is essential for normal brain function. While considerable progress has been made to define molecular and cellular mechanisms underlying the maintenance of axon ensheathment, less is known about molecular details of early events for the wrapping of axons by glial processes in the developing nervous system. In this study, we investigate the role of the transmembrane protein Borderless (Bdl) in the developing Drosophila visual system. Bdl belongs to the immunoglobulin (Ig) superfamily, and its in vivo function is unknown. We show that Bdl is expressed in wrapping glia (WG) in the developing eye disc. Cell-type-specific transgene rescue and knockdown indicate that Bdl is specifically required in WG for the extension of glial processes along photoreceptor axons in the optic lobe, and axon ensheathment. Our results identify Bdl as a novel glia-specific cell-surface recognition molecule in regulating glial extension and axon ensheathment. PMID:27131624

  7. Ultrastructural analysis of olfactory ensheathing cells derived from olfactory bulb and nerve of neonatal and juvenile rats.

    PubMed

    Gómez, Rosa M; Ghotme, Kemel; Botero, Lucía; Bernal, Jaime E; Pérez, Rosalía; Barreto, George E; Bustos, Rosa Helena

    2016-02-01

    Olfactory nerve derived and olfactory bulb derived olfactory ensheathing cells (OECs) have the ability to promote axonal regeneration and remyelination, both of which are essential in a successful cell transplant. Thus, morphological identification of OECs is a key aspect to develop an applicable cell therapy for injuries to the nervous system. However, there is no clear definition regarding which developmental stage or anatomical origin of OECs is more adequate for neural repair. In the present study, an ultrastructural comparison was made between OECs recovered from primary cultures of olfactory nerve and bulb in two developmental stages. The most notorious difference between cells obtained from olfactory nerve and bulb was the presence of indented nuclei in bulb derived OECs, suggesting a greater ability for possible chemotaxis. In neonatal OECs abundant mitochondria, lipid vacuoles, and smooth endoplasmic reticulum were detected, suggesting an active lipid metabolism, probably involved in synthesis of myelin. Our results suggest that neonatal OECs obtained from olfactory bulb have microscopic properties that could make them more suitable for neural repair. PMID:26254553

  8. Increased migration of olfactory ensheathing cells secreting the Nogo receptor ectodomain over inhibitory substrates and lesioned spinal cord.

    PubMed

    Reginensi, Diego; Carulla, Patricia; Nocentini, Sara; Seira, Oscar; Serra-Picamal, Xavier; Torres-Espín, Abel; Matamoros-Angles, Andreu; Gavín, Rosalina; Moreno-Flores, María Teresa; Wandosell, Francisco; Samitier, Josep; Trepat, Xavier; Navarro, Xavier; del Río, José Antonio

    2015-07-01

    Olfactory ensheathing cell (OEC) transplantation emerged some years ago as a promising therapeutic strategy to repair injured spinal cord. However, inhibitory molecules are present for long periods of time in lesioned spinal cord, inhibiting both OEC migration and axonal regrowth. Two families of these molecules, chondroitin sulphate proteoglycans (CSPG) and myelin-derived inhibitors (MAIs), are able to trigger inhibitory responses in lesioned axons. Mounting evidence suggests that OEC migration is inhibited by myelin. Here we demonstrate that OEC migration is largely inhibited by CSPGs and that inhibition can be overcome by the bacterial enzyme Chondroitinase ABC. In parallel, we have generated a stable OEC cell line overexpressing the Nogo receptor (NgR) ectodomain to reduce MAI-associated inhibition in vitro and in vivo. Results indicate that engineered cells migrate longer distances than unmodified OECs over myelin or oligodendrocyte-myelin glycoprotein (OMgp)-coated substrates. In addition, they also show improved migration in lesioned spinal cord. Our results provide new insights toward the improvement of the mechanisms of action and optimization of OEC-based cell therapy for spinal cord lesion. PMID:25708702

  9. GFRalpha-3, a protein related to GFRalpha-1, is expressed in developing peripheral neurons and ensheathing cells.

    PubMed

    Widenfalk, J; Tomac, A; Lindqvist, E; Hoffer, B; Olson, L

    1998-04-01

    We report here the identification of a gene, termed GFRalpha-3 (glial cell line-derived neurotrophic factor family receptor alpha-3), related to GFRalpha-1 and GFRalpha-2 (also known as GDNFR-alpha and GDNFR-beta), and describe distribution of GDNFalpha-3 in the nervous system and other parts of the mouse body during development and in the adult. GFRalpha-3 in situ hybridization signals were found mainly in the peripheral nervous system, with prominent signals in developing dorsal root and trigeminal ganglia. Sympathetic ganglia were also positive. Developing nerves manifested strong GFRalpha-3 mRNA signals, presumably generated by the Schwann cells. Olfactory ensheathing cells were also positive. Other non-neuronal cells appearing positive during development included chromaffin cells in the adrenal gland and small clusters of cells in the intestinal epithelium. In the central nervous system no robust signals could be detected at any stage investigated with the present probes. Compared with the previously described GFRalpha-1 and GFRalpha-2 mRNAs, which are widely distributed in the central nervous system and peripheral organs, the expression of GFRalpha-3 mRNA is much more restricted. The prominent expression in Schwann cells during development suggests a key role for GFRalpha-3 in the development of the peripheral nervous system. As Schwann cells are known to lack expression of the transducing RET receptor, we propose that a possible function of GFRalpha-3 during development could be to bind Schwann cell-derived GDNF-like ligands, thus presenting such molecules to growing axons. PMID:9749804

  10. Global cellular responses to β-methyl-amino-L-alanine (BMAA) by olfactory ensheathing glial cells (OEC).

    PubMed

    Chiu, Alexander S; Braidy, Nady; Marçal, Helder; Welch, Jeffrey H; Gehringer, Michelle M; Guillemin, Gilles J; Neilan, Brett A

    2015-06-01

    This study utilised a proteomics approach to identify any differential protein expression in a glial cell line, rat olfactory ensheathing cells (OECs), treated with the cyanotoxin β-methylamino-l-alanine (BMAA). Five proteins of interest were identified, namely Rho GDP-dissociation inhibitor 1 (RhoGDP1), Nck-associated protein 1 (NCKAP1), voltage-dependent anion-selective channel protein 1 (VDAC1), 3-hydroxyacyl-CoA dehydrogenase type-2 (3hCoAdh2), and ubiquilin-4 (UBQLN4). Four of these candidates, nuclear receptor subfamily 4 group A member 1 (Nur77), cyclophilin A (CyPA), RhoGDP1 and VDAC1, have been reported to be involved in cell growth. A microarray identified UBQLN4, palladin and CyPA, which have been implicated to have roles in excitotoxicity. Moreover, the NCKAP1, UBQLN4, CyPA and 3hCoAdh2 genes have been associated with abnormal protein aggregation. Differential expression of genes involved in mitochondrial activity, Nur77, 3hCoAdh2, VDAC1 and UBQLN4, were also identified. Confirmatory reverse transcription quantitative PCR (RT-qPCR) analysis of transcripts generated from the genes of interest corroborated the differential expression trends identified in the global protein analysis. BMAA induced cell cycle arrest in the G2/M phase of OEC and apoptosis after 48 h at concentrations of 250 μM and 500 μM. Collectively, this work advances our understanding of the mechanism of BMAA-mediated glial-toxicity in vitro. PMID:25797319

  11. New serum-derived albumin scaffold seeded with adipose-derived stem cells and olfactory ensheathing cells used to treat spinal cord injured rats.

    PubMed

    Ferrero-Gutierrez, A; Menendez-Menendez, Y; Alvarez-Viejo, M; Meana, A; Otero, J

    2013-01-01

    Recent advances in spinal cord injury (SCI) research and cell culture techniques and biomaterials predict promising new treatments for patients with SCI or other nerve injuries. Biomaterial scaffolds form a substrate within which cells are instructed to form a tissue in a controlled manner. This study was designed to assess axon regeneration and locomotor recovery in rats with spinal cord injury treated with a novel serum-derived albumin scaffold seeded with adipose derived stem cells (ADSCs) and olfactory ensheathing cells (OECs). OECs are considered promising candidates for the treatment of SCI, and ADSCs have the ability to differentiate into neural lineages. In vitro experiments revealed that ADSCs and OECs adhered to the scaffold, remained viable and expressed specific markers of their cell types when cultured in the scaffold. Rats treated with scaffold plus cells showed locomotor skills at several time points from 45 days post-injury that were improved over those recorded in control injured, untreated animals. Astrocytic scars and tissue regeneration, identified using histological and immunohistochemical techniques, revealed that although the scaffold itself appeared to play a significant role in reducing glial scar formation and filling of the lesion cavity with cells, the presence of ADSCs and OECs in the scaffold led to the appearance of cells expressing markers of neurons and axons at the injury site. Our findings point to the clinical feasibility of an albumin scaffold seeded with ADSCs and OECs as a treatment candidate for use in spinal cord injury repair studies. PMID:23233062

  12. Myelin-associated proteins block the migration of olfactory ensheathing cells: an in vitro study using single-cell tracking and traction force microscopy.

    PubMed

    Nocentini, Sara; Reginensi, Diego; Garcia, Simón; Carulla, Patricia; Moreno-Flores, María Teresa; Wandosell, Francisco; Trepat, Xavier; Bribian, Ana; del Río, José A

    2012-05-01

    Newly generated olfactory receptor axons grow from the peripheral to the central nervous system aided by olfactory ensheathing cells (OECs). Thus, OEC transplantation has emerged as a promising therapy for spinal cord injuries and for other neural diseases. However, these cells do not present a uniform population, but instead a functionally heterogeneous population that exhibits a variety of responses including adhesion, repulsion, and crossover during cell-cell and cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. Here, we demonstrated that rodent OECs express all the components of the Nogo receptor complex and that their migration is blocked by myelin. Next, we used cell tracking and traction force microscopy to analyze OEC migration and its mechanical properties over myelin. Our data relate the decrease of traction force of OEC with lower migratory capacity over myelin, which correlates with changes in the F-actin cytoskeleton and focal adhesion distribution. Lastly, OEC traction force and migratory capacity is enhanced after cell incubation with the Nogo receptor inhibitor NEP1-40. PMID:22205212

  13. Transduction of an immortalized olfactory ensheathing glia cell line with the green fluorescent protein (GFP) gene: Evaluation of its neuroregenerative capacity as a proof of concept.

    PubMed

    Plaza, N; Simón, D; Sierra, J; Moreno-Flores, M T

    2016-01-26

    Olfactory ensheathing glia (OEG) cells are known to foster axonal regeneration of central nervous system (CNS) neurons. Several lines of reversibly immortalized human OEG (ihOEG) have been previously established that enabled to develop models for their validation in vitro and in vivo. In this work, a constitutively GFP-expressing ihOEG cell line was obtained, and named Ts14-GFP. Ts14-GFP neuroregenerative ability was similar to that found for the parental line Ts14 and it can be assayed using in vivo transplantation experimental paradigms, after spinal cord or optic nerve damage. Additionally, we have engineered a low-regenerative ihOEG line, hTL2, using lentiviral transduction of the large T antigen from SV40 virus, denominated from now on Ts12. Ts12 can be used as a low regeneration control in these experiments. PMID:26655478

  14. A prospective randomized double-blind clinical trial using a combination of olfactory ensheathing cells and Schwann cells for the treatment of chronic complete spinal cord injuries.

    PubMed

    Chen, Lin; Huang, Hongyun; Xi, Haitao; Zhang, Feng; Liu, Yancheng; Chen, Di; Xiao, Juan

    2014-01-01

    The aim of this prospective randomized double-blind clinical study is to examine the benefits of using olfactory ensheathing cells (OECs) combined with or without Schwann cells (SCs) in treating chronic complete spinal cord injuries (SCIs). This would offer patients a better alternative for neurological functional recovery. According to the initial design, 28 eligible participants with cervical chronic complete SCI were recruited and randomly allocated into four groups of seven participants each. The neurological assessments were to be performed according to the American Spinal Injury Association (ASIA) and International Association of Neurorestoratology (IANR) Functional Rating Scales, in combination with electrophysiological tests, for example, electromyography (EMG) and paraspinal somatosensory evoked potentials (PSSEPs). Here we have summarized the data from seven patients; three patients received an OEC intraspinal transplantation, one underwent SC implantation, and one received a combination of OECs and SCs. The remaining two patients were used as controls. The scores were evaluated independently by at least two neurologists in a blinded fashion for comparing the neurological functional changes during pre- and post-cell transplantation (6-month follow-up). All patients who received OECs, SCs alone, and a combination of them showed functional improvement. Mild fever occurred in one of the patients with OEC transplant that subsided after symptomatic treatments. All treated patients except one showed improvement in the electrophysiological tests. The functional improvement rate comprises 5/5 (100%) in the treated group, but 0/2 (0%) in the control group (p = 0.008). These preliminary findings show that transplanting OECs, SCs, or a combination of them is well tolerated and that they have beneficial effects in patients. Thus, further studies in larger patient cohorts are warranted to assess the benefits and risks of these intervention strategies. This

  15. Combined Bone Mesenchymal Stem Cell and Olfactory Ensheathing Cell Transplantation Promotes Neural Repair Associated With CNTF Expression in Traumatic Brain-Injured Rats.

    PubMed

    Fu, Xue-Mei; Liu, Su-Juan; Dan, Qi-Qin; Wang, Yan-Ping; Lin, Na; Lv, Long-Yun; Zou, Yu; Liu, Su; Zhou, Xue; Wang, Ting-Hua

    2015-01-01

    This study examined the role of bone mesenchymal stem cell (BMSC) and olfactory ensheathing cell (OEC) cografting on neural function and underlying molecular mechanisms in acute stage of traumatic brain injury (TBI) rats. Eighty Sprague-Dawley (SD) female rats were randomly divided into five groups (n = 16 per category): sham operated group (Sham), weight-drop-induced TBI group (TBI), BMSC transplantation group (BMSC), OEC transplantation group (OEC), and cotransplantation group (CO). Eight rats were randomly selected from each group for behavioral and morphological assessment. Another category (n = 8 rats) was employed in the genetic expression detection. BMSCs were isolated from GFP mice and identified by CD44 antibody. OECs were isolated from the SD rats, identified by P75 antibody and labeled by Hoechst 33342. They were then transplanted into the surrounding tissue of the epicenter of TBI rats. The result of neurological severity scores revealed that BMSC or OEC transplantation alone and BMSC and OEC cografting significantly ameliorated the neurological deficits of TBI rats. Quantitative immunohistochemical analysis showed that graft-recipient animals possessed dramatically more neurons and regenerated axons and smaller amounts of astrocytes than controls 14 days posttransplantation (p < 0.05). However, the expressional level of ciliary neurotrophic factor significantly decreased in the cografting group as determined by RT-PCR (p < 0.05), and the Janus kinase/signal transducer and activator of transcription pathway was significantly activated at 7 days after cell transplantation (p < 0.05). This study is the first to report the role of cotransplantation of BMSCs and OECs in the therapy of TBI and explore its potential molecular mechanisms, therefore providing the important morphological and molecular biological evidence for the clinical application of BMSC and/or OEC transplantation in TBI. PMID:24612678

  16. Axonal Ensheathment and Intercellular Barrier Formation in Drosophila

    PubMed Central

    Blauth, Kevin; Banerjee, Swati; Bhat, Manzoor A.

    2014-01-01

    Glial cells are critical players in every major aspect of nervous system development, function, and disease. Other than their traditional supportive role, glial cells perform a variety of important functions such as myelination, synapse formation and plasticity, and establishment of blood–brain and blood–nerve barriers in the nervous system. Recent studies highlight the striking functional similarities between Drosophila and vertebrate glia. In both systems, glial cells play an essential role in neural ensheathment thereby isolating the nervous system and help to create a local ionic microenvironment for conduction of nerve impulses. Here, we review the anatomical aspects and the molecular players that underlie ensheathment during different stages of nervous system development in Drosophila and how these processes lead to the organization of neuroglial junctions. We also discuss some key aspects of the invertebrate axonal ensheathment and junctional organization with that of vertebrate myelination and axon–glial interactions. Finally, we highlight the importance of intercellular junctions in barrier formation in various cellular contexts in Drosophila. We speculate that unraveling the genetic and molecular mechanisms of ensheathment across species might provide key insights into human myelin-related disorders and help in designing therapeutic interventions. PMID:20801419

  17. Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells.

    PubMed

    Dando, Samantha J; Ipe, Deepak S; Batzloff, Michael; Sullivan, Matthew J; Crossman, David K; Crowley, Michael; Strong, Emily; Kyan, Stephanie; Leclercq, Sophie Y; Ekberg, Jenny A K; St John, James; Beacham, Ifor R; Ulett, Glen C

    2016-07-01

    Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs. PMID:27091931

  18. Olfactory Ensheathing Cell Transplantation in Experimental Spinal Cord Injury: Effect size and Reporting Bias of 62 Experimental Treatments: A Systematic Review and Meta-Analysis

    PubMed Central

    Sena, Emily S.; Dirnagl, Ulrich

    2016-01-01

    Olfactory ensheathing cell (OEC) transplantation is a candidate cellular treatment approach for human spinal cord injury (SCI) due to their unique regenerative potential and autologous origin. The objective of this study was, through a meta-epidemiologic approach, (i) to assess the efficacy of OEC transplantation on locomotor recovery after traumatic experimental SCI and (ii) to estimate the likelihood of reporting bias and/or missing data. A study protocol was finalized before data collection. Embedded into a systematic review and meta-analysis, we conducted a literature research of databases including PubMed, EMBASE, and ISI Web of Science from 1949/01 to 2014/10 with no language restrictions, screened by two independent investigators. Studies were included if they assessed neurobehavioral improvement after traumatic experimental SCI, administrated no combined interventions, and reported the number of animals in the treatment and control group. Individual effect sizes were pooled using a random effects model. Details regarding the study design were extracted and impact of these on locomotor outcome was assessed by meta-regression. Missing data (reporting bias) was determined by Egger regression and Funnel-plotting. The primary study outcome assessed was improvement in locomotor function at the final time point of measurement. We included 49 studies (62 experiments, 1,164 animals) in the final analysis. The overall improvement in locomotor function after OEC transplantation, measured using the Basso, Beattie, and Bresnahan (BBB) score, was 20.3% (95% CI 17.8–29.5). One missing study was imputed by trim and fill analysis, suggesting only slight publication bias and reducing the overall effect to a 19.2% improvement of locomotor activity. Dose-response ratio supports neurobiological plausibility. Studies were assessed using a 9-point item quality score, resulting in a median score of 5 (interquartile range [IQR] 3–5). In conclusion, OEC transplantation exerts

  19. Olfactory Ensheathing Cell Transplantation in Experimental Spinal Cord Injury: Effect size and Reporting Bias of 62 Experimental Treatments: A Systematic Review and Meta-Analysis.

    PubMed

    Watzlawick, Ralf; Rind, Julian; Sena, Emily S; Brommer, Benedikt; Zhang, Tian; Kopp, Marcel A; Dirnagl, Ulrich; Macleod, Malcolm R; Howells, David W; Schwab, Jan M

    2016-05-01

    Olfactory ensheathing cell (OEC) transplantation is a candidate cellular treatment approach for human spinal cord injury (SCI) due to their unique regenerative potential and autologous origin. The objective of this study was, through a meta-epidemiologic approach, (i) to assess the efficacy of OEC transplantation on locomotor recovery after traumatic experimental SCI and (ii) to estimate the likelihood of reporting bias and/or missing data. A study protocol was finalized before data collection. Embedded into a systematic review and meta-analysis, we conducted a literature research of databases including PubMed, EMBASE, and ISI Web of Science from 1949/01 to 2014/10 with no language restrictions, screened by two independent investigators. Studies were included if they assessed neurobehavioral improvement after traumatic experimental SCI, administrated no combined interventions, and reported the number of animals in the treatment and control group. Individual effect sizes were pooled using a random effects model. Details regarding the study design were extracted and impact of these on locomotor outcome was assessed by meta-regression. Missing data (reporting bias) was determined by Egger regression and Funnel-plotting. The primary study outcome assessed was improvement in locomotor function at the final time point of measurement. We included 49 studies (62 experiments, 1,164 animals) in the final analysis. The overall improvement in locomotor function after OEC transplantation, measured using the Basso, Beattie, and Bresnahan (BBB) score, was 20.3% (95% CI 17.8-29.5). One missing study was imputed by trim and fill analysis, suggesting only slight publication bias and reducing the overall effect to a 19.2% improvement of locomotor activity. Dose-response ratio supports neurobiological plausibility. Studies were assessed using a 9-point item quality score, resulting in a median score of 5 (interquartile range [IQR] 3-5). In conclusion, OEC transplantation exerts

  20. A large fraction of neocortical myelin ensheathes axons of local inhibitory neurons

    PubMed Central

    Micheva, Kristina D; Wolman, Dylan; Mensh, Brett D; Pax, Elizabeth; Buchanan, JoAnn; Smith, Stephen J; Bock, Davi D

    2016-01-01

    Myelin is best known for its role in increasing the conduction velocity and metabolic efficiency of long-range excitatory axons. Accordingly, the myelin observed in neocortical gray matter is thought to mostly ensheath excitatory axons connecting to subcortical regions and distant cortical areas. Using independent analyses of light and electron microscopy data from mouse neocortex, we show that a surprisingly large fraction of cortical myelin (half the myelin in layer 2/3 and a quarter in layer 4) ensheathes axons of inhibitory neurons, specifically of parvalbumin-positive basket cells. This myelin differs significantly from that of excitatory axons in distribution and protein composition. Myelin on inhibitory axons is unlikely to meaningfully hasten the arrival of spikes at their pre-synaptic terminals, due to the patchy distribution and short path-lengths observed. Our results thus highlight the need for exploring alternative roles for myelin in neocortical circuits. DOI: http://dx.doi.org/10.7554/eLife.15784.001 PMID:27383052

  1. The function of RhoGTPases in axon ensheathment and myelination

    PubMed Central

    Feltri, M. Laura; Suter, Ueli; Relvas, João B.

    2008-01-01

    RhoGTPases are molecular switches that integrate extracellular signals to perform diverse cellular responses. This ability relies on the network of proteins regulating RhoGTPases activity and localization, and on the interaction of RhoGTPases with many different cellular effectors. Myelination is an ideal place for RhoGTPases regulation, as it is the result of fine orchestration of many stimuli from at least two cell types. Recent work has revealed that RhoGTPases are required for Schwann cells to sort, ensheath and myelinate axons. Here we will review recent advances showing the critical roles for RhoGTPases in various aspects of Schwann development and myelination, including the recent discovery of their involvement in Charcot-Marie-Tooth disease. Comparison with potential roles of RhoGTPases in central nervous system myelination will be drawn. PMID:18803320

  2. How Cells Endure Extreme Conditions

    SciTech Connect

    2009-01-01

    One of natures most gripping feats of survival is now better understood. For the first time, Berkeley Lab scientists observed the chemical changes in individual cells that enable them to survive in conditions that should kill them. http://newscenter.lbl.gov/feature-stories/2009/07/07/cells-endure-extremes/

  3. Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment

    NASA Technical Reports Server (NTRS)

    Leiserson, W. M.; Harkins, E. W.; Keshishian, H.

    2000-01-01

    Fray is a serine/threonine kinase expressed by the peripheral glia of Drosophila, whose function is required for normal axonal ensheathment. Null fray mutants die early in larval development and have nerves with severe swelling and axonal defasciculation. The phenotype is associated with a failure of the ensheathing glia to correctly wrap peripheral axons. When the fray cDNA is expressed in the ensheathing glia of fray mutants, normal nerve morphology is restored. Fray belongs to a novel family of Ser/Thr kinases, the PF kinases, whose closest relatives are the PAK kinases. Rescue of the Drosophila mutant phenotype with PASK, the rat homolog of Fray, demonstrates a functional homology among these proteins and suggests that the Fray signaling pathway is widely conserved.

  4. Fuel-cell engine stream conditioning system

    DOEpatents

    DuBose, Ronald Arthur

    2002-01-01

    A stream conditioning system for a fuel cell gas management system or fuel cell engine. The stream conditioning system manages species potential in at least one fuel cell reactant stream. A species transfer device is located in the path of at least one reactant stream of a fuel cell's inlet or outlet, which transfer device conditions that stream to improve the efficiency of the fuel cell. The species transfer device incorporates an exchange media and a sorbent. The fuel cell gas management system can include a cathode loop with the stream conditioning system transferring latent and sensible heat from an exhaust stream to the cathode inlet stream of the fuel cell; an anode humidity retention system for maintaining the total enthalpy of the anode stream exiting the fuel cell related to the total enthalpy of the anode inlet stream; and a cooling water management system having segregated deionized water and cooling water loops interconnected by means of a brazed plate heat exchanger.

  5. Axon Regeneration Can Facilitate or Suppress Hindlimb Function after Olfactory Ensheathing Glia Transplantation

    PubMed Central

    Takeoka, Aya; Jindrich, Devin L.; Muñoz-Quiles, Cintia; Zhong, Hui; van den Brand, Rubia; Pham, Daniel L.; Ziegler, Matthias D.; Ramón-Cueto, Almudena; Roy, Roland R.; Edgerton, V. Reggie

    2011-01-01

    Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function. PMID:21411671

  6. Methods of conditioning direct methanol fuel cells

    DOEpatents

    Rice, Cynthia; Ren, Xiaoming; Gottesfeld, Shimshon

    2005-11-08

    Methods for conditioning the membrane electrode assembly of a direct methanol fuel cell ("DMFC") are disclosed. In a first method, an electrical current of polarity opposite to that used in a functioning direct methanol fuel cell is passed through the anode surface of the membrane electrode assembly. In a second method, methanol is supplied to an anode surface of the membrane electrode assembly, allowed to cross over the polymer electrolyte membrane of the membrane electrode assembly to a cathode surface of the membrane electrode assembly, and an electrical current of polarity opposite to that in a functioning direct methanol fuel cell is drawn through the membrane electrode assembly, wherein methanol is oxidized at the cathode surface of the membrane electrode assembly while the catalyst on the anode surface is reduced. Surface oxides on the direct methanol fuel cell anode catalyst of the membrane electrode assembly are thereby reduced.

  7. Stem cell therapy in spinal cord injury: Hollow promise or promising science?

    PubMed Central

    Goel, Aimee

    2016-01-01

    Spinal cord injury (SCI) remains one of the most physically, psychologically and socially debilitating conditions worldwide. While rehabilitation measures may help limit disability to some extent, there is no effective primary treatment yet available. The efficacy of stem cells as a primary therapeutic option in spinal cord injury is currently an area under much scrutiny and debate. Several laboratory and some primary clinical studies into the use of bone marrow mesenchymal stem cells or embryonic stem cell-derived oligodentrocyte precursor cells have shown some promising results in terms of remyelination and regeneration of damaged spinal nerve tracts. More recently,laboratory and early clinical experiments into the use of Olfactory Ensheathing Cells, a type of glial cell derived from olfactory bulb and mucosa have provided some phenomenal preliminary evidence as to their neuroregenerative and neural bridging capacity. This report compares and evaluates some current research into selected forms of embryonic and mesenchymal stem cell therapy as well as olfactory ensheathing cell therapy in SCI, and also highlights some legal and ethical issues surrounding their use. While early results shows promise, more rigorous large scaleclinical trials are needed to shed light on the safety, efficacy and long term viability of stem cell and cellular transplant techniques in SCI.

  8. Stem cell therapy in spinal cord injury: Hollow promise or promising science?

    PubMed

    Goel, Aimee

    2016-01-01

    Spinal cord injury (SCI) remains one of the most physically, psychologically and socially debilitating conditions worldwide. While rehabilitation measures may help limit disability to some extent, there is no effective primary treatment yet available. The efficacy of stem cells as a primary therapeutic option in spinal cord injury is currently an area under much scrutiny and debate. Several laboratory and some primary clinical studies into the use of bone marrow mesenchymal stem cells or embryonic stem cell-derived oligodentrocyte precursor cells have shown some promising results in terms of remyelination and regeneration of damaged spinal nerve tracts. More recently,laboratory and early clinical experiments into the use of Olfactory Ensheathing Cells, a type of glial cell derived from olfactory bulb and mucosa have provided some phenomenal preliminary evidence as to their neuroregenerative and neural bridging capacity. This report compares and evaluates some current research into selected forms of embryonic and mesenchymal stem cell therapy as well as olfactory ensheathing cell therapy in SCI, and also highlights some legal and ethical issues surrounding their use. While early results shows promise, more rigorous large scaleclinical trials are needed to shed light on the safety, efficacy and long term viability of stem cell and cellular transplant techniques in SCI. PMID:27217662

  9. Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment.

    PubMed

    MacNamee, Sarah E; Liu, Kendra E; Gerhard, Stephan; Tran, Cathy T; Fetter, Richard D; Cardona, Albert; Tolbert, Leslie P; Oland, Lynne A

    2016-07-01

    Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. Here, we show the first electrophysiological recordings from Drosophila astrocytes and characterize their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic premotor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via excitatory amino acid transporter 1 (Eaat1), and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory postsynaptic currents in motor neurons. The neuronal synapses were always located within 1 μm of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters. J. Comp. Neurol. 524:1979-1998, 2016. © 2016 Wiley Periodicals, Inc. PMID:27073064

  10. Chronic Spinal Injury Repair by Olfactory Bulb Ensheathing Glia and Feasibility for Autologous Therapy

    PubMed Central

    Muñoz-Quiles, Cintia; Santos-Benito, Fernando F.; Llamusí, M. Beatriz; Ramón-Cueto, Almudena

    2009-01-01

    Olfactory bulb ensheathing glia (OB-OEG) promote repair of spinal cord injury (SCI) in rats after transplantation at acute or subacute (up to 45 days) stages. The most relevant clinical scenario in humans, however, is chronic SCI, in which no more major cellular or molecular changes occur at the injury site; this occurs after the third month in rodents. Whether adult OB-OEG grafts promote repair of severe chronic SCI has not been previously addressed. Rats with complete SCI that were transplanted with OB-OEG 4 months after injury exhibited progressive improvement in motor function and axonal regeneration from different brainstem nuclei across and beyond the SCI site. A positive correlation between motor outcome and axonal regeneration suggested a role for brainstem neurons in the recovery. Functional and histological outcomes did not differ at subacute or chronic stages. Thus, autologous transplantation is a feasible approach as there is time for patient stabilization and OEG preparation in human chronic SCI; the healing effects of OB-OEG on established injuries may offer new therapeutic opportunities for chronic SCI patients. PMID:19915486

  11. Mast Cells Condition Dendritic Cells to Mediate Allograft Tolerance

    PubMed Central

    de Vries, Victor C.; Pino-Lagos, Karina; Nowak, Elizabeth C.; Bennett, Kathy A.; Oliva, Carla; Noelle, Randolph J.

    2013-01-01

    SUMMARY Peripheral tolerance orchestrated by regulatory T cells, dendritic cells (DCs), and mast cells (MCs) has been studied in several models including skin allograft tolerance. We now define a role for MCs in controlling DC behavior (“conditioning”) to facilitate tolerance. Under tolerant conditions, we show that MCs mediated a marked increase in tumor necrosis factor (TNFα)-dependent accumulation of graft-derived DCs in the dLN compared to nontolerant conditions. This increase of DCs in the dLN is due to the local production of granulocyte macrophage colony-stimulating factor (GM-CSF) by MCs that induces a survival advantage of graft-derived DCs. DCs that migrated to the dLN from the tolerant allograft were tolerogenic; i.e., they dominantly suppress T cell responses and control regional immunity. This study underscores the importance of MCs in conditioning DCs to mediate peripheral tolerance and shows a functional impact of peripherally produced TNFα and GM-CSF on the migration and function of tolerogenic DCs. PMID:22035846

  12. A Global In Vivo Drosophila RNAi Screen Identifies a Key Role of Ceramide Phosphoethanolamine for Glial Ensheathment of Axons

    PubMed Central

    Ghosh, Aniket; Kling, Tina; Snaidero, Nicolas; Sampaio, Julio L.; Shevchenko, Andrej; Gras, Heribert; Geurten, Bart; Göpfert, Martin C.; Schulz, Jörg B.; Voigt, Aaron; Simons, Mikael

    2013-01-01

    Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Here, using glial-specific RNAi knockdown in Drosophila, we silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among our screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, our RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia. PMID:24348263

  13. Are Purkinje Cell Pauses Drivers of Classically Conditioned Blink Responses?

    PubMed

    Jirenhed, Dan-Anders; Hesslow, Germund

    2016-08-01

    Several lines of evidence show that classical or Pavlovian conditioning of blink responses depends on the cerebellum. Recordings from cerebellar Purkinje cells that control the eyelid and the conditioned blink show that during training with a conditioning protocol, a Purkinje cell develops a pause response to the conditional stimulus. This conditioned cellular response has many of the properties that characterise the overt blink. The present paper argues that the learned Purkinje cell pause response is the memory trace and main driver of the overt conditioned blink and that it explains many well-known behavioural phenomena. PMID:26400585

  14. N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination

    PubMed Central

    Jin, Fuzi; Dong, Baoxia; Georgiou, John; Jiang, Qiuhong; Zhang, Jinyi; Bharioke, Arjun; Qiu, Frank; Lommel, Silvia; Feltri, M. Laura; Wrabetz, Lawrence; Roder, John C.; Eyer, Joel; Chen, Xiequn; Peterson, Alan C.; Siminovitch, Katherine A.

    2011-01-01

    Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation. PMID:21385763

  15. [Effect of Conditioned Medium from Endothelial Cells on Cancer Stem Cell Phenotype of Hepatoma Cells].

    PubMed

    Feng, Chuan; Yang, Xianjiong; Sun, Jinghui; Luo, Qing; Song, Guanbin

    2015-10-01

    In this study, we aimed to investigate the influences of conditioned medium from human umbilical vein endothelial cells (HUVEC) on cancer stem cell phenotype of human hepatoma cells. HUVEC and human hepatoma cells (MHCC97H) were cultured, respectively, and then the MHCC97H cells were co-cultured with conditioned medium from HUVEC (EC-CM) with Transwell system. Anti-cancer drug sensitivity, colony-formation, migration/invasion ability, expression of cancer stem cell marker and sphere formation were performed to determine the cancer stem cell phenotype in MHCC97H cells. We found that MHCC97H cells co-cultured with EC-CM exhibited significantly higher colony-formation ability and lower sensitivity of anti-cancer drugs 5-FU and Cis. Transwell assay showed that treatment with EC-CM obviously increased migration and invasion of MHCC97H cells. Moreover, increased sphere forming capability and expression of CD133 in MHCC97H cells were observed after co-cultured with EC-CM. These results suggested that EC-CM could promote cancer stem cell phenotype of hepatoma cells. PMID:26964312

  16. Test facility for solar-cell reference conditions

    NASA Technical Reports Server (NTRS)

    Klucher, T. M.

    1976-01-01

    A test facility, intended primarily for long-term monitoring of the global insolation and its components and the concurrent solar cell performance under a wide variety of measureable atmospheric and weather conditions, is described. Instruments for the measurement of insolation, cell performance, turbidity, water vapor, and cloud cover are described. Preliminary evaluation of the hourly data base generated over a two-month period for a range of sky conditions from clear to overcast is presented.

  17. Measurement of multijunction cells under close-match conditions

    SciTech Connect

    Wilkinson, V.A.; Goodbody, C.; Williams, W.G.

    1997-12-31

    This paper presents details of a new close-match solar simulator developed for DERA`s Space Power Laboratory for the accurate characterization of multijunction solar cells. The authors present data on the simulator measurements of dual and triple junction cells. The measurements are compared with those made under less ideal spectral conditions.

  18. PULMONARY CELL POPULATIONS IN HAMSTERS MAINTAINED UNDER EGYPTIAN LABORATORY CONDITIONS

    EPA Science Inventory

    The study was conducted to obtain baseline values for pulmonary cells in golden hamsters (Mesocricetus auratus) bred and maintained under the laboratory conditions of Al-Azhar University in Egypt. An improvised technique is presented for measuring pulmonary cells obtained by lung...

  19. The appropriateness of organic solar cells for indoor lighting conditions

    NASA Astrophysics Data System (ADS)

    Minnaert, B.; Veelaert, P.

    2010-05-01

    Most commercially available photovoltaic solar cells are crystalline silicon cells. However, in indoor environments, the efficiency of silicon solar cells is poor. Typically, the light intensity under artificial lighting conditions is less than 10 W/m2 as compared to 100-1000 W/m2 under outdoor conditions. Moreover, the spectrum is different from the outdoor solar spectrum and there is more diffuse than direct light. Taken into account the predicted cheaper costs for the production of organic solar cells, a possible niche market for organic PV can be indoor applications. In this article, we study the influence of the narrow absorption window, characteristic for organic solar cells, for different indoor conditions. This comparison is made for typical artificial light sources, i.e. a common incandescent lamp, an LED lamp and a "warm" and a "cool" fluorescent tube, which are compared to the outdoor AM 1.5 spectrum as reference. The comparisons are done by simulation based on the quantum efficiencies of the solar cells and the light spectra of the different light sources. A classical silicon solar cell is used as reference. In this way we determine the appropriateness for indoor use of organic solar cells.

  20. Path dependence of lithium ion cells aging under storage conditions

    NASA Astrophysics Data System (ADS)

    Su, Laisuo; Zhang, Jianbo; Huang, Jun; Ge, Hao; Li, Zhe; Xie, Fengchao; Liaw, Bor Yann

    2016-05-01

    This work investigates path dependence of lithium ion cells that are stored under static and non-static conditions. In the static storage tests, the levels of temperature and state of charge (SOC) are kept constant. The results of 12 tests from a combination of three temperatures and four SOCs show that, as expected, the cell ages faster at higher temperature and higher SOC. However, the cell aging mode, while consistent for all the evaluated temperatures, is different at 95% SOC from that at lower SOCs. In the non-static storage tests, the levels of temperature and SOC vary with time during the test process. The effect of the sequence of stress levels on cell aging is studied statistically using the statistical method of analysis of variation (ANOVA). It is found that cell capacity fade is path independent of both SOC and temperature, while cell resistance increase is path dependent on SOC and path independent of temperature. Finally, rate-based empirical aging models are adopted to fit the cell aging in the static storage tests. The aging model for capacity fade is demonstrated to be applicable to the non-static tests with errors between -3% and +3% for all the tested conditions over 180 days.

  1. Improved Boundary Conditions for Cell-centered Difference Schemes

    NASA Technical Reports Server (NTRS)

    VanderWijngaart, Rob F.; Klopfer, Goetz H.; Chancellor, Marisa K. (Technical Monitor)

    1997-01-01

    Cell-centered finite-volume (CCFV) schemes have certain attractive properties for the solution of the equations governing compressible fluid flow. Among others, they provide a natural vehicle for specifying flux conditions at the boundaries of the physical domain. Unfortunately, they lead to slow convergence for numerical programs utilizing them. In this report a method for investigating and improving the convergence of CCFV schemes is presented, which focuses on the effect of the numerical boundary conditions. The key to the method is the computation of the spectral radius of the iteration matrix of the entire demoralized system of equations, not just of the interior point scheme or the boundary conditions.

  2. Embryonic stem cells conditioned medium enhances Wharton's jelly-derived mesenchymal stem cells expansion under hypoxic condition.

    PubMed

    Prasajak, Patcharee; Rattananinsruang, Piyaporn; Chotinantakul, Kamonnaree; Dechsukhum, Chavaboon; Leeanansaksiri, Wilairat

    2015-05-01

    Mesenchymal stem cells (MSCs) are accepted as a promising tool for therapeutic purposes. However, low proliferation and early senescence are still main obstacles of MSCs expansion for using as cell-based therapy. Thus, clinical scale of cell expansion is needed to obtain a large number of cells serving for further applications. In this study, we investigated the value of embryonic stem cells conditioned medium (ESCM) for in vitro expansion of Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) as compared to typical culture medium for MSCs, Dulbecco's modified Eagle's medium with 1.0 g/l glucose (DMEM-LG) supplemented with 10 % FBS, under hypoxic condition. The expanded cells from ESCM (ESCM-MSCs) and DMEM-LG (DMEM-MSCs) were characterized for both phenotype and biological activities including proliferation rate, population doubling time, cell cycle distribution and MSCs characteristics. ESCM and DMEM-LG could enhance WJ-MSCs proliferation as 204.66 ± 10.39 and 113.77 ± 7.89 fold increase at day 12, respectively. ESCM-MSCs could express pluripotency genes including Oct-4, Oct-3/4, Nanog, Klf-4, C-Myc and Sox-2 both in early and late passages whereas the downregulations of Oct-4 and Nanog were detected in late passage cells of DMEM-MSCs. The 2 cell populations also showed common MSCs characteristics including normal cell cycle, fibroblastic morphology, cell surface markers expressions (CD29(+), CD44(+), CD90(+), CD34(-), CD45(-)) and differentiation capacities into adipogenic, chondrogenic and osteogenic lineages. Moreover, our results revealed that ESCM exhibited as a rich source of several factors which are required for supportive WJ-MSCs proliferation. In conclusion, ESCM under hypoxic condition could accelerate WJ-MSCs expansion while maintaining their pluripotency properties. Our knowledge provide short term and cost-saving in WJ-MSCs expansion which has benefit to overcome insufficient cell numbers for clinical applications by reusing the

  3. Primary cancer cell culture: mammary-optimized vs conditional reprogramming.

    PubMed

    Alamri, Ahmad M; Kang, Keunsoo; Groeneveld, Svenja; Wang, Weisheng; Zhong, Xiaogang; Kallakury, Bhaskar; Hennighausen, Lothar; Liu, Xuefeng; Furth, Priscilla A

    2016-07-01

    The impact of different culture conditions on biology of primary cancer cells is not always addressed. Here, conditional reprogramming (CRC) was compared with mammary-optimized EpiCult-B (EpiC) for primary mammary epithelial cell isolation and propagation, allograft generation, and genome-wide transcriptional consequences using cancer and non-cancer mammary tissue from mice with different dosages of Brca1 and p53 Selective comparison to DMEM was included. Primary cultures were established with all three media, but CRC was most efficient for initial isolation (P<0.05). Allograft development was faster using cells grown in EpiC compared with CRC (P<0.05). Transcriptome comparison of paired CRC and EpiC cultures revealed 1700 differentially expressed genes by passage 20. CRC promoted Trp53 gene family upregulation and increased expression of epithelial differentiation genes, whereas EpiC elevated expression of epithelial-mesenchymal transition genes. Differences did not persist in allografts where both methods yielded allografts with relatively similar transcriptomes. Restricting passage (<7) reduced numbers of differentially expressed genes below 50. In conclusion, CRC was most efficient for initial cell isolation but EpiC was quicker for allograft generation. The extensive culture-specific gene expression patterns that emerged with longer passage could be limited by reducing passage number when both culture transcriptomes were equally similar to that of the primary tissue. Defining impact of culture condition and passage on the transcriptome of primary cells could assist experimental design and interpretation. For example, differences that appear with passage and culture condition are potentially exploitable for comparative studies targeting specific biological networks in different transcriptional environments. PMID:27267121

  4. The Use of Cell Transplantation in Spinal Cord Injuries.

    PubMed

    Schroeder, Gregory D; Kepler, Christopher K; Vaccaro, Alexander R

    2016-04-01

    Acute spinal cord injuries are life-changing events that lead to substantial morbidity and mortality, but the role of cell-based treatment for these injuries is unclear. Cell therapy is a rapidly evolving treatment methodology, with basic science and early phase I/II human trials showing promise. Multiple cell lines can be used in cell therapy, including adult or embryonic stem cells, Schwann cells, olfactory ensheathing cells, and induced pluripotent stem cells. Adult stem cells, Schwann cells, and olfactory ensheathing cells are readily available but lack the ability to differentiate into cells of the central nervous system. Mesenchymal stem cells can decrease cell death by modifying the local environment into which they are introduced. Peripheral nerve cells, such as Schwann cells and olfactory ensheathing cells, can myelinate existing axons and foster axonal growth in the central nervous system, and embryonic stem cells can differentiate into neural progenitor stem cells of the central nervous system. Induced pluripotent stem cells are the basis of an emerging technology that has yet to be implemented in human trials but may offer a means of cell therapy without the ethical dilemmas associated with embryonic cells. PMID:26945167

  5. Hyperglycemic Conditions Prime Cells for RIP1-dependent Necroptosis.

    PubMed

    LaRocca, Timothy J; Sosunov, Sergey A; Shakerley, Nicole L; Ten, Vadim S; Ratner, Adam J

    2016-06-24

    Necroptosis is a RIP1-dependent programmed cell death (PCD) pathway that is distinct from apoptosis. Downstream effector pathways of necroptosis include formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS), both of which depend on glycolysis. This suggests that increased cellular glucose may prime necroptosis. Here we show that exposure to hyperglycemic levels of glucose enhances necroptosis in primary red blood cells (RBCs), Jurkat T cells, and U937 monocytes. Pharmacologic or siRNA inhibition of RIP1 prevented the enhanced death, confirming it as RIP1-dependent necroptosis. Hyperglycemic enhancement of necroptosis depends upon glycolysis with AGEs and ROS playing a role. Total levels of RIP1, RIP3, and mixed lineage kinase domain-like (MLKL) proteins were increased following treatment with high levels of glucose in Jurkat and U937 cells and was not due to transcriptional regulation. The observed increase in RIP1, RIP3, and MLKL protein levels suggests a potential positive feedback mechanism in nucleated cell types. Enhanced PCD due to hyperglycemia was specific to necroptosis as extrinsic apoptosis was inhibited by exposure to high levels of glucose. Hyperglycemia resulted in increased infarct size in a mouse model of brain hypoxia-ischemia injury. The increased infarct size was prevented by treatment with nec-1s, strongly suggesting that increased necroptosis accounts for exacerbation of this injury in conditions of hyperglycemia. This work reveals that hyperglycemia represents a condition in which cells are extraordinarily susceptible to necroptosis, that local glucose levels alter the balance of PCD pathways, and that clinically relevant outcomes may depend on glucose-mediated effects on PCD. PMID:27129772

  6. Conditional IL-2 Gene Deletion: Consequences for T Cell Proliferation

    PubMed Central

    Popmihajlov, Zoran; Xu, Dong; Morgan, Heather; Milligan, Zoie; Smith, Kendall A.

    2012-01-01

    To explore the role of interleukin-2 (IL-2) in T cell proliferation, and to circumvent the IL-2 deficiency autoimmune syndrome of conventional il2 gene deletion, mice were created to allow conditional il2 gene deletion when treated with the estrogen analog, tamoxifen (TAM) as adults. Splenocytes from four different mouse strains, C57Bl/6 wild type (WT), conventional IL-2(−/−), TAM-treated Cre recombinase-negative (Cre−)/IL2fl/fl, and Cre recombinase-positive (Cre+)/IL2fl/fl, were activated with anti-CD3 and anti-CD28, and monitored for CD4+ and CD8+ T cell lymphocyte blastogenesis, aerobic glycolysis, BrdU incorporation into newly synthesized DNA, and CFSE dye dilution to monitor cell division. IL-2 production was monitored by quantitative ELISA and multiple additional cytokines were monitored by quantitative protein-bead arrays. Splenocytes from conventional IL-2(−/−) and TAM-treated Cre+ mice resulted in undetectable IL-2 production by ELISA, so that both strains were IL-2-deficient. As monitored by flow cytometry, activated CD4+ and CD8+ T cells from WT, Cre+, and Cre− mice all underwent blastogenesis, whereas far fewer cells from conventional IL-2(−/−) mice did so. By comparison, only cells from IL-2 sufficient WT and Cre− mice switched to aerobic glycolysis as evidenced by a drop in media pH. Blastogenesis was mirrored by BrdU incorporation and CFSE dye dilution by CD4+ and CD8+ T cells from WT, Cre+, and Cre− mice, which were all equivalent, while proliferation of cells from conventional IL-2(−/−) mice was compromised. Splenocytes from IL-2 deficient conventional IL-2(−/−) mice produced low or undetectable other γc-chain cytokines (IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21), whereas production of these γc-chain cytokines from IL-2-deficient conditional IL-2(−/−) Cre+ mice were comparable with WT and Cre− mice. These results indicate that CD4+ and CD8+ T cell blastogenesis cannot be attributable to IL-2 alone, but a switch

  7. Cell Mechanisms of Bone Tissue Loss Under Space Flight Conditions

    NASA Astrophysics Data System (ADS)

    Rodionova, Natalia

    bone tissue. The macrophages are incorporated into resorption lacunaes and utilize the organic matrix and cellular detritus. The products are secreted to remodeling zones and act as haemoattractants for recruiting and subsequent differentiation here of the osteogenic precursor cells. However, as shown by our results with 3H-glycine, in absence of mechanical stimulus the activization of osteoblastogenesis either doesn't occur, or takes place on a smaller scale. According to our electron-microscopic data a load deficit leads to an adaptive differentiation of fibroblasts and adipocytes in this remodeling zones. This sequence of events is considered as a mechanism of bone tissue loss which underlies the development of osteopenia and osteoporosis under space flight condition.

  8. Effects of ambient conditions on fuel cell vehicle performance

    NASA Astrophysics Data System (ADS)

    Haraldsson, K.; Alvfors, P.

    Ambient conditions have considerable impact on the performance of fuel cell hybrid vehicles. Here, the vehicle fuel consumption, the air compressor power demand, the water management system and the heat loads of a fuel cell hybrid sport utility vehicle (SUV) were studied. The simulation results show that the vehicle fuel consumption increases with 10% when the altitude increases from 0 m up to 3000 m to 4.1 L gasoline equivalents/100 km over the New European Drive Cycle (NEDC). The increase is 19% on the more power demanding highway US06 cycle. The air compressor is the major contributor to this fuel consumption increase. Its load-following strategy makes its power demand increase with increasing altitude. Almost 40% of the net power output of the fuel cell system is consumed by the air compressor at the altitude of 3000 m with this load-following strategy and is thus more apparent in the high-power US06 cycle. Changes in ambient air temperature and relative humidity effect on the fuel cell system performance in terms of the water management rather in vehicle fuel consumption. Ambient air temperature and relative humidity have some impact on the vehicle performance mostly seen in the heat and water management of the fuel cell system. While the heat loads of the fuel cell system components vary significantly with increasing ambient temperature, the relative humidity did not have a great impact on the water balance. Overall, dimensioning the compressor and other system components to meet the fuel cell system requirements at the minimum and maximum expected ambient temperatures, in this case 5 and 40 °C, and high altitude, while simultaneously choosing a correct control strategy are important parameters for efficient vehicle power train management.

  9. Multiscale molecular simulations of proteins in cell-like conditions

    NASA Astrophysics Data System (ADS)

    Samiotakis, Antonios

    Proteins are the workhorses of all living organisms, performing a broad range of functions in the crowded cellular interior. However, little is known about how proteins function in cell-like conditions since most studies focus in dilute aqueous environments. In order to address this problem we incorporated molecular simulations and coarse-grained models that capture the protein dynamics in the cellular interior. We study the macromolecular crowding effects of cell-like environments on protein Borrelia Burgdorferi VlsE (variable major protein-like sequence-expressed), an aspherical membrane protein, and the enzyme Phosphoglycerate kinase. We show that protein conformation can be significantly perturbed under crowded cell-like conditions which, in turn, can have dramatic effects to the proteins' function. In addition, we look into the effects of mutations in the folding pathways of the topologically frustrated protein apoflavodoxin while correlation with experiments is also achieved. We further developed a multiscale simulation scheme that combines the sampling efficiency of low-resolution models with the detail of all-atomistic simulations. An algorithm that reconstructs all-atomistic conformations from coarse-grained representations was developed, in addition to an energy function that accounts for chemical interference based on the Boltzamn inversion method. The multiscale simulation scheme manages to sample all-atomistic structures of the protein Trp-cage that match very well with experiments. The folding kinetic behavior of Trp-cage was also studied in the combined presence of urea denaturant and macromolecular crowding.

  10. Influence of cell growth conditions and medium composition on EGFP photostability in live cells.

    PubMed

    Mamontova, Anastasia V; Bogdanov, Alexey M; Lukyanov, Konstantin A

    2015-05-01

    Photostability is a key characteristic of fluorescent proteins. It was recently demonstrated that green fluorescent protein (GFP) photobleaching in live cells can be suppressed by changes in medium composition. Here we show that Ham's F12 medium provides very high enhanced GFP (EGFP) photostability during fluorescence microscopy of live cells. This property of Ham's F12 medium is associated with decreased concentrations of riboflavin and pyridoxine, and increased concentrations of FeSO4, cyanocobalamine, lipoic acid, hypoxanthine, and thymidine compared with DMEM. We also found that the rate of EGFP photobleaching strongly depends on cell growth conditions such as cell density and the concentration of serum. We conclude that both imaging medium composition and the physiological state of the cells can strongly affect the photostability of fluorescent proteins. Thus, accurate comparison of the photostabilities of fluorescent proteins should be performed only in side-by-side analysis in identical cell growth conditions and media. PMID:25967905

  11. Graft rejection after hematopoietic cell transplantation with nonmyeloablative conditioning.

    PubMed

    Masmas, Tania N; Petersen, Søren L; Madsen, Hans O; Ryder, Lars P; Kornblit, Brian; Svejgaard, Arne; Andersen, Pernille; Dickmeiss, Ebbe; Vindeløv, Lars L

    2008-07-01

    Graft rejection after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning is a rare but serious clinical problem. Graft rejection and salvage therapy in eight patients in a retrospective analysis of 124 consecutive patients is reported. The patients were conditioned with low-dose fludarabine and total body irradiation (TBI). The association of pretransplantation risk factors with rejection and the effect of chimerism and graft-versus-host disease on rejection were analyzed. Overall survival (OS) and progression free survival (PFS) were compared between patients with and without rejection. Retransplantation was performed with increased TBI conditioning for all patients, and with increased mycophenolate mofetil doses for recipients with HLA-identical sibling donors. No known pretransplantation risk factors were confirmed in this study. Rejection episodes were unevenly distributed over time. The storage temperature of the apheresis products was identified as a risk factor for rejection. Storage of the apheresis products at 5 degrees C diminished the risk of rejection. Low donor T cell chimerism at Day +14 significantly increased the risk of rejection. Seven patients were retransplanted. All but one engrafted successfully, but with decreased OS and PFS. Two patients received pentostatin infusion prior to donor lymphocyte infusions in unsuccessful attempts at reversing rejection. Storage temperature and donor chimerism had a significant effect on rejection. Following rejection, patients are at greater risk of dying from infections and progression/relapse of their malignancy. Retransplantation is feasible and well tolerated after HCT with nonmyeloablative conditioning and should be performed without delay in patients with imminent and manifest graft rejection. PMID:18383319

  12. Effect of metformin on Schwann cells under hypoxia condition

    PubMed Central

    Ma, Junxiong; Liu, Jun; Yu, Hailong; Chen, Yu; Wang, Qi; Xiang, Liangbi

    2015-01-01

    Metformin, which is the first-line drug for the treatment of diabetes mellitus type 2, has been proved to possess beneficial effects on nerve regeneration in many studies. However, the underlying mechanism is currently unclear. The present study was designed to investigate the potential beneficial effect of metformin on SCs under hypoxia condition, which is a biological process at the injury site. The cell number and cell viability of SCs were examined using fluorescence observation and MTT assay. The migration of SCs was evaluated using a Transwell chamber. The expression and secretion of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell derived neurotrophic factor (GDNF) and neural cell adhesion molecule (N-CAM) in SCs were assayed by RT-PCR and ELISA method. The results showed that metformin could help SCs recover from hypoxia injury and inhibit hypoxia-induced apoptosis. In addition, metformin could partially reverse the detrimental effect of hypoxia on cell number, viability, migration and adhesion. Metformin is also capable of maintaining the biological activities of SCs after hypoxia injury, such as increasing the expression and secretion of BDNF, NGF, GDNF, and N-CAM. Further studies showed that pre-incubation with AMPK (5’-AMP-activated protein kinase) inhibitor Compound C might partially inhibit the effect of metformin mentioned above, indicating the possible involvement of AMPK pathway in the beneficial effects of metformin on peripheral nervous system. In conclusion, metformin is capable of alleviating hypoxia-induced injury to SCs and AMPK pathway might be involved in this process. PMID:26261558

  13. Cell-Free Protein Expression under Macromolecular Crowding Conditions

    PubMed Central

    Ge, Xumeng; Luo, Dan; Xu, Jianfeng

    2011-01-01

    Background Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process. Methodology/Principal Findings Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments. Conclusions/Significance Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE. PMID:22174874

  14. Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

    SciTech Connect

    Kawata, Shigehisa; Suzuki, Jun; Maruoka, Masahiro; Mizutamari, Megumi; Ishida-Kitagawa, Norihiro; Yogo, Keiichiro; Jat, Parmjit S.; Shishido, Tomoyuki . E-mail: shishid@bs.naist.jp

    2006-11-10

    Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.

  15. A microfluidic live cell assay to study anthrax toxin induced cell lethality assisted by conditioned medium

    PubMed Central

    Shen, Jie; Cai, Changzu; Yu, Zhilong; Pang, Yuhong; Zhou, Ying; Qian, Lili; Wei, Wensheng; Huang, Yanyi

    2015-01-01

    It is technically challenging to investigate the function of secreted protein in real time by supply of conditioned medium that contains secreted protein of interest. The internalization of anthrax toxin is facilitated by a secreted protein Dickkopf-1 (DKK1) and its receptor, and eventually leads to cell lethality. To monitor the dynamic interplay between these components in live cells, we use an integrated microfluidic device to perform the cell viability assays with real-time controlled culture microenvironment in parallel. Conditioned medium, which contains the secreted proteins from specific cell lines, can be continuously pumped towards the cells that exposed to toxin. The exogenous DKK1 secreted from distant cells is able to rescue the sensitivity to toxin for those DKK1-knocked-down cells. This high-throughput assay allows us to precisely quantify the dynamic interaction between key components that cause cell death, and provide independent evidence of the function of DKK1 in the complex process of anthrax toxin internalization. PMID:25731605

  16. Smooth muscle cell conditioned medium elevates angiotensin-converting enzyme of bovine pulmonary artery endothelial cells.

    PubMed

    Yu, F S; Lee, S L; Fanburg, B L

    1989-11-01

    Conditioned medium obtained from bovine pulmonary artery smooth muscle cells (SMC) in culture was found to elevate angiotensin-I-converting enzyme (ACE) of bovine pulmonary artery endothelial cells (EC) by 2- to 3-fold upon incubation for 24 to 48 h. The elevation in ACE was time dependent and inhibited by 10(-6) M cycloheximide, making it likely that the elevation was related to new protein synthesis by EC. Conditioned medium from EC failed to produce the same effect. The stimulatory effect of SMC conditioned medium on EC ACE was uninfluenced by exposures to anoxia as compared with room air, either during conditioning of medium or while assaying the effect of conditioned medium on EC ACE. Inhibitors of prostaglandin metabolism and calcium transport failed to influence the stimulatory effect of SMC conditioned medium on EC ACE. The stimulatory effect on EC ACE by conditioned medium was additive to that produced by Ca2+ ionophore A23187 and 3-isobutyl-1-methylxanthine and, similar to baseline ACE, was inhibited by 3 x 10(-8) M ouabain. Thus, SMC produce a factor that regulates the level of EC ACE. PMID:2484059

  17. Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis

    PubMed Central

    Lee, Esak; Fertig, Elana J.; Jin, Kideok; Sukumar, Saraswati; Pandey, Niranjan B.; Popel, Aleksander S.

    2014-01-01

    Breast cancer metastasis involves lymphatic dissemination in addition to hematogenous spreading. Although stromal lymphatic vessels (LVs) serve as initial metastatic routes, roles of organ-residing LVs are under-investigated. Here we show that lymphatic endothelial cells (LECs), a component of LVs within pre-metastatic niches, are conditioned by triple-negative breast cancer (TNBC) cells to accelerate metastasis. LECs within the lungs and lymph nodes, conditioned by tumor-secreted factors express CCL5 that is not expressed either in normal LECs or cancer cells, and direct tumor dissemination into these tissues. Moreover, tumor-conditioned LECs promote angiogenesis in these organs, allowing tumor extravasation and colonization. Mechanistically, tumor cell-secreted IL6 causes Stat3 phosphorylation in LECs. This pStat3 induces HIF-1α and VEGF, and a pStat3-pc-Jun-pATF-2 ternary complex induces CCL5 expression in LECs. This study demonstrates anti-metastatic activities of multiple repurposed drugs, blocking a self-reinforcing paracrine loop between breast cancer cells and LECs. PMID:25178650

  18. Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

    PubMed Central

    Lu, Xiaoyan; Chen, Dong; Liu, Zhiping; Li, Chaoyang; Liu, Ying; Zhou, Jin; Wan, Pengxia; Mou, Yong-gao

    2010-01-01

    Purpose To determine whether mouse embryonic stem cell conditioned medium (ESC-CM) increases the proliferative capacity of human corneal endothelial cells (HCECs) in vitro. Methods Primary cultures of HCECs were established from explants of the endothelial cell layer, including the Descemet’s membrane. Cells were cultured in human corneal endothelium medium (CEM) containing 25% ESC-CM for the experimental group and CEM alone for the control group. Phase-contrast microscopy and reverse-transcription polymerase chain reaction (RT–PCR) were used to identify HCECs. The eruption time and HCEC morphology were observed under phase-contrast microscopy. We detected the protein expression of zona occludens protein-1 (ZO-1; a tight junction protein) and the Na+-K+-ATPase by western blot analysis and immunocytochemistry. The mRNA expression of the Na+-K+-ATPase, voltage-dependent anion channel 3 (VDAC3), solute carrier family 4, sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel protein 3 (CLCN3) were detected by RT–PCR. To explore the proliferation capacity of HCECs, the colony forming efficiency (CFE) was determined by Giemsa staining and the cellular proliferation marker of Ki-67 protein (Ki-67) positive cells were detected by immunocytochemistry and flow cytometry. Progression of the cell cycle and apoptosis were analyzed by flow cytometry. Negative regulation of the cell cycle, as measured by cyclin-dependent kinase inhibitor p21 (p21) levels, was detected by western blot analysis and immunocytochemistry. Results In primary culture, HCECs in the 25%ESC-CM group erupted with polygonal appearance on day 2, while those in the CEM group erupted with slightly larger cells on day 3–4. HCECs in the 25%ESC-CM group could be subcultured until passage 6 without enlargement of cell volume, while those in the CEM group were enlarged and lost their polygonal appearance by passage 2. HCECs in both the 25%ESC-CM and CEM groups expressed ZO-1, Na

  19. Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions

    PubMed Central

    Jung, Juwon; Baek, Jin Ah; Seol, Hye Won; Choi, Young Min

    2016-01-01

    Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies. PMID:27294211

  20. Haematopoietic Stem Cell Transplantation for Refractory Langerhans Cell Histiocytosis: Outcome by Intensity of Conditioning

    PubMed Central

    Veys, Paul A.; Nanduri, Vasanta; Baker, K. Scott; He, Wensheng; Bandini, Giuseppe; Biondi, Andrea; Dalissier, Arnaud; Davis, Jeffrey H.; Eames, Gretchen M.; Egeler, R. Maarten; Filipovich, Alexandra H.; Fischer, Alain; Jürgens, Herbert; Krance, Robert; Lanino, Edoardo; Leung, Wing H.; Matthes, Susanne; Michel, Gérard; Orchard, Paul J.; Pieczonka, Anna; Ringdén, Olle; Schlegel, Paul G.; Sirvent, Anne; Vettenranta, Kim; Eapen, Mary

    2015-01-01

    Summary Patients with Langerhans cell histiocytosis (LCH) refractory to conventional chemotherapy have a poor outcome. There are currently two promising treatment strategies for high-risk patients: the first involves the combination of 2-chlorodeoxyadenosine and cytarbine; the other approach is allogeneic haematopoietic stem cell transplantation (HSCT). Here we evaluated 87 patients with high-risk LCH who were transplanted between 1990–2013. Prior to the year 2000, most patients underwent HSCT following myeloablative conditioning (MAC): only 5 of 20 patients (25%) survived with a high rate (55%) of transplant-related mortality (TRM). After the year 2000 an increasing number of patients underwent HSCT with reduced intensity conditioning (RIC): 49/67 (73%) patients survived, however, the improved survival was not overtly achieved by the introduction of RIC regimens with similar 3-year probability of survival after MAC (77%) and RIC transplantation (71%). There was no significant difference in TRM by conditioning regimen intensity but relapse rates were higher after RIC compared to MAC regimens (28% vs. 8%, p=0.02), although most patients relapsing after RIC transplantation could be salvaged with further chemotherapy. HSCT may be a curative approach in 3 out of 4 patients with high risk LCH refractory to chemotherapy: the optimal choice of HSCT conditioning remains uncertain. PMID:25817915

  1. Conditions of steady switching in phase-transition memory cells

    SciTech Connect

    Popov, A. I. Salnikov, S. M.; Anufriev, Yu. V.

    2015-04-15

    Three types of non-volatile memory cells of different designs based on phase transitions are developed and implemented. The effect of the design features of the cells and their active-region sizes on the switching characteristics and normal operation of the cells is considered as a whole. The causes of failure of the cells are analyzed from the obtained series of scanning electron images upon level-by-level etching of the samples. It is shown that the cell design is the most critical factor from the viewpoint of switching to the high-resistance state. The causes of this fact are analyzed and the criterion for providing the steady operation of cells of non-volatile memory based on phase transitions is formulated.

  2. Production of Plasminogen Activator in Cultures of Superior Cervical Ganglia and Isolated Schwann Cells

    NASA Astrophysics Data System (ADS)

    Alvarez-Buylla, Arturo; Valinsky, Jay E.

    1985-05-01

    Plasminogen activator has been implicated in tissue remodeling and cell migration during embryogenesis. In the developing nervous system, these processes are evident in the migration of neurons, axonal extension, Schwann cell migration, and the ensheathment and myelination of nerves. We have studied the production of plasminogen activator in cultures of superior cervical ganglia under conditions in which both neurons and glia are present. We have found that a principal source of the enzyme in these cultures is the glial cells and that the enzyme could not be detected at the growing tips of neurites. Plasminogen activator is also produced by Schwann cells isolated from neonatal rat sciatic nerve. The production of the enzyme by these cells is stimulated 6- to 10-fold by cholera toxin. Isolated Schwann cells and glial cells in the ganglion explant cultures produce the tissue form of plasminogen activator, a form of the enzyme not often found in nonmalignant cells. Preliminary experiments suggest that neuronal-glial interactions may regulate enzyme production by Schwann cells.

  3. Thermal imaging of solid oxide cells operating under electrolysis conditions

    NASA Astrophysics Data System (ADS)

    Cumming, D. J.; Elder, R. H.

    2015-04-01

    Solid oxide fuel cells remain at the forefront of research into electrochemical energy conversion technology. More recent interest has focused on operating in electrolyser mode to convert steam or carbon dioxide into hydrogen or carbon monoxide, respectively. The mechanism of these reactions is not fully understood, particularly when operated in co-electrolysis mode using both steam and CO2. This contribution reports the use of a thermal camera to directly observe changes in the cell temperature during operation, providing a remote, non-contact and highly sensitive method for monitoring an operational cell.

  4. Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin.

    PubMed

    Palchaudhuri, Rahul; Saez, Borja; Hoggatt, Jonathan; Schajnovitz, Amir; Sykes, David B; Tate, Tiffany A; Czechowicz, Agnieszka; Kfoury, Youmna; Ruchika, Fnu; Rossi, Derrick J; Verdine, Gregory L; Mansour, Michael K; Scadden, David T

    2016-07-01

    Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45-saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45-SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases. PMID:27272386

  5. The Induction of Metformin Inhibitory Effects on Tumor Cell Growth in Hypoxic Condition.

    PubMed

    Safari, Zohreh; Safaralizadeh, Reza; Seyedzadeh, Mir Hadi; Valinezad Orang, Ayla; Zare, Ahad; Hosseinpour Feizi, Mohammad Ali; Kardar, Gholam Ali

    2015-12-01

    It is aimed to evaluate the actual anti-cancerous effects of metformin on cancer cells in hypoxic condition. Non-cancerous cells (HEK293) and cancer cells (MCF-7) were cultured in both hypoxia and normoxia conditions and treated with different concentrations of metformin. The proliferation, apoptosis, and necrosis rate were assessed using MTT test and Annexin V assay. The S6K1 phosphorylation was assessed using western blotting. Zymography was used to measure the activity of metalloproteinase-9 (MMP-9). Metformin treatment inhibited proliferation of cancer cells in the optimal concentration of 10 mM under hypoxia condition, while it showed no effects on non-cancerous cell viability. The statistical analysis of MTT assay indicated that the pro-apoptotic function of metformin for cancer cells under hypoxia condition compared to normoxia was significant with different metformin concentrations (p<0.01). However, the effect of metformin treatments for non-cancerous cells under hypoxia condition compared to normoxia was not significant. Western-blot analysis indicated a significant decrease in S6K1 phosphorylation in cancer cells under hypoxia condition (p<0.05). Nevertheless, there was no considerable difference between normoxia and hypoxia conditions in non-cancerous cells. MMP-9 zymography analysis revealed that the highest inhibition of MMP-9 activity was observed in hypoxia condition by 20mM of metformin concentration only in cancer cell. The results indicate that in hypoxia condition metformin exerts its anti-cancerous function by inhibiting proliferation and tumor progression and inducing cell apoptosis more effectively than normoxia condition. In line with cancer cell conditions, most importantly hypoxic condition, metformin can be considered as a potential anti-cancerous drug. PMID:26725558

  6. Defining cell culture conditions to improve human norovirus infectivity assays.

    PubMed

    Straub, T M; Hutchison, J R; Bartholomew, R A; Valdez, C O; Valentine, N B; Dohnalkova, A; Ozanich, R M; Bruckner-Lea, C J

    2013-01-01

    Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log(10) increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus. PMID:23306266

  7. Defining cell culture conditions to improve human norovirus infectivity assays

    SciTech Connect

    Straub, Tim M.; Hutchison, Janine R.; Bartholomew, Rachel A.; Valdez, Catherine O.; Valentine, Nancy B.; Dohnalkova, Alice; Ozanich, Richard M.; Bruckner-Lea, Cindy J.

    2013-01-10

    Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that leads to more reproducible hNoV infectivity in vitro requires that the cell line be 1) of human gastrointestinal origin, 2) expresses apical microvilli, and 3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both reverse transcription quantitative PCR (qRT-PCR) and microscopy. Using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using quantitative reverse transcription PCR (qRT-PCR) that measures all RNA vs. plaque assays that measure infectious virus.

  8. Natural Treg cells spontaneously differentiate into pathogenic helper cells in lymphopenic conditions.

    PubMed

    Duarte, João H; Zelenay, Santiago; Bergman, Marie-Louise; Martins, Ana C; Demengeot, Jocelyne

    2009-04-01

    Induction of Forkhead-box p3 (Foxp3) expression in developing T cells upon peptide-MHC encountering has been proposed to define a lineage of committed Treg cells. However, sustained expression of Foxp3 is required for Treg function and what maintains Foxp3 expression in peripheral Treg remains obscure. To address this issue, we monitored natural Treg phenotype and function upon adoptive transfer into lymphocyte-deficient mice. We first show that about 50% of Foxp3-GFP(+) Treg isolated from Foxp3(gfp) KI animals loose Foxp3 expression in severe lymphopenic conditions. We next evidence that the cytokine IL-2, either produced by co-transferred conventional T cells or administrated i.v. prevents Foxp3 downregulation. Moreover, we document that Treg that lost Foxp3 expression upon adoptive transfer produce IL-2 are not suppressive and promote tissue infiltration and damage upon secondary transfer into alymphoid mice. Our findings that Treg convert into pathogenic Th cells in absence of IL-2 provide new clues to the success of Treg-based immune therapies. PMID:19291701

  9. Proliferation of differentiated glial cells in the brain stem.

    PubMed

    Barradas, P C; Cavalcante, L A

    1998-02-01

    Classical studies of macroglial proliferation in muride rodents have provided conflicting evidence concerning the proliferating capabilities of oligodendrocytes and microglia. Furthermore, little information has been obtained in other mammalian orders and very little is known about glial cell proliferation and differentiation in the subclass Metatheria although valuable knowledge may be obtained from the protracted period of central nervous system maturation in these forms. Thus, we have studied the proliferative capacity of phenotypically identified brain stem oligodendrocytes by tritiated thymidine radioautography and have compared it with known features of oligodendroglial differentiation as well as with proliferation of microglia in the opossum Didelphis marsupialis. We have detected a previously undescribed ephemeral, regionally heterogeneous proliferation of oligodendrocytes expressing the actin-binding, ensheathment-related protein 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), that is not necessarily related to the known regional and temporal heterogeneity of expression of CNPase in cell bodies. On the other hand, proliferation of microglia tagged by the binding of Griffonia simplicifolia B4 isolectin, which recognizes an alpha-D-galactosyl-bearing glycoprotein of the plasma membrane of macrophages/microglia, is known to be long lasting, showing no regional heterogeneity and being found amongst both ameboid and differentiated ramified cells, although at different rates. The functional significance of the proliferative behavior of these differentiated cells is unknown but may provide a low-grade cell renewal in the normal brain and may be augmented under pathological conditions. PMID:9686148

  10. Can manipulation of differentiation conditions eliminate proliferative cells from a population of ES cell-derived forebrain cells?

    PubMed

    Precious, Sophie V; Kelly, Claire M; Allen, Nicholas D; Rosser, Anne E

    2016-01-01

    There is preliminary evidence that implantation of primary fetal striatal cells provides functional benefit in patients with Huntington's disease, a neurodegenerative condition resulting in loss of medium-sized spiny neurons (MSN) of the striatum. Scarcity of primary fetal tissue means it is important to identify a renewable source of cells from which to derive donor MSNs. Embryonic stem (ES) cells, which predominantly default to telencephalic-like precursors in chemically defined medium (CDM), offer a potentially inexhaustible supply of cells capable of generating the desired neurons. Using an ES cell line, with the forebrain marker FoxG1 tagged to the LacZ reporter, we assessed effects of known developmental factors on the yield of forebrain-like precursor cells in CDM suspension culture. Addition of FGF2, but not DKK1, increased the proportion of FoxG1-expressing cells at day 8 of neural induction. Oct4 was expressed at day 8, but was undetectable by day 16. Differentiation of day 16 precursors generated GABA-expressing neurons, with few DARPP32 positive MSNs. Transplantation of day 8 precursor cells into quinolinic acid-lesioned striata resulted in generation of teratomas. However, transplantation of day 16 precursors yielded grafts expressing neuronal markers including NeuN, calbindin and parvalbumin, but no DARPP32 6 weeks post-transplantation. Manipulation of fate of ES cells requires optimization of both concentration and timing of addition of factors to culture systems to generate the desired phenotypes. Furthermore, we highlight the value of increasing the precursor phase of ES cell suspension culture when directing differentiation toward forebrain fate, so as to dramatically reduce the risk of teratoma formation. PMID:27606335

  11. Purkinje cell activity during classical conditioning with different conditional stimuli explains central tenet of Rescorla–Wagner model

    PubMed Central

    Rasmussen, Anders; Zucca, Riccardo; Johansson, Fredrik; Jirenhed, Dan-Anders; Hesslow, Germund

    2015-01-01

    A central tenet of Rescorla and Wagner’s model of associative learning is that the reinforcement value of a paired trial diminishes as the associative strength between the presented stimuli increases. Despite its fundamental importance to behavioral sciences, the neural mechanisms underlying the model have not been fully explored. Here, we present findings that, taken together, can explain why a stronger association leads to a reduced reinforcement value, within the context of eyeblink conditioning. Specifically, we show that learned pause responses in Purkinje cells, which trigger adaptively timed conditioned eyeblinks, suppress the unconditional stimulus (US) signal in a graded manner. Furthermore, by examining how Purkinje cells respond to two distinct conditional stimuli and to a compound stimulus, we provide evidence that could potentially help explain the somewhat counterintuitive overexpectation phenomenon, which was derived from the Rescorla–Wagner model. PMID:26504227

  12. Gene targeting in embryonic stem cells, II: conditional technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  13. EXAMINATION OF CULTURE CONDITIONS ON ESTERASE ACTIVITIES IN HUMAN AND MOUSE NEUROBLASTOMA CELLS

    EPA Science Inventory

    Because neuroblastoma cell lines have potential to be used as in vitro alternatives for screening of antiesterase compounds (e.g., organophosphates (OPs) and carbamates), information is needed on conditions under which the cells are grown as these conditions may contribute to exp...

  14. Nonlinear Dielectric Properties of Yeast Cells Cultured in Different Environmental Conditions

    NASA Astrophysics Data System (ADS)

    Kawanishi, Gomon; Fukuda, Naoki; Muraji, Masafumi

    The harmonics of the electric current through yeast suspensions, the nonlinear dielectric properties of yeast cells, have particular patterns according to the biological activity of the cells and the measurement of these patterns is a technique for determining the activity of living cells. The concentration of glucose and oxygen in yeast culture medium influences the manifestation of fermentation or respiration of yeast cells. Measurements were made with yeast cells (Saccharomyces cerevisiae) cultured aerobically and anaerobically in sufficient glucose concentration, aerobic fermentation and anaerobic fermentation, and aerobically in limited glucose concentration, respiration. The results showed that the harmonics were barely apparent for yeast cells in aerobic fermentation and respiratory; however, cells in the anaerobic fermentation displayed substantial third and fifth harmonics. We can say that environmental condition affects the yeast cells' nonlinear properties, from another viewpoint, the measurements of the nonlinear properties are available to determine the activity of yeast cells adjusted to the conditions of their cultivation.

  15. Differentiation of embryonic stem cells conditionally expressing neurogenin 3.

    PubMed

    Treff, Nathan R; Vincent, Robert K; Budde, Melisa L; Browning, Victoria L; Magliocca, Joseph F; Kapur, Vivek; Odorico, Jon S

    2006-11-01

    Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets. To better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profiles of murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3. An mESC line was created in order to induce Ngn3 by adding doxycycline to the culture medium. Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of contexts, including undifferentiated ESCs and differentiating embryoid bodies (EBs). Genes regulated by Ngn3 in a context-independent manner were identified and analyzed using systematic gene ontology tools. This analysis revealed Notch signaling as the most significantly regulated signaling pathway (p = .009). This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and, conversely, more sensitive to Notch signaling inhibition. Indeed, EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor-mediated Notch signaling inhibition (p < .0001) when compared with uninduced EBs. Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin expression. PMID:16809427

  16. Selective advantage of trisomic human cells cultured in non-standard conditions

    PubMed Central

    Rutledge, Samuel D.; Douglas, Temple A.; Nicholson, Joshua M.; Vila-Casadesús, Maria; Kantzler, Courtney L.; Wangsa, Darawalee; Barroso-Vilares, Monika; Kale, Shiv D.; Logarinho, Elsa; Cimini, Daniela

    2016-01-01

    An abnormal chromosome number, a condition known as aneuploidy, is a ubiquitous feature of cancer cells. A number of studies have shown that aneuploidy impairs cellular fitness. However, there is also evidence that aneuploidy can arise in response to specific challenges and can confer a selective advantage under certain environmental stresses. Cancer cells are likely exposed to a number of challenging conditions arising within the tumor microenvironment. To investigate whether aneuploidy may confer a selective advantage to cancer cells, we employed a controlled experimental system. We used the diploid, colorectal cancer cell line DLD1 and two DLD1-derived cell lines carrying single-chromosome aneuploidies to assess a number of cancer cell properties. Such properties, which included rates of proliferation and apoptosis, anchorage-independent growth, and invasiveness, were assessed both under standard culture conditions and under conditions of stress (i.e., serum starvation, drug treatment, hypoxia). Similar experiments were performed in diploid vs. aneuploid non-transformed human primary cells. Overall, our data show that aneuploidy can confer selective advantage to human cells cultured under non-standard conditions. These findings indicate that aneuploidy can increase the adaptability of cells, even those, such as cancer cells, that are already characterized by increased proliferative capacity and aggressive tumorigenic phenotypes. PMID:26956415

  17. Selective advantage of trisomic human cells cultured in non-standard conditions.

    PubMed

    Rutledge, Samuel D; Douglas, Temple A; Nicholson, Joshua M; Vila-Casadesús, Maria; Kantzler, Courtney L; Wangsa, Darawalee; Barroso-Vilares, Monika; Kale, Shiv D; Logarinho, Elsa; Cimini, Daniela

    2016-01-01

    An abnormal chromosome number, a condition known as aneuploidy, is a ubiquitous feature of cancer cells. A number of studies have shown that aneuploidy impairs cellular fitness. However, there is also evidence that aneuploidy can arise in response to specific challenges and can confer a selective advantage under certain environmental stresses. Cancer cells are likely exposed to a number of challenging conditions arising within the tumor microenvironment. To investigate whether aneuploidy may confer a selective advantage to cancer cells, we employed a controlled experimental system. We used the diploid, colorectal cancer cell line DLD1 and two DLD1-derived cell lines carrying single-chromosome aneuploidies to assess a number of cancer cell properties. Such properties, which included rates of proliferation and apoptosis, anchorage-independent growth, and invasiveness, were assessed both under standard culture conditions and under conditions of stress (i.e., serum starvation, drug treatment, hypoxia). Similar experiments were performed in diploid vs. aneuploid non-transformed human primary cells. Overall, our data show that aneuploidy can confer selective advantage to human cells cultured under non-standard conditions. These findings indicate that aneuploidy can increase the adaptability of cells, even those, such as cancer cells, that are already characterized by increased proliferative capacity and aggressive tumorigenic phenotypes. PMID:26956415

  18. Cell Motility Is Decreased in Macrophages Activated by Cancer Cell-Conditioned Medium

    PubMed Central

    Go, Ahreum; Ryu, Yun-Kyoung; Lee, Jae-Wook; Moon, Eun-Yi

    2013-01-01

    Macrophages play a role in innate immune responses to various foreign antigens. Many products from primary tumors influence the activation and transmigration of macrophages. Here, we investigated a migration of macrophages stimulated with cancer cell culture-conditioned medium (CM). Macrophage activation by treatment with CM of B16F10 cells were judged by the increase in protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). The location where macrophages were at 4 h-incubation with control medium or CM was different from where they were at 5 h-incubation in culture dish. Percentage of superimposed macrophages at every 1 h interval was gradually increased by CM treatment as compared to control. Total coverage of migrated track expressed in coordinates was smaller and total distance of migration was shorter in CM-treated macrophages than that in control. Rac1 activity in CM-treated macrophages was also decreased as compared to that in control. When macrophages were treated with CM in the presence of dexamethasone (Dex), an increase in COX2 protein levels, and a decrease in Rac1 activity and total coverage of migration were reversed. In the meanwhile, biphasic changes were detected by Dex treatment in section distance of migration at each time interval, which was more decreased at early time and then increased at later time. Taken together, data demonstrate that macrophage motility could be reduced in accordance with activation in response to cancer cell products. It suggests that macrophage motility could be a novel marker to monitor cancer-associated inflammatory diseases and the efficacy of anti-inflammatory agents. PMID:24404340

  19. ReNCell VM conditioned medium enhances the induction of dental pulp stem cells into dopaminergic like cells.

    PubMed

    Gnanasegaran, Nareshwaran; Govindasamy, Vijayendran; Musa, Sabri; Abu Kasim, Noor Hayaty

    2016-03-01

    Among the debilitating diseases, neurological related diseases are the most challenging ones to be treated using cell replacement therapies. Recently, dental pulp stem cells (SHED) were found to be most suitable cell choice for neurological related diseases as evidenced with many preclinical studies. To enhance the neurological potential of SHED, we recapitulated one of the pharmacological therapeutic tools in cell replacement treatment, we pre-conditioned dental pulp stem cells (SHED) with culture medium of ReNCell VM, an immortalized neuron progenitor cell, prior to neurogenesis induction and investigated whether this practice enhances their neurogenesis potential especially towards dopaminergic neurons. We hypothesed that the integration of pharmacological practices such as co-administration of various drugs, a wide range of doses and duration as well as pre-conditioning into cell replacement may enhance the efficacy of stem cell therapy. In particular, pre-conditioning is shown to be involved in the protective effect from some membrano-tropic drugs, thereby improving the resistance of cell structures and homing capabilities. We found that cells pre-treated with ReNCell VM conditioned medium displayed bipolar structures with extensive branches resembling putative dopaminergic neurons as compared to non-treated cells. Furthermore, many neuronal related markers such as NES, NR4A2, MSI1, and TH were highly expressed (fold changes > 2; p < 0.05) in pre-treated cells. Similar observations were detected at the protein level. The results demonstrate for the first time that SHED pre-conditioning enhances neurological potential and we suggest that cells should be primed to their respective environment prior to transplantation. PMID:25322895

  20. Cellular interactions via conditioned media induce in vivo nephron generation from tubular epithelial cells or mesenchymal stem cells

    SciTech Connect

    Machiguchi, Toshihiko Nakamura, Tatsuo

    2013-06-07

    Highlights: •We have attempted in vivo nephron generation using conditioned media. •Vascular and tubular cells do cross-talks on cell proliferation and tubular changes. •Tubular cells suppress these changes in mesenchymal stem cells. •Tubular cells differentiate mesenchymal stem cells into tubular cells. •Nephrons can be created from implanted tubular cells or mesenchymal stem cells. -- Abstract: There are some successful reports of kidney generation by utilizing the natural course of kidney development, namely, the use of an artificially treated metanephros, blastocyst or ureteric bud. Under a novel concept of cellular interactions via conditioned media (CMs), we have attempted in vivo nephron generation from tubular epithelial cells (TECs) or mesenchymal stem cells (MSCs). Here we used 10× CMs of vascular endothelial cells (VECs) and TECs, which is the first to introduce a CM into the field of organ regeneration. We first present stimulative cross-talks induced by these CMs between VECs and TECs on cell proliferation and morphological changes. In MSCs, TEC-CM suppressed these changes, however, induced cytokeratin expression, indicating the differentiation of MSCs into TECs. As a result, glomerular and tubular structures were created following the implantation of TECs or MSCs with both CMs. Our findings suggest that the cellular interactions via CMs might induce in vivo nephron generation from TECs or MSCs. As a promoting factor, CMs could also be applied to the regeneration of other organs and tissues.

  1. Investigating Microbial Fuel Cell Bioanode Performance Under Different Cathode Conditions

    SciTech Connect

    Borole, Abhijeet P; Hamilton, Choo Yieng; Aaron, D; Tsouris, Costas

    2009-01-01

    A compact, three-in-one, flow-through, porous, electrode design with minimal electrode spacing and minimal dead volume was implemented to develop a microbial fuel cell (MFC) with improved anode performance. A biofilm-dominated anode consortium enriched under a multimode, continuous-flow regime was used. The increase in the power density of the MFC was investigated by changing the cathode (type, as well as catholyte strength) to determine whether anode was limiting. The power density obtained with an air-breathing cathode was 56 W/m3 of net anode volume (590 mW/m2) and 203 W/m3 (2160 mW/m2) with a 50-mM ferricyanide- based cathode. Increasing the ferricyanide concentration and ionic strength further increased the power density, reaching 304 W/m3 (3220 mW/m2, with 200 mM ferricyanide and 200 mM buffer concentration). The increasing trend in the power density indicated that the anode was not limiting and that higher power densities could be obtained using cathodes capable of higher rates of oxidation. The internal solution resistance for the MFC was 5 6 X, which supported the improved performance of the anode design. A new parameter defined as the ratio of projected surface area to total anode volume is suggested as a design parameter to relate volumetric and area-based power densities and to enable comparison of various MFC configurations.

  2. T cells conditioned with MDSC show an increased anti-tumor activity after adoptive T cell based immunotherapy

    PubMed Central

    Raber, Patrick L.; Sierra, Rosa A.; Thevenot, Paul T.; Shuzhong, Zhang; Wyczechowska, Dorota D.; Kumai, Takumi; Celis, Esteban; Rodriguez, Paulo C.

    2016-01-01

    The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including T cell proliferation and the expression of various cytotoxic mediators. Paradoxically, the inhibition of CD8+ T cell differentiation into cytotoxic populations increased their efficacy after ACT into tumor-bearing hosts. Therefore, we aimed to test the impact of conditioning CD8+ T cells with MDSC on their differentiation potential and ACT efficacy. Our results indicate that MDSC impaired the progression of CD8+ T cells into effector populations, without altering their activation status, production of IL-2, or signaling through the T cell receptor. In addition, culture of CD8+ T cells with MDSC resulted in an increased ACT anti-tumor efficacy, which correlated with a higher frequency of the transferred T cells and elevated IFNγ production. Interestingly, activated CD62L+ CD8+ Tcells were responsible for the enhanced anti-tumor activity showed by MDSC-exposed T cells. Additional results showed a decreased protein synthesis rate and lower activity of the mammalian/mechanistic target of rapamycin (mTOR) in T cells conditioned with MDSC. Silencing of the negative mTOR regulator tuberous sclerosis complex-2 in T cells co-cultured with MDSC restored mTOR activity, but resulted in T cell apoptosis. These results indicate that conditioning of T cells with MDSC induces stress survival pathways mediated by a blunted mTOR signaling, which regulated T cell differentiation and ACT efficacy. Continuation of this research will enable the development of better strategies to increase ACT responses in cancer. PMID:27007050

  3. Distinct patterns of cell motion inside a micro-channel under different osmotic conditions.

    PubMed

    Tsai, Chia-Hung Dylan; Kaneko, Makoto; Sakuma, Shinya; Arai, Fumihito

    2013-01-01

    The effect of osmotic condition on a living cell inside a micro-channel is firstly studied in this work. By utilizing a high-speed camera, we observed distinct patterns of cell motion under different osmotic conditions, which are established by saline with different concentrations of sodium chloride (NaCl). The cell motions are tracked by a computer, and are presented by the coordinates of location and time (x-t chart). The motions of cells under hypotonic condition (NaCl% < 0.9%) are convex curves on the chart while the ones under isotonic and hypertonic conditions (NaCl% ≥ 0.9%) are concave curves. Since saline is widely used in both medical practices and cell-related researches, our results point out two important facts: 1) Cells are sensitive to the percentage of NaCl. One percent difference in overall concentration makes dramatic changes in cell characteristics, such as cell stiffness. 2) The micro-channel method can clearly tell the difference between hypotonic, isotonic and hypertonic conditions according to the pattern of cell motion. Interpretations of the phenomena from different perspectives are also discussed in this paper. PMID:24110988

  4. Sustainable landfilling in tropical conditions: comparison between open and closed cell approach.

    PubMed

    Visvanathan, C; Karthikeyan, Obuli P; Park, K H

    2011-04-01

    Two landfill test cells were constructed in a tropical climate using locally available low-cost materials. One cell was operated without a cover on the municipal solid waste to simulate 'open landfill' conditions and the other cell was covered to create 'closed landfill' conditions. Both test cells were monitored over a period of 290 days under rainy, dry and artificial wetting conditions. Due to the relatively high compaction density of waste in the closed test cell, the substrate settlement was gradual and comparatively lower than in the open test cell. Multiple top covers in the closed test cell resulted in significant run-off of incident precipitation during the rainy season, which delayed the waste stabilization and subsequently produced a lesser volume of leachate. On the other hand, operation of the open test cell was found to be advantageous in terms of leachate management and substrate settlement along with waste stabilization pattern. Infiltration of rain-water into the waste mass leached out the maximum organic pollutants and oxidized the nitrogen content, which is deemed to be a benefit of operating an open cell landfill under tropical conditions. Artificial wetting during dry periods by recirculation of stored leachate notably accelerated the waste stabilization and secondary substrate settlement in the open test cell. The continuous monitoring of ground-water quality from the site showed only seasonal variations. PMID:20855353

  5. Human Airway Primary Epithelial Cells Show Distinct Architectures on Membrane Supports Under Different Culture Conditions.

    PubMed

    Min, Kyoung Ah; Rosania, Gus R; Shin, Meong Cheol

    2016-06-01

    To facilitate drug development for lung delivery, it is highly demanding to establish appropriate airway epithelial cell models as transport barriers to evaluate pharmacokinetic profiles of drug molecules. Besides the cancer-derived cell lines, as the primary cell model, normal human bronchial epithelial (NHBE) cells have been used for drug screenings because of physiological relevance to in vivo. Therefore, to accurately interpret drug transport data in NHBE measured by different laboratories, it is important to know biophysical characteristics of NHBE grown on membranes in different culture conditions. In this study, NHBE was grown on the polyester membrane in a different medium and its transport barrier properties as well as cell architectures were fully characterized by functional assays and confocal imaging throughout the days of cultures. Moreover, NHBE cells on inserts in a different medium were subject to either of air-interfaced culture (AIC) or liquid-covered culture (LCC) condition. Cells in the AIC condition were cultivated on the membrane with medium in the basolateral side only, whereas cells with medium in apical and basolateral sides under the LCC condition. Quantitative microscopic imaging with biophysical examination revealed distinct multilayered architectures of differentiated NHBE cells, suggesting NHBE as functional cell barriers for the lung-targeting drug transport. PMID:26818810

  6. Glial expression of Swiss cheese (SWS), the Drosophila orthologue of neuropathy target esterase (NTE), is required for neuronal ensheathment and function

    PubMed Central

    Dutta, Sudeshna; Rieche, Franziska; Eckl, Nina; Duch, Carsten; Kretzschmar, Doris

    2016-01-01

    ABSTRACT Mutations in Drosophila Swiss cheese (SWS) or its vertebrate orthologue neuropathy target esterase (NTE), respectively, cause progressive neuronal degeneration in Drosophila and mice and a complex syndrome in humans that includes mental retardation, spastic paraplegia and blindness. SWS and NTE are widely expressed in neurons but can also be found in glia; however, their function in glia has, until now, remained unknown. We have used a knockdown approach to specifically address SWS function in glia and to probe for resulting neuronal dysfunctions. This revealed that loss of SWS in pseudocartridge glia causes the formation of multi-layered glial whorls in the lamina cortex, the first optic neuropil. This phenotype was rescued by the expression of SWS or NTE, suggesting that the glial function is conserved in the vertebrate protein. SWS was also found to be required for the glial wrapping of neurons by ensheathing glia, and its loss in glia caused axonal damage. We also detected severe locomotion deficits in glial sws-knockdown flies, which occurred as early as 2 days after eclosion and increased further with age. Utilizing the giant fibre system to test for underlying functional neuronal defects showed that the response latency to a stimulus was unchanged in knockdown flies compared to controls, but the reliability with which the neurons responded to increasing frequencies was reduced. This shows that the loss of SWS in glia impairs neuronal function, strongly suggesting that the loss of glial SWS plays an important role in the phenotypes observed in the sws mutant. It is therefore likely that changes in glia also contribute to the pathology observed in humans that carry mutations in NTE. PMID:26634819

  7. Temperature Characteristics Analysis of Triple-Junction Solar Cell under Concentrated Conditions using Spice Diode Model

    NASA Astrophysics Data System (ADS)

    Sakurada, Yuya; Ota, Yasuyuki; Nishioka, Kensuke

    2011-12-01

    Using spice diode model, the temperature characteristics of an InGaP/InGaAs/Ge triple-junction solar cell under concentrated light conditions were analyzed in detail. The current-voltage (I-V) characteristics of the single-junction solar cells (InGaP, InGaAs, and Ge solar cells) were measured at various temperatures. From dark I-V characteristics of each single-junction solar cell, the diode parameters and temperature exponents were extracted. The extracted diode parameters and temperature exponents were applied to the equivalent circuit model for the triple-junction solar cell, and the solar cell performance was calculated with considering the temperature characteristics of series resistance. There was good agreement between the measured and calculated I-V characteristics of the triple-junction solar cell at various temperatures under concentrated light conditions.

  8. Synapses lacking astrocyte appear in the amygdala during consolidation of Pavlovian threat conditioning

    PubMed Central

    Ostroff, Linnaea E.; Manzur, Mustfa K.; Cain, Christopher K.; LeDoux, Joseph E.

    2014-01-01

    There is growing evidence that astrocytes, long held to merely provide metabolic support in the adult brain, participate in both synaptic plasticity and learning and memory. Astrocytic processes are sometimes present at the synaptic cleft, suggesting that they might act directly at individual synapses. Associative learning induces synaptic plasticity and morphological changes at synapses in the lateral amygdala (LA). To determine whether astrocytic contacts are involved in these changes, we examined LA synapses after either threat conditioning (also called fear conditioning) or conditioned inhibition in adult rats using serial section transmission electron microscopy (ssTEM) reconstructions. There was a transient increase in the density of synapses with no astrocytic contact after threat conditioning, especially on enlarged spines containing both polyribosomes and a spine apparatus. In contrast, synapses with astrocytic contacts were smaller after conditioned inhibition. This suggests that during memory consolidation astrocytic processes are absent if synapses are enlarging but present if they are shrinking. We measured the perimeter of each synapse and its degree of astrocyte coverage, and found that only about 20–30% of each synapse was ensheathed. The amount of synapse perimeter surrounded by astrocyte did not scale with synapse size, giving large synapses a disproportionately long astrocyte-free perimeter and resulting in a net increase in astrocyte-free perimeter after threat conditioning. Thus astrocytic processes do not mechanically isolate LA synapses, but may instead interact through local signaling, possibly via cell-surface receptors. Our results suggest that contact with astrocytic processes opposes synapse growth during memory consolidation. PMID:24338694

  9. Efficient Conversion of Spermatogonial Stem Cells to Phenotypic and Functional Dopaminergic Neurons via the PI3K/Akt and P21/Smurf2/Nolz1 Pathway.

    PubMed

    Yang, Hao; Liu, Yang; Hai, Yanan; Guo, Ying; Yang, Shi; Li, Zheng; Gao, Wei-Qiang; He, Zuping

    2015-12-01

    Parkinson's disease (PD) is a common neurodegenerative syndrome characterized by loss of midbrain dopaminergic (DA) neurons. Generation of functional dopaminergic (DA) neurons is of unusual significance for treating Parkinson's disease (PD). However, direct conversion of spermatogonial stem cells (SSCs) to functional DA neurons without being reprogrammed to a pluripotent status has not been achieved. Here, we report an efficient approach to obtain morphological, phenotypic, and functional DA neurons from SSCs using a specific combination of olfactory ensheathing cell-conditioned medium (OECCM) and several defined growth factors (DGF). By following the current protocol, direct conversion of SSCs (both SSC line and primary SSCs) to neural cells and DA neurons was demonstrated by expression of numerous phenotypic genes and proteins for neural cells, as well as cell morphological features. More significantly, SSCs-derived DA neurons acquired neuronal functional properties such as synapse formation, electrophysiology activity, and dopamine secretion. Furthermore, PI3K/Akt pathway and p21/Nolz1 cascades were activated whereas Smurf2 was inactivated, leading to cell cycle exit during the conversion of SSCs into DA neurons. Collectively, this study could provide sufficient neural cells from SSCs for applications in the treatment of PD and offers novel insights into mechanisms underlying neural system development from the line of germ cells. PMID:25373443

  10. IL-12-conditioning improves retrovirally-mediated transduction efficiency of CD8+ T cells

    PubMed Central

    Andrijauskaite, Kristina; Suriano, Samantha; Cloud, Colleen A.; Li, Mingli; Kesarwani, Pravin; Stefanik, Leah S.; Moxley, Kelly M.; Salem, Mohamed L; Garrett-Mayer, Elizabeth; Paulos, Chrystal M.; Mehrotra, Shikhar; Kochenderfer, James N.; Cole, David J.; Rubinstein, Mark P.

    2016-01-01

    The ability to genetically modify T cells is a critical component to many immunotherapeutic strategies and research studies. However, the success of these approaches is often limited by transduction efficiency. Since retroviral vectors require cell division for integration, transduction efficiency is dependent on the appropriate activation and culture conditions for T cells. Naïve CD8+ T cells which are quiescent must be first activated to induce cell division to allow genetic modification. To optimize this process, we activated mouse T cells with a panel of different cytokines, including IL-2, IL-4, IL-6, IL-7, IL-12, IL-15 and IL-23, known to act on T cells. After activation, cytokines were removed, and activated T cells were retrovirally transduced. We found that IL-12 pre-conditioning of mouse T cells greatly enhanced transduction efficiency while preserving function and expansion potential. We also observed a similar transduction enhancing effect of IL-12 pre-conditioning on human T cells. These findings provide a simple method to improve the transduction efficiencies of CD8+ T cells. PMID:26182912

  11. NLRC5 shields T lymphocytes from NK-cell-mediated elimination under inflammatory conditions

    PubMed Central

    Ludigs, Kristina; Jandus, Camilla; Utzschneider, Daniel T.; Staehli, Francesco; Bessoles, Stéphanie; Dang, Anh Thu; Rota, Giorgia; Castro, Wilson; Zehn, Dietmar; Vivier, Eric; Held, Werner; Romero, Pedro; Guarda, Greta

    2016-01-01

    NLRC5 is a transcriptional regulator of MHC class I (MHCI), which maintains high MHCI expression particularly in T cells. Recent evidence highlights an important NK–T-cell crosstalk, raising the question on whether NLRC5 specifically modulates this interaction. Here we show that NK cells from Nlrc5-deficient mice exhibit moderate alterations in inhibitory receptor expression and responsiveness. Interestingly, NLRC5 expression in T cells is required to protect them from NK-cell-mediated elimination upon inflammation. Using T-cell-specific Nlrc5-deficient mice, we show that NK cells surprisingly break tolerance even towards ‘self' Nlrc5-deficient T cells under inflammatory conditions. Furthermore, during chronic LCMV infection, the total CD8+ T-cell population is severely decreased in these mice, a phenotype reverted by NK-cell depletion. These findings strongly suggest that endogenous T cells with low MHCI expression become NK-cell targets, having thus important implications for T-cell responses in naturally or therapeutically induced inflammatory conditions. PMID:26861112

  12. Conditioned medium as a strategy for human stem cells chondrogenic differentiation.

    PubMed

    Alves da Silva, M L; Costa-Pinto, A R; Martins, A; Correlo, V M; Sol, P; Bhattacharya, M; Faria, S; Reis, R L; Neves, Nuno M

    2015-06-01

    Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow-derived mesenchymal stem cells (hBSMCs) and human Wharton's jelly-derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three-dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage-related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications. PMID:24155167

  13. Mathematical model of quasistationary conditions of mass transfer in an electrodialysis cell

    NASA Astrophysics Data System (ADS)

    Khanmamedov, M. N.

    2000-07-01

    The author suggests a quasistationary mathematical model of the mass-transfer conditions in an electrodialysis cell in which the main operating parameters of the electrodialysis apparatus are expressed as a function of the dimensionless diluate concentration.

  14. Concise Review: Stem Cell Trials Using Companion Animal Disease Models.

    PubMed

    Hoffman, Andrew M; Dow, Steven W

    2016-07-01

    Studies to evaluate the therapeutic potential of stem cells in humans would benefit from more realistic animal models. In veterinary medicine, companion animals naturally develop many diseases that resemble human conditions, therefore, representing a novel source of preclinical models. To understand how companion animal disease models are being studied for this purpose, we reviewed the literature between 2008 and 2015 for reports on stem cell therapies in dogs and cats, excluding laboratory animals, induced disease models, cancer, and case reports. Disease models included osteoarthritis, intervertebral disc degeneration, dilated cardiomyopathy, inflammatory bowel diseases, Crohn's fistulas, meningoencephalomyelitis (multiple sclerosis-like), keratoconjunctivitis sicca (Sjogren's syndrome-like), atopic dermatitis, and chronic (end-stage) kidney disease. Stem cells evaluated in these studies included mesenchymal stem-stromal cells (MSC, 17/19 trials), olfactory ensheathing cells (OEC, 1 trial), or neural lineage cells derived from bone marrow MSC (1 trial), and 16/19 studies were performed in dogs. The MSC studies (13/17) used adipose tissue-derived MSC from either allogeneic (8/13) or autologous (5/13) sources. The majority of studies were open label, uncontrolled studies. Endpoints and protocols were feasible, and the stem cell therapies were reportedly safe and elicited beneficial patient responses in all but two of the trials. In conclusion, companion animals with naturally occurring diseases analogous to human conditions can be recruited into clinical trials and provide realistic insight into feasibility, safety, and biologic activity of novel stem cell therapies. However, improvements in the rigor of manufacturing, study design, and regulatory compliance will be needed to better utilize these models. Stem Cells 2016;34:1709-1729. PMID:27066769

  15. Combination of a peptide-modified gellan gum hydrogel with cell therapy in a lumbar spinal cord injury animal model.

    PubMed

    Gomes, Eduardo D; Mendes, Sofia S; Leite-Almeida, Hugo; Gimble, Jeffrey M; Tam, Roger Y; Shoichet, Molly S; Sousa, Nuno; Silva, Nuno A; Salgado, António J

    2016-10-01

    Spinal Cord Injury (SCI) is a highly incapacitating condition for which there is still no cure. Current clinical approaches are mainly based on palliative care, so there is a need to find possible treatments to SCI. Cellular transplantation is regarded with great expectation due to the therapeutic potential of cells such as Adipose tissue-derived Stromal/Stem Cells (ASCs) or Olfactory Ensheathing Cells (OECs). Both are accessible sources and present positive paracrine and cell-to-cell interactions, previously reported by our group. Additionally, biomaterials such as hydrogels have been applied in SCI repair with promising results. We propose to combine a GRGDS-modified gellan gum hydrogel with ASCs and OECs in order to promote SCI regeneration. In vitro, ASCs and OECs could be co-cultured within GG-GRGDS hydrogels inducing a more robust neurite outgrowth when compared to controls. In vivo experiments in a hemisection SCI rat model revealed that the administration of ASCs and OECs encapsulated in a GG-GRGDS hydrogel led to significant motor improvements when compared to both control (SCI) and hydrogel alone (GG-GRGDS) groups. This was accompanied by a decreased infiltration of inflammatory cells and astrocytes, and by an increased intensity of neurofilament. These results suggest evident gains induced by the encapsulation of ASCs and OECs in GG-GRGDS based hydrogels. PMID:27505621

  16. Initial conditioning of polymer eelectrolyte membrane fuel cell by temperature and potential cycling.

    PubMed

    Bezmalinović, Dario; Radošević, Jagoda; Barbir, Frano

    2015-01-01

    Polymer electrolyte membrane fuel cells need initial conditioning, activation or break-in the first time they are operated after being assembled. During this period performance of the fuel cell improves until it reaches its nominal performance. The exact mechanism of this initial conditioning is not completely understood, but it is assumed that during the conditioning process the polymer membrane, as well as the polymer in the catalyst layer, get humidified, and the number of active catalyst sites increases. Activation procedure proposed here consists of temperature and potential cycling. Temperature cycling is a new approach for the conditioning and the idea is to rapidly cool the running cell at some point to allow the membrane to equilibrate with condensed water which should result in higher intake of water within the membrane. The results show that proposed procedure is better or at least comparable to some conventional procedures for the initial conditioning. PMID:25830963

  17. Tumor-associated fibroblast-conditioned medium induces CDDP resistance in HNSCC cells

    PubMed Central

    Steinbichler, Teresa Bernadette; Metzler, Veronika; Pritz, Christian; Riechelmann, Herbert; Dudas, Jozsef

    2016-01-01

    Objective EMT (epithelial to mesenchymal transition) contributes to tumor progression and metastasis. We aimed to investigate the effects of EMT on CDDP resistance in HNSCC (head and neck squamous cell carcinoma)-cells. Methods EMT was induced using conditioned medium from a tumor cell/fibroblast co-culture. HNSCC cells were alternatively treated with TGF-β1. The response to CDDP was evaluated with viability and clonogenic assays. Results Treatment of SCC-25/Detroit 562 cells with conditioned medium increased viability of the tumor cells. Moreover, it doubled the IC50 of CDDP of SCC-25 cells from 6.2 μM to 13.1 μM (p < 0.001). The IC50 of CDDP of Detroit 562 cells was increased following treatment with conditioned medium from 13.1 μM to 26.8 μM (p < 0.01). Colony forming ability after treatment with 5 or 10 μM CDDP was significantly higher in HNSCC cells treated with co-culture conditioned medium than in controls (p < 0.05). Treatment with TGF-β1 had no effect on the IC50 of CDDP (p > 0.1). Conclusions Cell free medium from a co-culture was able to induce EMT in HNSCC cells. Co-culture treated HNSCC cells revealed increased viability and were less sensitive to CDDP treatment. TGF-β1 also induced a mesenchymal phenotype, but did not alter resistance to CDDP in HNSCC cells. PMID:26497215

  18. Hypergravity and hypobaric hypoxic conditions promote endothelial cell and platelet activation.

    PubMed

    Rubenstein, David A; Yin, Wei

    2014-09-01

    Cardiovascular disease risk is heightened during exposure to altered gravity and/or altered barometric conditions. Previous work has suggested that this heightened cardiovascular risk is due to enhancements of endothelial cell inflammatory and/or thrombogenic responses. In recent work, the role of platelets on instigating or inhibiting endothelial cell responses associated with cardiovascular disease has been found to be dependent on both biochemical and biophysical factors. In this work, we aimed to determine how two biophysical forces, gravity and atmospheric pressure, alter endothelial cell and platelet functions and their interactions to instigate or inhibit cardiovascular disease responses. To address this aim, endothelial cells and platelets were subjected to a force 8 times greater than the normal gravitational force, for up to 30 minutes. In separate experiments, endothelial cells and platelets were subjected to 50% of normal atmospheric pressure. Endothelial cell and platelet responses, associated with cardiovascular diseases, were measured as a time course during exposure. In general, the exposure of endothelial cells to either hypergravity or hypobaric conditions enhanced cardiovascular disease responses. However, the presence of platelets generally inhibited endothelial cell responses. Platelet activation was, however, somewhat enhanced under both hypergravity and hypobaric conditions. Our data suggest that altered biophysical forces can modulate endothelial cell and platelet responses that are salient for cardiovascular disease progression. However, the interaction of these two cells tends to restrain the progression of the pro-cardiovascular disease responses. PMID:25211651

  19. The Effect of Hypoxic Preconditioning on Induced Schwann Cells under Hypoxic Conditions

    PubMed Central

    Chen, Ou; Wu, Miaomiao; Jiang, Liangfu

    2015-01-01

    Object Our objective was to explore the protective effects of hypoxic preconditioning on induced Schwann cells exposed to an environment with low concentrations of oxygen. It has been observed that hypoxic preconditioning of induced Schwann cells can promote axonal regeneration under low oxygen conditions. Method Rat bone marrow mesenchymal stem cells (MSCs) were differentiated into Schwann cells and divided into a normal oxygen control group, a hypoxia-preconditioning group and a hypoxia group. The ultrastructure of each of these groups of cells was observed by electron microscopy. In addition, flow cytometry was used to measure changes in mitochondrial membrane potential. Annexin V-FITC/PI staining was used to detect apoptosis, and Western blots were used to detect the expression of Bcl-2/Bax. Fluorescence microscopic observations of axonal growth in NG-108 cells under hypoxic conditions were also performed. Results The hypoxia-preconditioning group maintained mitochondrial cell membrane and crista integrity, and these cells exhibited less edema than the hypoxia group. In addition, the cells in the hypoxia-preconditioning group were found to be in early stages of apoptosis, whereas cells from the hypoxia group were in the later stages of apoptosis. The hypoxia-preconditioning group also had higher levels of Bcl-2/Bax expression and longer NG-108 cell axons than were observed in the hypoxia group. Conclusion Hypoxic preconditioning can improve the physiological state of Schwann cells in a severe hypoxia environment and improve the ability to promote neurite outgrowth. PMID:26509259

  20. Unimpaired trace classical eyeblink conditioning in Purkinje cell degeneration (pcd) mutant mice

    PubMed Central

    Brown, Kevin L.; Agelan, Alexis; Woodruff-Pak, Diana S.

    2009-01-01

    Young adult Purkinje cell degeneration (pcd) mutant mice, with complete loss of cerebellar cortical Purkinje cells, are impaired in delay eyeblink classical conditioning. In the delay paradigm, the conditioned stimulus (CS) overlaps and coterminates with the unconditioned stimulus (US), and the cerebellar cortex supports normal acquisition. The ability of pcd mutant mice to acquire trace eyeblink conditioning in which the CS and US do not overlap has not been explored. Recent evidence suggests that cerebellar cortex may not be necessary for trace eyeblink classical conditioning. Using a 500 ms trace paradigm for which forebrain structures are essential in mice, we assessed the performance of homozygous male pcd mutant mice and their littermates in acquisition and extinction. In contrast to results with delay conditioning, acquisition of trace conditioning was unimpaired in pcd mutant mice. Extinction to the CS alone did not differ between pcd and littermate control mice, and timing of the conditioned response was not altered by the absence of Purkinje cells during acquisition or extinction. The ability of pcd mutant mice to acquire and extinguish trace eyeblink conditioning at levels comparable to controls suggests that the cerebellar cortex is not a critical component of the neural circuitry underlying trace conditioning. Results indicate that the essential neural circuitry for trace eyeblink conditioning involves connectivity that bypasses cerebellar cortex. PMID:19931625

  1. RSS1 regulates the cell cycle and maintains meristematic activity under stress conditions in rice

    PubMed Central

    Ogawa, Daisuke; Abe, Kiyomi; Miyao, Akio; Kojima, Mikiko; Sakakibara, Hitoshi; Mizutani, Megumi; Morita, Haruka; Toda, Yosuke; Hobo, Tokunori; Sato, Yutaka; Hattori, Tsukaho; Hirochika, Hirohiko; Takeda, Shin

    2011-01-01

    Plant growth and development are sustained by continuous cell division in the meristems, which is perturbed by various environmental stresses. For the maintenance of meristematic functions, it is essential that cell division be coordinated with cell differentiation. However, it is unknown how the proliferative activities of the meristems and the coordination between cell division and differentiation are maintained under stressful conditions. Here we show that a rice protein, RSS1, whose stability is controlled by cell cycle phases, contributes to the vigour of meristematic cells and viability under salinity conditions. These effects of RSS1 are exerted by regulating the G1–S transition, possibly through an interaction of RSS1 with protein phosphatase 1, and are mediated by the phytohormone, cytokinin. RSS1 is conserved widely in plant lineages, except eudicots, suggesting that RSS1-dependent mechanisms might have been adopted in specific lineages during the evolutionary radiation of angiosperms. PMID:21505434

  2. Characterization and optimization of cathodic conditions for H2O2 synthesis in microbial electrochemical cells

    EPA Science Inventory

    Cathode potential and O2 supply methods were investigated to improve H2O2 synthesis in an electrochemical cell, and optimal cathode conditions were applied for microbial electrochemical cells (MECs). Using aqueous O2 for the cathode significantly improved current density, but H2...

  3. Purkinje Cell Activity in the Cerebellar Anterior Lobe after Rabbit Eyeblink Conditioning

    ERIC Educational Resources Information Center

    Green, John T.; Steinmetz, Joseph E.

    2005-01-01

    The cerebellar anterior lobe may play a critical role in the execution and proper timing of learned responses. The current study was designed to monitor Purkinje cell activity in the rabbit cerebellar anterior lobe after eyeblink conditioning, and to assess whether Purkinje cells in recording locations may project to the interpositus nucleus.…

  4. Hedgehog pathway maintains cell survival under stress conditions, and drives drug resistance in lung adenocarcinoma.

    PubMed

    Lin, Erh-Hsuan; Kao, Yu-Rung; Lin, Chih-An; Kuo, Ting-Yu; Yang, Sheng-Ping; Hsu, Chiung-Fang; Chou, Teh-Ying; Ho, Chao-Chi; Wu, Cheng-Wen

    2016-04-26

    Hedgehog (HH) pathway plays an important role in embryonic development, but is largely inactive in adult except for tissue repair. Aberrant activation of HH pathway has been found in a variety of cancer types. In non-small cell lung cancer, however, the role and importance of HH pathway remain controversial. In the current study, we found that HH pathway was maintained in low activity in lung adenocarcinoma (LAC) cells under normal culture condition, but was highly induced in response to stress conditions. Activation of HH pathway promoted cell survival, growth, and invasion partially through HGF and MET signaling. Hedgehog-Interacting Protein (HHIP), a cell-surface negative regulator of HH pathway, was epigenetically silenced in LAC. Overexpression of HHIP blocked the activation of HH and HGF/MET pathways, and made cells significantly more susceptible to stress conditions. In LAC cells with acquired resistance to Epidermal Growth Factor Receptor Tyrosin Kinase Inhibitor (EGFR-TKI), we found that a part of tumor cells were much more sensitive to HH or HGF/MET inhibitors, suggesting an oncogenic addiction shift from EGFR to HH and HGF/MET pathways. In conclusion, this study showed that HH pathway is a survival signaling that drives LAC cell growth under stress conditions, and HHIP is a key regulator to block the induction of HH pathway. Targeting the HH pathway through inhibitors or HHIP thus holds promise to address EGFR-TKI resistance in LAC in clinic. PMID:27015549

  5. Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

    PubMed

    Galbraith, R M; Goust, J M; Fudenberg, H H

    1977-12-01

    The presence of phytohemagglutinin or pokeweed mitogen in cultures of human peripheral blood mononuclear cells in agar is known to stimulate the formation of lymphoid colonies. We now report that similar colonies can be induced in the absence of plant lectins upon addition of filtered and ultracentrifuged conditioned medium (CM) obtained from certain human lymphoblastoid cell lines. Colony formation required at least 6 X 10(5) mononuclear cells per milliliter, and optimum results were obtained at concentrations of 1 X 10(6) cells/ml in the presence of 20% CM (50-500 colonies per 10(6) cells cultured). Individual cells within colonies displayed uniform morphological characteristics of lymphoid cells, and the majority formed rosettes with sheep erythrocytes, suggesting that they were of T-cell type. PMID:303689

  6. Engraftment of embryonic stem cells and differentiated progeny by host conditioning with total lymphoid irradiation and regulatory T cells.

    PubMed

    Pan, Yuqiong; Leveson-Gower, Dennis B; de Almeida, Patricia E; Pierini, Antonio; Baker, Jeanette; Florek, Mareike; Nishikii, Hidekazu; Kim, Byung-Su; Ke, Rong; Wu, Joseph C; Negrin, Robert S

    2015-03-24

    Embryonic stem cells (ESCs) hold promise for the treatment of many medical conditions; however, their utility is limited by immune rejection. The objective of our study is to establish tolerance or promote engraftment of transplanted ESCs as well as mature cell populations derived from ESCs. Luciferase (luc(+))-expressing ESCs were utilized to monitor the survival of the ESCs and differentiated progeny in living recipients. Allogeneic recipients conditioned with fractioned total lymphoid irradiation (TLI) and anti-thymocyte serum (ATS) or TLI plus regulatory T cells (T(reg)) promoted engraftment of ESC allografts after transplantation. Following these treatments, the engraftment of transplanted terminally differentiated endothelial cells derived from ESCs was also significantly enhanced. Our findings provide clinically translatable strategies of inducing tolerance to adoptively transferred ESCs for cell replacement therapy of medical disorders. PMID:25801020

  7. An integrated system for synchronous culture of animal cells under controlled conditions.

    PubMed

    Mendoza-Pérez, Elena; Hernández, Vanessa; Palomares, Laura A; Serrato, José A

    2016-01-01

    The cell cycle has fundamental effects on cell cultures and their products. Tools to synchronize cultured cells allow the study of cellular physiology and metabolism at particular cell cycle phases. However, cells are most often arrested by methods that alter their homeostasis and are then cultivated in poorly controlled environments. Cell behavior could then be affected by the synchronization method and culture conditions used, and not just by the particular cell cycle phase under study. Moreover, only a few viable cells are recovered. Here, we designed an integrated system where a large number of cells from a controlled bioreactor culture is separated by centrifugal elutriation at high viabilities. In contrast to current elutriation methods, cells are injected directly from a bioreactor into an injection loop, allowing the introduction of a large number of cells into the separation chamber without stressful centrifugation. A low pulsation peristaltic pump increases the stability of the elutriation chamber. Using this approach, a large number of healthy cells at each cell cycle phase were obtained, allowing their direct inoculation into fully instrumented bioreactors. Hybridoma cells synchronized and cultured in this system behaved as expected for a synchronous culture. PMID:27625207

  8. Platelet adhesion and fusion to endothelial cell facilitate the metastasis of tumor cell in hypoxia-reoxygenation condition.

    PubMed

    Zhang, Na; Zhang, Wen-Jian; Cai, Han-Qing; Liu, Hong-Lin; Peng, Liang; Li, Cheng-Hui; Ye, Li-Ya; Xu, Shi-Qing; Yang, Zhi-Hua; Lou, Jin-Ning

    2011-01-01

    To investigate the relevant molecular mechanisms of platelet in promoting metastasis of tumor cell. The adhesion of fluorescence dye labeled-platelet to human liver sinusoidal endothelial cell (LSEC) line and tumor cell lines were detected by fluorescence microscope and fluorescence plate reader or laser scanning confocal microscope. The relevant adhesion molecules were analyzed by the antibody blockage experiment. The immune colloidal gold transmission electron microscope (TEM), flow cytometry and dye transfer were used to decipher the adhesion and fusion of platelet and LSEC. The tumor cells adhesion to vessels in ischemia condition was analyzed on mouse mesenteric vessels and the metastasis and neovascularization of metastatic foci in pulmonary tissue were also detected after tumor cells injected into nude mice via tail veil. After hypoxia-reoxygenation, tumor cell or LSEC markedly increased its adhesion with platelet, which could be blocked by different antibodies to platelet adhesion molecules. Platelet increased adhesion of tumor cell to LSEC in dose-dependent manner. The fusion of platelet and LSEC was demonstrated by translocation of fluorescent dye from platelet into the adherent LSEC; gpIIb emerged on the LSEC; and confirmed by TEM. The morphological examination found platelet presented between tumor cell and LSEC. Animal experiment indicated that the tumor adhesion to vessels was seldom in normal condition, but increased in ischemia-reperfusion condition, and further significantly enhanced by platelets. The number of tumor metastatic foci and the density of blood vessels within metastatic foci in lung were markedly increased by tumor cell pre-adhered with platelet. The adhesion or fusion of platelet to endothelial cell mediated by platelet surface adhesion molecules, which could promote the adhesion of tumor cell with endothelial cells and the tumor metastasis. PMID:21061145

  9. Viable mononuclear cell stability study for implementation in a proficiency testing program: impact of shipment conditions.

    PubMed

    Kofanova, Olga A; Davis, Kristine; Glazer, Barbara; De Souza, Yvonne; Kessler, Joseph; Betsou, Fotini

    2014-06-01

    The impact of shipping temperatures and preservation media used during transport of either peripheral blood mononuclear cells (PBMCs) or Jurkat cells was assessed, in view of implementing of a proficiency testing scheme on mononuclear cell viability. Samples were analyzed before and after shipment at different temperatures (ambient temperature, dry ice, and liquid nitrogen) and in different preservation media (serum with cryoprotectant, commercial cryopreservation solution, and room temperature transport medium). Sample quality was assessed by viability assays (Trypan Blue dye exclusion, flow cytometry, Cell Analysis System cell counting (CASY)), and by ELISpot functional assay. The liquid nitrogen storage and shipment were found to be the most stable conditions to preserve cell viability and functionality. However, we show that alternative high quality shipment conditions for viable cells are dry ice shipment and commercial cryopreservation solution. These were also cost-efficient shipment conditions, satisfying the requirements of a proficiency testing scheme for viable mononuclear cells. Room temperature transport medium dramatically and adversely affected the integrity of mononuclear cells. PMID:24955735

  10. Human-induced pluripotent stem cells produced under xeno-free conditions.

    PubMed

    Ross, Pablo Juan; Suhr, Steven Thomas; Rodriguez, Ramon Maria; Chang, Eun-Ah; Wang, Kai; Siripattarapravat, Kannika; Ko, Tak; Cibelli, Jose Bernardo

    2010-08-01

    Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. While cell differentiation protocols have been successfully developed, and animal models of human disease have proved that these cells have the potential to treat human diseases and conditions produced as a consequence of aging, degeneration, injury, and birth defects, logistical issues still remain unsolved and hamper the possibility of testing these cells in human clinical trials. Among them is the widely spread use of animal products for the generation and culture of iPSCs. We report here a xeno-free iPSC generation system that addresses all the steps of iPSCs production including the isolation and culture of adult skin fibroblasts, and iPSCs generation, expansion, and maintenance. iPSCs generated with a polycistronic lentiviral vector under xeno-free conditions displayed markers of pluripotency and gave rise to embryoid bodies (EBs) displaying indicators of the 3 primary germ layers. Xeno-free iPSCs injected into nude mice produced classic teratomas, and teratoma explants cultured under conditions favoring fibroblastic cells gave rise to cells morphologically indistinguishable from input cells. Protocols here described will facilitate the implementation of new cellular therapies for preclinical and clinical studies, potentially reducing the regulatory burden without compromising the differentiation potential of the cells. PMID:20030562

  11. Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions

    PubMed Central

    2011-01-01

    Background Betulinic acid (BA) is a novel antineoplastic agent under evaluation for tumor therapy. Because of the selective cytotoxic effects of BA in tumor cells (including gliomas), the combination of this agent with conservative therapies (such as radiotherapy and chemotherapy) may be useful. Previously, the combination of BA with irradiation under hypoxic conditions had never been studied. Methods In this study, the effects of 3 to 30 μM BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1α, as well as radiosensitivity under normoxic and hypoxic conditions were analyzed in the human malignant glioma cell lines U251MG and U343MG. Cytotoxicity and radiosensitivity were analyzed with clonogenic survival assays, migration was analyzed with Boyden chamber assays (or scratch assays) and protein expression was examined with Western blot analyses. Results Under normoxic conditions, a half maximal inhibitory concentration (IC50) of 23 μM was observed in U251MG cells and 24 μM was observed in U343MG cells. Under hypoxic conditions, 10 μM or 15 μM of BA showed a significantly increased cytotoxicity in U251MG cells (p = 0.004 and p = 0.01, respectively) and U343MG cells (p < 0.05 and p = 0.01, respectively). The combination of BA with radiotherapy resulted in an additive effect in the U343MG cell line under normoxic and hypoxic conditions. Weak radiation enhancement was observed in U251MG cell line after treatment with BA under normoxic conditions. Furthermore, under hypoxic conditions, the incubation with BA resulted in increased radiation enhancement. The enhancement factor, at an irradiation dose of 15 Gy after treatment with 10 or 15 μM BA, was 2.20 (p = 0.02) and 4.50 (p = 0.03), respectively. Incubation with BA led to decreased cell migration, cleavage of PARP and decreased expression levels of survivin in both cell lines. Additionally, BA treatment resulted in a reduction of HIF-1α protein under hypoxic conditions. Conclusion Our

  12. Hair Regeneration Treatment Using Adipose-Derived Stem Cell Conditioned Medium: Follow-up With Trichograms

    PubMed Central

    Suga, Hirotaka

    2015-01-01

    Objective: Adipose-derived stem cells secrete various growth factors that promote hair growth. This study examined the effects of adipose-derived stem cell-conditioned medium on alopecia. Methods: Adipose-derived stem cell-conditioned medium was intradermally injected in 22 patients (11 men and 11 women) with alopecia. Patients received treatment every 3 to 5 weeks for a total of 6 sessions. Hair numbers were counted using trichograms before and after treatment. A half-side comparison study was also performed in 10 patients (8 men and 2 women). Results: Hair numbers were significantly increased after treatment in both male (including those without finasteride administration) and female patients. In the half-side comparison study, the increase in hair numbers was significantly higher on the treatment side than on the placebo side. Conclusion: Treatment using adipose-derived stem cell-conditioned medium appears highly effective for alopecia and may represent a new therapy for hair regeneration. PMID:25834689

  13. Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency.

    PubMed

    Martin Gonzalez, Javier; Morgani, Sophie M; Bone, Robert A; Bonderup, Kasper; Abelchian, Sahar; Brakebusch, Cord; Brickman, Joshua M

    2016-08-01

    Embryonic stem cells (ESCs) are cell lines derived from the mammalian pre-implantation embryo. Here we assess the impact of derivation and culture conditions on both functional potency and ESC transcriptional identity. Individual ESCs cultured in either two small-molecule inhibitors (2i) or with knockout serum replacement (KOSR), but not serum, can generate high-level chimeras regardless of how these cells were derived. ESCs cultured in these conditions showed a transcriptional correlation with early pre-implantation embryos (E1.5-E3.5) and contributed to development from the 2-cell stage. Conversely, the transcriptome of serum-cultured ESCs correlated with later stages of development (E4.5), at which point embryonic cells are more restricted in their developmental potential. Thus, ESC culture systems are not equivalent, but support cell types that resemble distinct developmental stages. Cells derived in one condition can be reprogrammed to another developmental state merely by adaptation to another culture condition. PMID:27509134

  14. Derivation of rigorous conditions for high cell-type diversity by algebraic approach.

    PubMed

    Yoshida, Hiroshi; Anai, Hirokazu; Horimoto, Katsuhisa

    2007-01-01

    The development of a multicellular organism is a dynamic process. Starting with one or a few cells, the organism develops into different types of cells with distinct functions. We have constructed a simple model by considering the cell number increase and the cell-type order conservation, and have assessed conditions for cell-type diversity. This model is based on a stochastic Lindenmayer system with cell-to-cell interactions for three types of cells. In the present model, we have successfully derived complex but rigorous algebraic relations between the proliferation and transition rates for cell-type diversity by using a symbolic method: quantifier elimination (QE). Surprisingly, three modes for the proliferation and transition rates have emerged for large ratios of the initial cells to the developed cells. The three modes have revealed that the equality between the development rates for the highest cell-type diversity is reduced during the development process of multicellular organisms. Furthermore, we have found that the highest cell-type diversity originates from order conservation. PMID:17293029

  15. Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation.

    PubMed

    Wingender, Gerhard; Birkholz, Alysia M; Sag, Duygu; Farber, Elisa; Chitale, Sampada; Howell, Amy R; Kronenberg, Mitchell

    2015-10-15

    Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness. PMID:26355152

  16. In vitro differentiation of mouse embryonic stem cells into inner ear hair cell-like cells using stromal cell conditioned medium.

    PubMed

    Ouji, Y; Ishizaka, S; Nakamura-Uchiyama, F; Yoshikawa, M

    2012-01-01

    Hearing loss is mainly caused by loss of sensory hair cells (HCs) in the organ of Corti or cochlea. Although embryonic stem (ES) cells are a promising source for cell therapy, little is known about the efficient generation of HC-like cells from ES cells. In the present study, we developed a single-medium culture method for growing embryoid bodies (EBs), in which conditioned medium (CM) from cultures of ST2 stromal cells (ST2-CM) was used for 14-day cultures of 4-day EBs. At the end of the 14-day cultures, up to 20% of the cells in EB outgrowths expressed HC-related markers, including Math1 (also known as Atoh1), myosin6, myosin7a, calretinin, α9AchR and Brn3c (also known as Pou4f3), and also showed formation of stereocilia-like structures. Further, we found that these cells were incorporated into the developing inner ear after transplantation into chick embryos. The present inner ear HC induction method using ST2-CM (HIST2 method) is quite simple and highly efficient to obtain ES-derived HC-like cells with a relatively short cultivation time. PMID:22622133

  17. Differentiation of P19 embryonal carcinoma stem cells into insulin-producing cells promoted by pancreas-conditioned medium.

    PubMed

    Mansouri, Akram; Esmaeili, Fariba; Nejatpour, Azadeh; Houshmand, Fariba; Shabani, Leila; Ebrahimie, Esmaeil

    2016-07-01

    The ability of embryonal carcinoma )EC (stem cells to generate insulin-producing cells (IPCs) is still unknown. We examined the trophic effects of pancreas-conditioned medium (PCM) on in vitro production of IPCs. Initially, P19 EC cells were characterized by the expression of stem cell markers, Oct3/4, Sox-2 and Nanog. To direct differentiation, P19-derived embryoid bodies (EBs) were induced by selection of nestin-positive cells and treatment with different concentrations of PCM. Morphological studies documented the presence of islet-like cell IPCs clusters. The differentiated cells were immunoreactive for β cell-specific proteins, including insulin, proinsulin, C-peptide and insulin receptor-β. The expression of genes related to pancreatic β cell development and function (PDX-1, INS1, INS2, EP300 and CREB1) was confirmed by qPCR. During differentiation, the expression of EP300 and CREB1 increased by 2.5 and 3.1 times, respectively. In contrast, a sharp decrease in the expression of Oct3/4, Sox-2 and Nanog by 4, 1.5 and 1.5 times, respectively, was observed. The differentiated cells were functionally active, synthesizing and secreting insulin in a glucose-regulated manner. Network prediction highlighted crosstalk between PDX-1 transcription factor and INS2 ligand in IPC generation and revealed positive regulatory effects of EP300, CREB1, PPARA, EGR, KIT, GLP1R, and PKT2 on activation of PDX-1 and INS2. This is the first report of the induction of IPC differentiation from EC cells by using neonate mouse PCM. Since P19 EC cells are widely available, easily cultured without feeders and do not require special growth conditions, they would provide a valuable tool for studying pancreatic β cell differentiation and development. Copyright © 2016 John Wiley & Sons, Ltd. PMID:25044225

  18. Studies on culture and osteogenic induction of human mesenchymal stem cells under CO2-independent conditions.

    PubMed

    Chen, Jian; Zhang, Cui; Feng, Yiding; Zong, Chen; Chen, Jiarong; Tang, Zihua; Jia, Bingbing; Tong, Xiangming; Zheng, Qiang; Wang, Jinfu

    2013-04-01

    Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce the

  19. Efficient passage of human pluripotent stem cells on spider silk matrices under xeno-free conditions.

    PubMed

    Wu, Siqin; Johansson, Jan; Hovatta, Outi; Rising, Anna

    2016-04-01

    Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine and pharmaceutical development. Such applications require cell culture methods and reagents that are chemically defined, xeno-free, scalable, and low-cost. Herein, we describe non-mechanical passaging of hPSCs on spider silk films under chemically defined and xeno-free conditions. The cells were dissociated into single cells or small aggregates using Accutase or enzyme-free dissociation buffer and then passaged to spider silk films, where they expanded in monolayers until they covered the surface. Cells cultured over 10 passages on spider silk film remained karyotypically normal and pluripotent. In conclusion, a novel method for passaging dissociated hPSCs under conditions that are compatible with clinical applications is presented. The method is cost-efficient and may be useful for both research and clinical applications. PMID:26427704

  20. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    SciTech Connect

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-08-26

    Highlights: {yields} Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. {yields} The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. {yields} Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  1. Conditional Density-based Analysis of T cell Signaling in Single Cell Data

    PubMed Central

    Krishnaswamy, Smita; Spitzer, Matthew H.; Mingueneau, Michael; Bendall, Sean C; Litvin, Oren; Stone, Erica; Pe’er, Dana; Nolan, Garry P

    2015-01-01

    Cellular circuits sense the environment, process signals, and compute decisions using networks of interacting proteins. To model such a system, the abundance of each activated protein species can be described as a stochastic function of the abundance of other proteins. High-dimensional single-cell technologies, like mass cytometry, offer an opportunity to characterize signaling circuit-wide. However, the challenge of developing and applying computational approaches to interpret such complex data remains. Here, we developed computational methods, based on established statistical concepts, to characterize signaling network relationships by quantifying the strengths of network edges and deriving signaling response functions. In comparing signaling between naïve and antigen-exposed CD4+ T-lymphocytes, we find that although these two cell subtypes had similarly-wired networks, naïve cells transmitted more information along a key signaling cascade than did antigen-exposed cells. We validated our characterization on mice lacking the extracellular-regulated MAP kinase (ERK2), which showed stronger influence of pERK on pS6 (phosphorylated-ribosomal protein S6), in naïve cells compared to antigen-exposed cells, as predicted. We demonstrate that by using cell-to-cell variation inherent in single cell data, we can algorithmically derive response functions underlying molecular circuits and drive the understanding of how cells process signals. PMID:25342659

  2. Simulated conditions of microgravity suppress progesterone production by luteal cells of the pregnant rat

    NASA Technical Reports Server (NTRS)

    Bhat, G. K.; Yang, H.; Sridaran, R.

    2001-01-01

    The purpose of this study was to assess whether simulated conditions of microgravity induce changes in the production of progesterone by luteal cells of the pregnant rat ovary using an in vitro model system. The microgravity environment was simulated using either a high aspect ratio vessel (HARV) bioreactor with free fall or a clinostat without free fall of cells. A mixed population of luteal cells isolated from the corpora lutea of day 8 pregnant rats was attached to cytodex microcarrier beads (cytodex 3). These anchorage dependent cells were placed in equal numbers in the HARV or a spinner flask control vessel in culture conditions. It was found that HARV significantly reduced the daily production of progesterone from day 1 through day 8 compared to controls. Scanning electron microscopy showed that cells attached to the microcarrier beads throughout the duration of the experiment in both types of culture vessels. Cells cultured in chamber slide flasks and placed in a clinostat yielded similar results when compared to those in the HARV. Also, when they were stained by Oil Red-O for lipid droplets, the clinostat flasks showed a larger number of stained cells compared to control flasks at 48 h. Further, the relative amount of Oil Red-O staining per milligram of protein was found to be higher in the clinostat than in the control cells at 48 h. It is speculated that the increase in the level of lipid content in cells subjected to simulated conditions of microgravity may be due to a disruption in cholesterol transport and/or lesions in the steroidogenic pathway leading to a fall in the synthesis of progesterone. Additionally, the fall in progesterone in simulated conditions of microgravity could be due to apoptosis of luteal cells.

  3. Some tests of flat plate photovoltaic module cell temperatures in simulated field conditions

    NASA Technical Reports Server (NTRS)

    Griffith, J. S.; Rathod, M. S.; Paslaski, J.

    1981-01-01

    The nominal operating cell temperature (NOCT) of solar photovoltaic (PV) modules is an important characteristic. Typically, the power output of a PV module decreases 0.5% per deg C rise in cell temperature. Several tests were run with artificial sun and wind to study the parametric dependencies of cell temperature on wind speed and direction and ambient temperature. It was found that the cell temperature is extremely sensitive to wind speed, moderately so to wind direction and rather insensitive to ambient temperature. Several suggestions are made to obtain data more typical of field conditions.

  4. Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

    PubMed Central

    Kishino, Yoshikazu; Seki, Tomohisa; Yuasa, Shinsuke; Fujita, Jun; Fukuda, Keiichi

    2015-01-01

    Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus. PMID:26650709

  5. Regenerative Potential of TGFβ3 + Dex and Notochordal Cell Conditioned Media on Degenerated Human Intervertebral Disc Cells

    PubMed Central

    Abbott, Rosalyn Delia; Purmessur, Devina; Monsey, Robert Daniel; Iatridis, James Christopher

    2011-01-01

    Injection of soluble cell signaling factors into degenerated intervertebral discs (IVDs) offers a minimally invasive treatment that could limit the processes of degeneration by stimulating native matrix repair. This study evaluated the regenerative capacity of degenerated nucleus pulposus (NP) cells obtained from patients undergoing anterior interbody fusions by measuring metabolic activity, DNA content, glycosaminoglycan (GAG) content, and cellular phenotype using qRT-PCR profiling with a custom array of 42 genes. NP cells were cultured in alginate for 7 days with 4 treatment groups: transforming growth factor beta 3 (TGFβ3) + dexamethasone (Dex), soluble factors released from notochordal cells (NCs) cultured in alginate (NCA), soluble factors released from NCs in their native tissue environment (NCT), and basal media. TGFβ3 + Dex stimulated degenerated human NP cells to proliferate and exhibit an anti-catabolic gene expression profile (with a decrease in ADAMTS5 and MMP1 compared to basal, and an increase in SOX9, decrease in ADAMTS5, MMP1, collagen I and collagen III compared to day 0), while NCA stimulated the greatest GAG per cell. We conclude that degenerated human NP cells exhibit regenerative potential, and that an optimal treatment will likely require treatments, such as TGFβ3 + Dex, which were able to increase cell metabolism and reduce catabolism, as well as treatments with factors found in NC conditioned medium, that were able to produce high amounts of GAG per cell. Additional studies to optimize NC culture conditions are required to determine if NC conditioned medium can be made with the capacity to enhance NP cell proliferation and metabolism. PMID:21866573

  6. Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient-specific neural cells.

    PubMed

    Fernandes, Tiago G; Duarte, Sofia T; Ghazvini, Mehrnaz; Gaspar, Cláudia; Santos, Diana C; Porteira, Ana R; Rodrigues, Gonçalo M C; Haupt, Simone; Rombo, Diogo M; Armstrong, Judith; Sebastião, Ana M; Gribnau, Joost; Garcia-Cazorla, Àngels; Brüstle, Oliver; Henrique, Domingos; Cabral, Joaquim M S; Diogo, Maria Margarida

    2015-10-01

    Standardization of culture methods for human pluripotent stem cell (PSC) neural differentiation can greatly contribute to the development of novel clinical advancements through the comprehension of neurodevelopmental diseases. Here, we report an approach that reproduces neural commitment from human induced pluripotent stem cells using dual-SMAD inhibition under defined conditions in a vitronectin-based monolayer system. By employing this method it was possible to obtain neurons derived from both control and Rett syndrome patients' pluripotent cells. During differentiation mutated cells displayed alterations in the number of neuronal projections, and production of Tuj1 and MAP2-positive neurons. Although investigation of a broader number of patients would be required, these observations are in accordance with previous studies showing impaired differentiation of these cells. Consequently, our experimental methodology was proved useful not only for the generation of neural cells, but also made possible to compare neural differentiation behavior of different cell lines under defined culture conditions. This study thus expects to contribute with an optimized approach to study the neural commitment of human PSCs, and to produce patient-specific neural cells that can be used to gain a better understanding of disease mechanisms. PMID:26123315

  7. From microgravity to osmotic conditions: mechanical integration of plant cells in response to stress

    NASA Astrophysics Data System (ADS)

    Wojtaszek, Przemyslaw; Kasprowicz, Anna; Michalak, Michal; Janczara, Renata; Volkmann, Dieter; Baluska, Frantisek

    Chemical reactions and interactions between molecules are commonly thought of as being at the basis of Life. Research of recent years, however, is more and more evidently indicating that physical forces are profoundly affecting the functioning of life at all levels of its organiza-tion. To detect and to respond to such forces, plant cells need to be integrated mechanically. Cell walls are the outermost functional zone of plant cells. They surround the individual cells, and also form a part of the apoplast. In cell suspensions, cell walls are embedded in the cul-ture medium which can be considered as a superapoplast. Through physical and chemical interactions they provide a basis for the structural and functional cell wall-plasma membrane-cytoskeleton (WMC) continuum spanning the whole cell. Here, the working of WMC contin-uum, and the participation of signalling molecules, like NO, would be presented in the context of plant responses to stress. In addition, the effects of the changing composition of WMC continuum will be considered, with particular attention paid to the modifications of the WMC components. Plant cells are normally adapted to changing osmotic conditions, resulting from variable wa-ter availability. The appearance of the osmotic stress activates adaptory mechanisms. If the strength of osmotic stress grows relatively slowly over longer period of time, the cells are able to adapt to conditions that are lethal to non-adapted cells. During stepwise adaptation of tobacco BY-2 suspension cells to the presence of various osmotically active agents, cells diverged into independent, osmoticum type-specific lines. In response to ionic agents (NaCl, KCl), the adhe-sive properties were increased and randomly dividing cells formed clumps, while cells adapted to nonionic osmotica (mannitol, sorbitol, PEG) revealed ordered pattern of precisely positioned cell divisions, resulting in the formation of long cell files. Changes in the growth patterns were accompanied by

  8. Hsc70 Contributes to Cancer Cell Survival by Preventing Rab1A Degradation under Stress Conditions

    PubMed Central

    Tanaka, Masako; Mun, Saya; Harada, Akihito; Ohkawa, Yasuyuki; Inagaki, Azusa; Sano, Soichi; Takahashi, Katsuyuki; Izumi, Yasukatsu; Osada-Oka, Mayuko; Wanibuchi, Hideki; Yamagata, Masayo; Yukimura, Tokihito; Miura, Katsuyuki; Shiota, Masayuki; Iwao, Hiroshi

    2014-01-01

    Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology. PMID:24801886

  9. Lysophosphatidic acid enhances survival of human CD34+ cells in ischemic conditions

    PubMed Central

    Kostic, Ivana; Fidalgo-Carvalho, Isabel; Aday, Sezin; Vazão, Helena; Carvalheiro, Tiago; Grãos, Mário; Duarte, António; Cardoso, Carla; Gonçalves, Lino; Carvalho, Lina; Paiva, Artur; Ferreira, Lino

    2015-01-01

    Several clinical trials are exploring therapeutic effect of human CD34+ cells in ischemic diseases, including myocardial infarction. Unfortunately, most of the cells die few days after delivery. Herein we show that lysophosphatidic acid (LPA)-treated human umbilical cord blood-derived CD34+ cells cultured under hypoxic and serum-deprived conditions present 2.2-fold and 1.3-fold higher survival relatively to non-treated cells and prostaglandin E2-treated cells, respectively. The pro-survival effect of LPA is concentration- and time-dependent and it is mediated by the activation of peroxisome proliferator-activator receptor γ (PPARγ) and downstream, by the activation of pro-survival ERK and Akt signaling pathways and the inhibition of mitochondrial apoptotic pathway. In hypoxia and serum-deprived culture conditions, LPA induces CD34+ cell proliferation without maintaining the their undifferentiating state, and enhances IL-8, IL-6 and G-CSF secretion during the first 12 h compared to non-treated cells. LPA-treated CD34+ cells delivered in fibrin gels have enhanced survival and improved cardiac fractional shortening at 2 weeks on rat infarcted hearts as compared to hearts treated with placebo. We have developed a new platform to enhance the survival of CD34+ cells using a natural and cost-effective ligand and demonstrated its utility in the preservation of the functionality of the heart after infarction. PMID:26553339

  10. Conditioned medium from alternatively activated macrophages induce mesangial cell apoptosis via the effect of Fas

    SciTech Connect

    Huang, Yuan; Luo, Fangjun; Li, Hui; Jiang, Tao; Zhang, Nong

    2013-11-15

    During inflammation in the glomerulus, the proliferation of myofiroblast-like mesangial cells is commonly associated with the pathological process. Macrophages play an important role in regulating the growth of resident mesangial cells in the glomeruli. Alternatively activated macrophage (M2 macrophage) is a subset of macrophages induced by IL-13/IL-4, which is shown to play a repair role in glomerulonephritis. Prompted by studies of development, we performed bone marrow derived macrophage and rat mesangial cell co-culture study. Conditioned medium from IL-4 primed M2 macrophages induced rat mesangial cell apoptosis. The pro-apoptotic effect of M2 macrophages was demonstrated by condensed nuclei stained with Hoechst 33258, increased apoptosis rates by flow cytometry analysis and enhanced caspase-3 activation by western blot. Fas protein was up-regulated in rat mesangial cells, and its neutralizing antibody ZB4 partly inhibited M2 macrophage-induced apoptosis. The up-regulated arginase-1 expression in M2 macrophage also contributed to this apoptotic effect. These results indicated that the process of apoptosis triggered by conditioned medium from M2 macrophages, at least is partly conducted through Fas in rat mesangial cells. Our findings provide compelling evidence that M2 macrophages control the growth of mesangial cells in renal inflammatory conditions. - Highlights: • Conditioned-medium from M2 macrophages induces rat mesangial cell (MsC) apoptosis. • M2 macrophage conditioned medium exerts its pro-apoptotic effects via Fas ligand. • Arginase-1 activity in M2 macrophages plays a role in inducing apoptosis in rat MsC.

  11. Mesenchymal Stem Cells Adopt Lung Cell Phenotype in Normal and Radiation-induced Lung Injury Conditions.

    PubMed

    Maria, Ola M; Maria, Ahmed M; Ybarra, Norma; Jeyaseelan, Krishinima; Lee, Sangkyu; Perez, Jessica; Shalaby, Mostafa Y; Lehnert, Shirley; Faria, Sergio; Serban, Monica; Seuntjens, Jan; El Naqa, Issam

    2016-04-01

    Lung tissue exposure to ionizing irradiation can invariably occur during the treatment of a variety of cancers leading to increased risk of radiation-induced lung disease (RILD). Mesenchymal stem cells (MSCs) possess the potential to differentiate into epithelial cells. However, cell culture methods of primary type II pneumocytes are slow and cannot provide a sufficient number of cells to regenerate damaged lungs. Moreover, effects of ablative radiation doses on the ability of MSCs to differentiate in vitro into lung cells have not been investigated yet. Therefore, an in vitro coculture system was used, where MSCs were physically separated from dissociated lung tissue obtained from either healthy or high ablative doses of 16 or 20 Gy whole thorax irradiated rats. Around 10±5% and 20±3% of cocultured MSCs demonstrated a change into lung-specific Clara and type II pneumocyte cells when MSCs were cocultured with healthy lung tissue. Interestingly, in cocultures with irradiated lung biopsies, the percentage of MSCs changed into Clara and type II pneumocytes cells increased to 40±7% and 50±6% at 16 Gy irradiation dose and 30±5% and 40±8% at 20 Gy irradiation dose, respectively. These data suggest that MSCs to lung cell differentiation is possible without cell fusion. In addition, 16 and 20 Gy whole thorax irradiation doses that can cause varying levels of RILD, induced different percentages of MSCs to adopt lung cell phenotype compared with healthy lung tissue, providing encouraging outlook for RILD therapeutic intervention for ablative radiotherapy prescriptions. PMID:26200842

  12. Cell Death Conversion under Hypoxic Condition in Tumor Development and Therapy

    PubMed Central

    Qiu, Yu; Li, Peng; Ji, Chunyan

    2015-01-01

    Hypoxia, which is common during tumor progression, plays important roles in tumor biology. Failure in cell death in response to hypoxia contributes to progression and metastasis of tumors. On the one hand, the metabolic and oxidative stress following hypoxia could lead to cell death by triggering signal cascades, like LKB1/AMPK, PI3K/AKT/mTOR, and altering the levels of effective components, such as the Bcl-2 family, Atg and p62. On the other hand, hypoxia-induced autophagy can serve as a mechanism to turn over nutrients, so as to mitigate the adverse condition and then avoid cell death potentially. Due to the effective role of hypoxia, this review focuses on the crosstalk in cell death under hypoxia in tumor progression. Additionally, the illumination of cell death in hypoxia could shed light on the clinical applications of cell death targeted therapy. PMID:26512660

  13. Dissociation and Re-Aggregation of Multicell-Ensheathed Fragments Responsible for Rapid Production of Massive Clumps of Leptothrix Sheaths.

    PubMed

    Kunoh, Tatsuki; Nagaoka, Noriyuki; McFarlane, Ian R; Tamura, Katsunori; El-Naggar, Mohamed Y; Kunoh, Hitoshi; Takada, Jun

    2016-01-01

    Species of the Fe/Mn-oxidizing bacteria Leptothrix produce tremendous amounts of microtubular, Fe/Mn-encrusted sheaths within a few days in outwells of groundwater that can rapidly clog water systems. To understand this mode of rapid sheath production and define the timescales involved, behaviors of sheath-forming Leptothrix sp. strain OUMS1 were examined using time-lapse video at the initial stage of sheath formation. OUMS1 formed clumps of tangled sheaths. Electron microscopy confirmed the presence of a thin layer of bacterial exopolymer fibrils around catenulate cells (corresponding to the immature sheath). In time-lapse videos, numerous sheath filaments that extended from the periphery of sheath clumps repeatedly fragmented at the apex of the same fragment, the fragments then aggregated and again elongated, eventually forming a large sheath clump comprising tangled sheaths within two days. In this study, we found that fast microscopic fragmentation, dissociation, re-aggregation and re-elongation events are the basis of the rapid, massive production of Leptothrix sheaths typically observed at macroscopic scales. PMID:27490579

  14. Optimization of Cell Adhesion on Mg Based Implant Materials by Pre-Incubation under Cell Culture Conditions

    PubMed Central

    Willumeit, Regine; Möhring, Anneke; Feyerabend, Frank

    2014-01-01

    Magnesium based implants could revolutionize applications where orthopedic implants such as nails, screws or bone plates are used because they are load bearing and degrade over time. This prevents a second surgery to remove conventional implants. To improve the biocompatibility we studied here if and for how long a pre-incubation of the material under cell culture conditions is favorable for cell attachment and proliferation. For two materials, Mg and Mg10Gd1Nd, we could show that 6 h pre-incubation are already enough to form a natural protective layer suitable for cell culture. PMID:24857908

  15. The Immune-Metabolic Basis of Effector Memory CD4+ T Cell Function under Hypoxic Conditions.

    PubMed

    Dimeloe, Sarah; Mehling, Matthias; Frick, Corina; Loeliger, Jordan; Bantug, Glenn R; Sauder, Ursula; Fischer, Marco; Belle, Réka; Develioglu, Leyla; Tay, Savaş; Langenkamp, Anja; Hess, Christoph

    2016-01-01

    Effector memory (EM) CD4(+) T cells recirculate between normoxic blood and hypoxic tissues to screen for cognate Ag. How mitochondria of these cells, shuttling between normoxia and hypoxia, maintain bioenergetic efficiency and stably uphold antiapoptotic features is unknown. In this study, we found that human EM CD4(+) T cells had greater spare respiratory capacity (SRC) than did naive counterparts, which was immediately accessed under hypoxia. Consequently, hypoxic EM cells maintained ATP levels, survived and migrated better than did hypoxic naive cells, and hypoxia did not impair their capacity to produce IFN-γ. EM CD4(+) T cells also had more abundant cytosolic GAPDH and increased glycolytic reserve. In contrast to SRC, glycolytic reserve was not tapped under hypoxic conditions, and, under hypoxia, glucose metabolism contributed similarly to ATP production in naive and EM cells. However, both under normoxic and hypoxic conditions, glucose was critical for EM CD4(+) T cell survival. Mechanistically, in the absence of glycolysis, mitochondrial membrane potential (ΔΨm) of EM cells declined and intrinsic apoptosis was triggered. Restoring pyruvate levels, the end product of glycolysis, preserved ΔΨm and prevented apoptosis. Furthermore, reconstitution of reactive oxygen species (ROS), whose production depends on ΔΨm, also rescued viability, whereas scavenging mitochondrial ROS exacerbated apoptosis. Rapid access of SRC in hypoxia, linked with built-in, oxygen-resistant glycolytic reserve that functionally insulates ΔΨm and mitochondrial ROS production from oxygen tension changes, provides an immune-metabolic basis supporting survival, migration, and function of EM CD4(+) T cells in normoxic and hypoxic conditions. PMID:26621861

  16. Mineralization of bone-related SaOS-2 cells under physiological hypoxic conditions.

    PubMed

    Müller, Werner E G; Schröder, Heinz C; Tolba, Emad; Diehl-Seifert, Bärbel; Wang, Xiaohong

    2016-01-01

    Inorganic polyphosphate (polyP) is a physiological energy-rich polymer with multiple phosphoric anhydride bonds. In cells such as bone-forming osteoblasts, glycolysis is the main pathway generating metabolic energy in the form of ATP. In the present study, we show that, under hypoxic culture conditions, the growth/viability of osteoblast-like SaOS-2 cells is not impaired. The addition of polyP to those cells, administered as amorphous calcium polyP nanoparticles (aCa-polyP-NP; approximate size 100 nm), significantly increased the proliferation of the cells. In the presence of polyP, the cells produce significant levels of lactate, the end product of anaerobic glycolysis. Under those conditions, an eight-fold increase in the steady-state level of the membrane-associated carbonic anhydrase IX is found, as well as a six-fold induction of the hypoxia-inducible factor 1. Consequently, biomineral formation onto the SaOS-2 cells decreases under low oxygen tension. If the polyP nanoparticles are added to the cells, the degree of mineralization is enhanced. These changes had been measured also in human mesenchymal stem cells. The assumption that the bicarbonate, generated by the carbonic anhydrase in the presence of polyP under low oxygen, is deposited as a constituent of the bioseeds formed during initial hydroxyapatite formation is corroborated by the identification of carbon besides of calcium, oxygen and phosphorus in the initial biomineral deposit onto the cells using the sensitive technology of high-resolution energy dispersive spectrometry mapping. Based on these data, we conclude that polyP is required for the supply of metabolic energy during bone mineral formation under physiological, hypoxic conditions, acting as a 'metabolic fuel' for the cells to grow. PMID:26453899

  17. Endothelial cell differentiation into capillary-like structures in response to tumour cell conditioned medium: a modified chemotaxis chamber assay.

    PubMed Central

    Garrido, T.; Riese, H. H.; Aracil, M.; Pérez-Aranda, A.

    1995-01-01

    We have developed a modified chemotaxis chamber assay in which bovine aortic endothelial (BAE) cells degrade Matrigel basement membrane and migrate and form capillary-like structures on type I collagen. This capillary formation occurs in the presence of conditioned media from highly metastatic tumour cell lines, such as B16F10 murine melanoma or MDA-MD-231 human breast adenocarcinoma, but not in the presence of conditioned medium (CM) from the less invasive B16F0 cell line. Replacement of tumour cell CM by 10 ng ml-1 basic fibroblast growth factor (bFGF) also results in capillary-like structure formation by BAE cells. An anti-bFGF antibody blocks this effect, showing that bFGF is one of the factors responsible for the angiogenic response induced by B16F10 CM in our assay. Addition of an anti-laminin antibody reduces significantly the formation of capillary-like structures, probably by blocking the attachment of BAE cells to laminin present in Matrigel. The anti-angiogenic compound suramin inhibits in a dose-dependent manner (complete inhibition with 100 microM suramin) the migration and differentiation of BAE cells on type I collagen in response to B16F10 CM. This assay represents a new model system to study tumour-induced angiogenesis in vitro. Images Figure 2 Figure 3 PMID:7536021

  18. Amelioration of murine sickle cell disease by nonablative conditioning and γ-globin gene-corrected bone marrow cells

    PubMed Central

    Pestina, Tamara I; Hargrove, Phillip W; Zhao, Huifen; Mead, Paul E; Smeltzer, Matthew P; Weiss, Mitchell J; Wilber, Andrew; Persons, Derek A

    2015-01-01

    Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.0. Fetal hemoglobin (HbF) levels ranged from 20 to 44% of total hemoglobin and approximately two-thirds of circulating red blood cells expressed HbF detected by immunofluorescence (F-cells). Gene therapy treatment of SCD mice ameliorated anemia, reduced hyperleukocytosis, improved renal function, and reduced iron accumulation in liver, spleen, and kidneys. Thus, modest levels of chimerism with donor cells expressing high levels of HbF from an insulated γ-globin lentiviral vector can improve the pathology of SCD in mice, thereby illustrating a potentially safe and effective strategy for gene therapy in humans. PMID:26665131

  19. Use of an active wire Bθ cell for electron beam conditioning

    NASA Astrophysics Data System (ADS)

    Murphy, D. P.; Myers, M. C.; Weidman, D. J.; Antoniades, J. A.; Fernsler, R. F.; Meger, R. A.

    1996-10-01

    The propagation of intense, relativistic electron beams in air is subject to the resistive hose instability. Conditioning the beam prior to injecting it into the air can extend its range by reducing the hose growth rate and by reducing the initial spatial perturbations that seed the hose instability. Experiments have been performed using the SuperIBEX accelerator (Ipeak=10-30 kA, E=4.5 MeV, 40 ns full width at half-maximum) to develop conditioning cells that suppress the hose. This paper describes the performance of an active wire Bθ cell that is used in conjunction with an ion focused regime (IFR) cell. The IFR cell detunes the instability by producing a head-to-tail radius taper on the beam. The wire cell maintains this radius taper while producing an emittance taper that is necessary to suppress the hose growth. In addition, the wire cell reduces the initial beam perturbations through the anharmonic centering force associated with the wire current and its azimuthal magnetic field Bθ. The ability of the Bθ cell to reduce the beam offset with a minimal increase in the beam radius gives it several advantages over the use of a simple, thick scattering foil to perform the radius taper to emittance taper conversion. The SuperIBEX beam propagation distance, in terms of the betatron oscillation scale length, was extended to ˜10λβ using these cells.

  20. Effectiveness of dye sensitised solar cell under low light condition using wide band dye

    SciTech Connect

    Sahmer, Ahmad Zahrin Mohamed, Norani Muti Zaine, Siti Nur Azella

    2015-07-22

    Dye sensistised solar cell (DSC) based on nanocrystalline TiO{sub 2} has the potential to be used in indoor consumer power application. In realizing this, the DSC must be optimized to generate power under low lighting condition and under wider visible light range. The use of wide band dye N749 which has a wider spectrum sensitivity increases the photon conversion to electron between the visible light spectrums of 390nm to 700nm. This paper reports the study on the effectiveness of the dye solar cell with N749 dye under low light condition in generating usable power which can be used for indoor consumer application. The DSC was fabricated using fluorine doped tin oxide (FTO) glass with screen printing method and the deposited TiO{sub 2} film was sintered at 500°C. The TiO{sub 2} coated FTO glass was then soaked in the N749 dye, assembled into test cell, and tested under the standard test condition at irradiance of 1000 W/m{sup 2} with AM1.5 solar soaker. The use of the 43T mesh for the dual pass screen printing TiO{sub 2} paste gives a uniform TiO{sub 2} film layer of 16 µm. The low light condition was simulated using 1/3 filtered irradiance with the solar soaker. The fabricated DSC test cell with the N749 dye was found to have a higher efficiency of 6.491% under low light condition compared to the N719 dye. Under the standard test condition at 1 sun the N749 test cell efficiency is 4.55%. The increases in efficiency is attributed to the wider spectral capture of photon of the DSC with N749 dye. Furthermore, the use of N749 dye is more effective under low light condition as the V{sub OC} decrement is less significant compared to the latter.

  1. Effectiveness of dye sensitised solar cell under low light condition using wide band dye

    NASA Astrophysics Data System (ADS)

    Sahmer, Ahmad Zahrin; Mohamed, Norani Muti; Zaine, Siti Nur Azella

    2015-07-01

    Dye sensistised solar cell (DSC) based on nanocrystalline TiO2 has the potential to be used in indoor consumer power application. In realizing this, the DSC must be optimized to generate power under low lighting condition and under wider visible light range. The use of wide band dye N749 which has a wider spectrum sensitivity increases the photon conversion to electron between the visible light spectrums of 390nm to 700nm. This paper reports the study on the effectiveness of the dye solar cell with N749 dye under low light condition in generating usable power which can be used for indoor consumer application. The DSC was fabricated using fluorine doped tin oxide (FTO) glass with screen printing method and the deposited TiO2 film was sintered at 500°C. The TiO2 coated FTO glass was then soaked in the N749 dye, assembled into test cell, and tested under the standard test condition at irradiance of 1000 W/m2 with AM1.5 solar soaker. The use of the 43T mesh for the dual pass screen printing TiO2 paste gives a uniform TiO2 film layer of 16 µm. The low light condition was simulated using 1/3 filtered irradiance with the solar soaker. The fabricated DSC test cell with the N749 dye was found to have a higher efficiency of 6.491% under low light condition compared to the N719 dye. Under the standard test condition at 1 sun the N749 test cell efficiency is 4.55%. The increases in efficiency is attributed to the wider spectral capture of photon of the DSC with N749 dye. Furthermore, the use of N749 dye is more effective under low light condition as the VOC decrement is less significant compared to the latter.

  2. Conditioning with α-emitter based radioimmunotherapy in canine allogeneic hematopoietic cell transplantation

    PubMed Central

    Kornblit, Brian; Chen, Yun; Sandmaier, Brenda M.

    2012-01-01

    With the introduction of nonmyeloablative conditioning, hematopoietic cell transplantation (HCT) has become a viable treatment option for patients who due to age or comorbidities are ineligible for high dose conditioning. However, relapse and toxicities are still major problems in HCT. Radioimmunotherapy (RIT)-based conditioning is a promising approach that has the ability to specifically target radiation to hematopoietic cells. The most widely investigated isotopes are the β-emitters, but because of long path lengths and low linear energy transfer, α-emitters which have more favorable physical characteristics, might prove to be a better alternative. In the current study we have investigated the efficacy and safety of α-emitter based RIT as the only form of conditioning in a preclinical model of canine allogeneic HCT. PMID:22772070

  3. Effect of adipose-derived stem cell-conditioned medium on the proliferation and migration of B16 melanoma cells

    PubMed Central

    LEE, JU-HEE; PARK, CHUL HONG; CHUN, KWANG-HOON; HONG, SOON-SUN

    2015-01-01

    Adipose-derived stem cells (ASCs) are a population of cells derived from adipose tissue. ASCs exhibit multilineage development potential and are able to secrete various factors, which influence adjacent cells. Previous studies have reported the effectiveness of ASC-conditioned medium (ASC-CM) in wound healing, anti-melanogenesis, wrinkle improvement and hair growth. In the present study, the anticancer function of ASC-CM was investigated in vitro and in vivo. An MTT assay revealed that ASC-CM significantly decreased the proliferation of B16 melanoma cells in a time- and dose-dependent manner (P<0.01). Cell cycle analysis indicated that ASC-CM significantly increased the number of cells in G1 phase while reducing the number of cells in the S and G2/M phases (P<0.01). Furthermore, a wound migration model demonstrated that ASC-CM treatment significantly decreased the migration ability of B16 melanoma cells (P<0.01). In addition, C57BL/6 mice were administered with a single intratumoral injection of ASC-CM, daily or every other day, and a significant reduction in the volume of the tumor mass was observed compared with that of the control group (P<0.01). Thus, the findings of the present study indicated that ASC-CM has an anti-tumorigenic effect on B16 melanoma cells in vitro and in vivo, and may potentially be used to support the treatment of melanoma in the future. PMID:26622561

  4. Reduced Toxicity Conditioning and Allogeneic Hematopoietic Progenitor Cell Transplantation for Recessive Dystrophic Epidermolysis Bullosa.

    PubMed

    Geyer, Mark B; Radhakrishnan, Kavita; Giller, Roger; Umegaki, Noriko; Harel, Sivan; Kiuru, Maija; Morel, Kimberly D; LeBoeuf, Nicole; Kandel, Jessica; Bruckner, Anna; Fabricatore, Sandra; Chen, Mei; Woodley, David; McGrath, John; Baxter-Lowe, LeeAnn; Uitto, Jouni; Christiano, Angela M; Cairo, Mitchell S

    2015-09-01

    Recessive dystrophic epidermolysis bullosa is a severe, incurable, inherited blistering disease caused by COL7A1 mutations. Emerging evidence suggests hematopoietic progenitor cells (HPCs) can be reprogrammed into skin; HPC-derived cells can restore COL7 expression in COL7-deficient mice. We report two children with recessive dystrophic epidermolysis bullosa treated with reduced-toxicity conditioning and HLA-matched HPC transplantation. PMID:26148662

  5. Culture conditions affect the cholinergic development of an isolated subpopulation of chick mesencephalic neural crest cells.

    PubMed

    Barald, K F

    1989-10-01

    Although neural crest cells are known to be very responsive to environmental cues during their development, recent evidence indicates that at least some subpopulations may be committed to a specific differentiation program prior to migration. Because the neural crest is composed of a heterogeneous mixture of cells that contributes to many vertebrate cell lineages, assessing the properties of specific subpopulations and the effect of the environment on their development has been difficult. To address this problem, we have isolated a pure subpopulation of chick mesencephalic neural crest cells by fluorescence no-flow cytometry after labeling them with monoclonal antibodies (Mabs) to a 75-kDa cell surface antigen that is associated with high affinity choline uptake. When cultures of chick mesencephalic neural crest cells are labeled with these Mabs and a fluorescent second step antibody, approximately 5% of the cells are antigen-positive (A+). After sorting, 100% of the resulting cultured mesencephalic neural crest cells are A+. The Mabs we used also label all of the neurons of the embryonic chick and quail ciliary ganglion in vivo and in vitro. We have compared the effect of various cell culture media on the isolated neural crest subpopulation and the heterogeneous chick mesencephalic neural crest from which it was derived. A+ cells were passaged and grown in a variety of media, each of which differently affected its characteristics and development. A+ cells proliferated in the presence of 15% fetal bovine serum (FBS) and high concentrations (10-15%) of chick embryo extract, but did not differentiate, although they retained basal levels of choline acetyltransferase (ChAT) activity. However, in chick serum and high (25 mM as opposed to 7 mM) K+, and heart-, iris-, or lung-conditioned medium, all of which are known to promote survival and/or cholinergic development of ciliary ganglion neurons, the cells ceased to proliferate and all of the cells in the culture became

  6. Conditional Cytotoxic Anti-HIV Gene Therapy for Selectable Cell Modification.

    PubMed

    Garg, Himanshu; Joshi, Anjali

    2016-05-01

    Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy. PMID:26800572

  7. L-selectin-mediated lymphocyte-cancer cell interactions under low fluid shear conditions.

    PubMed

    Resto, Vicente A; Burdick, Monica M; Dagia, Nilesh M; McCammon, Susan D; Fennewald, Susan M; Sackstein, Robert

    2008-06-01

    Cell migration in blood flow is mediated by engagement of specialized adhesion molecules that function under hemodynamic shear conditions, and many of the effectors of these adhesive interactions, such as the selectins and their ligands, are well defined. However, in contrast, our knowledge of the adhesion molecules operant under lymphatic flow conditions is incomplete. Among human malignancies, head and neck squamous cell cancer displays a marked predilection for locoregional lymph node metastasis. Based on this distinct tropism, we hypothesized that these cells express adhesion molecules that promote their binding to lymphoid tissue under lymphatic fluid shear stress. Accordingly, we investigated adhesive interactions between these and other cancer cells and the principal resident cells of lymphoid organs, lymphocytes. Parallel plate flow chamber studies under defined shear conditions, together with biochemical analyses, showed that human head and neck squamous cell cancer cells express heretofore unrecognized L-selectin ligand(s) that mediate binding to lymphocyte L-selectin at conspicuously low shear stress levels of 0.07-0.08 dynes/cm(2), consistent with lymphatic flow. The binding of head and neck squamous cancer cells to L-selectin displays canonical biochemical features, such as requirements for sialylation, sulfation, and N-glycosylation, but displays a novel operational shear threshold differing from all other L-selectin ligands, including those expressed on colon cancer and leukemic cells (e.g. HCELL). These data define a novel class of L-selectin ligands and expand the scope of function for L-selectin within circulatory systems to now include a novel activity within shear stresses characteristic of lymphatic flow. PMID:18385135

  8. Human Umbilical Cord Wharton's Jelly Stem Cell Conditioned Medium Induces Tumoricidal Effects on Lymphoma Cells Through Hydrogen Peroxide Mediation.

    PubMed

    Lin, Hao Daniel; Fong, Chui-Yee; Biswas, Arijit; Choolani, Mahesh; Bongso, Ariff

    2016-09-01

    Several groups have reported that human umbilical cord Wharton's jelly stem cells (hWJSCs) possess unique tumoricidal properties against many cancers. However, the exact mechanisms as to how hWJSCs inhibit tumor growth are not known. Recent evidence suggests that exposure of cancer cells to high hydrogen peroxide (H2 O2 ) levels from H2 O2 -releasing drugs causes their death. We therefore explored whether the tumoricidal effect of hWJSCs on lymphoma cells was mediated via H2 O2 . We first exposed lymphoma cells to six different molecular weight cut-off (MWCO) concentrates of hWJSC-conditioned medium (hWJSC-CM) (3, 5, 10, 30, 50, 100 kDa) for 48 h. Since, the 3 kDa-MWCO concentrate showed the greatest cell inhibition we then investigated whether the tumoricidal effect of the specific 3 kDa-MWCO concentrate on two different lymphoma cell lines (Ramos and Toledo) was mediated via accumulation of H2 O2 . We used a battery of assays (MTT, propidium iodide, mitochondria membrane potential, apoptosis, cell cycle, oxidative stress enzymes, hydrogen peroxide, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation) to test this mechanism. The hWJSC-CM-3 kDa MWCO concentrate significantly decreased cell viability and mitochondrial membrane potential and increased cell death and apoptosis in both lymphoma cell lines. There were significant increases in superoxide dismutase with concomitant decreases in glutathione peroxidase, catalase, and thioredoxin peroxidase activities. H2 O2 levels, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation were also significantly increased in both lymphoma cell lines. The results suggested that the hWJSC-CM-3 kDa MWCO concentrate regulates cellular H2 O2 leading to a tumoricidal effect and may thus be a promising anti-lymphoma agent. J. Cell. Biochem. 117: 2045-2055, 2016. © 2016 Wiley Periodicals, Inc. PMID:27392313

  9. Conditioning of Parsley (Petroselinum crispum L.) Suspension Cells Increases Elicitor-Induced Incorporation of Cell Wall Phenolics.

    PubMed Central

    Kauss, H.; Franke, R.; Krause, K.; Conrath, U.; Jeblick, W.; Grimmig, B.; Matern, U.

    1993-01-01

    The elicitor-induced incorporation of phenylpropanoid derivatives into the cell wall and the secretion of soluble coumarin derivatives (phytoalexins) by parsley (Petroselinum crispum L.) suspension cultures can be potentiated by pretreatment of the cultures with 2,6-dichloroisonicotinic acid or derivatives of salicylic acid. To investigate this phenomenon further, the cell walls and an extracellular soluble polymer were isolated from control cells or cells treated with an elicitor from Phytophthora megasperma f. sp. glycinea. After alkaline hydrolysis, both fractions from elicited cells showed a greatly increased content of 4-coumaric, ferulic, and 4-hydroxybenzoic acid, as well as 4-hydroxybenzaldehyde and vanillin. Two minor peaks were identified as tyrosol and methoxytyrosol. The pretreatment effect is most pronounced at a low elicitor concentration. Its specificity was elaborated for coumarin secretion. When the parsley suspension cultures were preincubated for 1 d with 2,6-dichloroisonicotinic, 4- or 5-chlorosalicylic, or 3,5- dichlorosalicylic acid, the cells exhibited a greatly increased elicitor response. Pretreatment with isonicotinic, salicylic, acetylsalicylic, or 2,6-dihydroxybenzoic acid was less efficient in enhancing the response, and some other isomers were inactive. This increase in elicitor response was also observed for the above-mentioned monomeric phenolics, which were liberated from cell walls upon alkaline hydrolysis and for "lignin-like" cell wall polymers determined by the thioglycolic acid method. It was shown for 5-chlorosalicylic acid that conditioning most likely improves the signal transduction leading to the activation of genes encoding phenylalanine ammonia lyase and 4-coumarate: coenzyme A ligase. The conditioning thus sensitizes the parsley suspension cells to respond to lower elicitor concentrations. If a similar mechanism were to apply to whole plants treated with 2,6-dichloroisonicotinic acid, a known inducer of systemic

  10. Leukemia cell mobilization with G-CSF plus plerixafor during busulfan-fludarabine conditioning in allogeneic stem cell transplantation

    PubMed Central

    Thall, Peter F.; Zeng, Zhihong; Shpall, Elizabeth; Ciurea, Stefan; Kebriaei, Partow; Alousi, Amin; Popat, Uday; Anderlini, Paolo; Nieto, Yago; Parmar, Simrit; Qiao, Wei; Chen, Julianne; Rondon, Gabriela; McMullin, Becky; Wang, Rui-Yu; Lu, Hongbo; Schober, Wendy; Woodworth, Glenda; Gulbis, Alison; Cool, Rita; Andreeff, Michael; Champlin, Richard

    2015-01-01

    We hypothesized that during conditioning chemotherapy for allogeneic stem cell transplant (allo-SCT), disruption of stromal-leukemia interactions using granulocyte-colony stimulating factor (G-CSF) in combination with the CXCR4-specific inhibitor plerixafor, may promote release of leukemic cells from the niche and increase tumor elimination. In a phase 1/2 investigation, we treated 45 AML/MDS/CML patients (34 AML, 7 MDS, and 4 CML) with G-CSF (10 μg/kg daily for 6 days starting on day −9) plus plerixafor (doses of 0, 80, 160 or 240 μg/kg daily for 4 days starting on day −7) along with the busulfan-fludarabine (Bu-Flu) conditioning regimen. In the phase 1 part, we determined that G-CSF plus plerixafor is safe in this setting. We compared clinical effects and outcomes of AML/MDS study patients (n = 40) to 164 patients from a historical data set who received Bu-Flu alone prior to allo-SCT by stratifying on cytogenetics and disease status to correct for bias. Study patients had increased myeloid chimerism and lower rates of GvHD. There was no significant difference in relapse free survival or overall survival. The G-CSF plus plerixafor combination increased circulating white blood cells, CD34+ cells, and CXCR4+ cells, and preferentially mobilized FISH+ leukemic cells. ClinicalTrials.gov identifier is NCT00822770. PMID:25867648

  11. Expansion of brain T cells in homeostatic conditions in lymphopenic Rag2(-/-) mice.

    PubMed

    Song, Chang; Nicholson, James D; Clark, Sarah M; Li, Xin; Keegan, Achsah D; Tonelli, Leonardo H

    2016-10-01

    The concept of the brain as an immune privileged organ is rapidly evolving in light of new findings outlining the sophisticated relationship between the central nervous and the immune systems. The role of T cells in brain development and function, as well as modulation of behavior has been demonstrated by an increasing number of studies. Moreover, recent studies have redefined the existence of a brain lymphatic system and the presence of T cells in specific brain structures, such as the meninges and choroid plexus. Nevertheless, much information is needed to further the understanding of brain T cells and their relationship with the central nervous system under non-inflammatory conditions. In the present study we employed the Rag2(-/-) mouse model of lymphocyte deficiency and reconstitution by adoptive transfer to study the temporal and anatomical expansion of T cells in the brain under homeostatic conditions. Lymphopenic Rag2(-/-) mice were reconstituted with 10 million lymphoid cells and studied at one, two and four weeks after transfer. Moreover, lymphoid cells and purified CD4(+) and CD8(+) T cells from transgenic GFP expressing mice were used to define the neuroanatomical localization of transferred cells. T cell numbers were very low in the brain of reconstituted mice up to one week after transfer and significantly increased by 2weeks, reaching wild type values at 4weeks after transfer. CD4(+) T cells were the most abundant lymphocyte subtype found in the brain followed by CD8(+) T cells and lastly B cells. Furthermore, proliferation studies showed that CD4(+) T cells expand more rapidly than CD8(+) T cells. Lymphoid cells localize abundantly in meningeal structures, choroid plexus, and circumventricular organs. Lymphocytes were also found in vascular and perivascular spaces and in the brain parenchyma across several regions of the brain, in particular in structures rich in white matter content. These results provide proof of concept that the brain meningeal

  12. Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses1

    PubMed Central

    Wu, Gregory F.; Corbo, Evann; Schmidt, Michelle; Smith-Garvin, Jennifer E.; Riese, Matthew J.; Jordan, Martha S.; Laufer, Terri M.; Brown, Eric J.; Maltzman, Jonathan S.

    2011-01-01

    SUMMARY The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of wild-type SLP-76 protein in thymocytes. Following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T cell mediated autoimmune disease experimental autoimmune encephalomyelitis. Our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects. PMID:21469089

  13. Metabolite profiling of microfluidic cell culture conditions for droplet based screening

    PubMed Central

    Bjork, Sara M.; Sjostrom, Staffan L.; Andersson-Svahn, Helene; Joensson, Haakan N.

    2015-01-01

    We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format. PMID:26392830

  14. Conditioned Media from Microvascular Endothelial Cells Cultured in Simulated Microgravity Inhibit Osteoblast Activity

    PubMed Central

    Cazzaniga, Alessandra; Castiglioni, Sara; Maier, Jeanette A. M.

    2014-01-01

    Background and Aims. Gravity contributes to the maintenance of bone integrity. Accordingly, weightlessness conditions during space flight accelerate bone loss and experimental models in real and simulated microgravity show decreased osteoblastic and increased osteoclastic activities. It is well known that the endothelium and bone cells cross-talk and this intercellular communication is vital to regulate bone homeostasis. Because microgravity promotes microvascular endothelial dysfunction, we anticipated that the molecular cross-talk between endothelial cells exposed to simulated microgravity and osteoblasts might be altered. Results. We cultured human microvascular endothelial cells in simulated microgravity using the rotating wall vessel device developed by NASA. Endothelial cells in microgravity show growth inhibition and release higher amounts of matrix metalloproteases type 2 and interleukin-6 than controls. Conditioned media collected from microvascular endothelial cells in simulated microgravity were used to culture human osteoblasts and were shown to retard osteoblast proliferation and inhibit their activity. Discussion. Microvascular endothelial cells in microgravity are growth retarded and release high amounts of matrix metalloproteases type 2 and interleukin-6, which might play a role in retarding the growth of osteoblasts and impairing their osteogenic activity. Conclusions. We demonstrate that since simulated microgravity modulates microvascular endothelial cell function, it indirectly impairs osteoblastic function. PMID:25210716

  15. Low-level laser irradiation effect on endothelial cells under conditions of hyperglycemia.

    PubMed

    Góralczyk, Krzysztof; Szymańska, Justyna; Szot, Katarzyna; Fisz, Jacek; Rość, Danuta

    2016-07-01

    Diabetes mellitus is considered to be a very serious lifestyle disease leading to cardiovascular complications and impaired wound healing observed in the diabetic foot syndrome. Chronic hyperglycemia is the source of the endothelial activation. The inflammatory process in diabetes is associated with the secretion of inflammatory cytokines by endothelial cells, e.g., tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). The method of phototherapy using laser beam of low power (LLLT-low-level laser therapy) effectively supports the conventional treatment of diabetic vascular complications such as diabetic foot syndrome. The aim of our study was to evaluate the effect of low-power laser irradiation at two wavelengths (635 and 830 nm) on the secretion of inflammatory factors (TNF-α and IL-6) by the endothelial cell culture-HUVEC line (human umbilical vein endothelial cell)-under conditions of hyperglycemia. It is considered that adverse effects of hyperglycemia on vascular endothelial cells may be corrected by the action of LLLT, especially with the wavelength of 830 nm. It leads to the reduction of TNF-α concentration in the supernatant and enhancement of cell proliferation. Endothelial cells play an important role in the pathogenesis of diabetes; however, a small number of studies evaluate an impact of LLLT on these cells under conditions of hyperglycemia. Further work on this subject is warranted. PMID:26861982

  16. Numerical analysis of molten carbonate fuel cell stack performance: diagnosis of internal conditions using cell voltage profiles

    NASA Astrophysics Data System (ADS)

    Yoshiba, F.; Abe, T.; Watanabe, T.

    A numerical model to diagnose the internal conditions of a molten carbonate fuel cell (MCFC) has been developed to calculate both the temperature and performance of stacks. The performance of the stack is evaluated by applying a `formula for MCFC performance' which has been derived from tests on single small cells with the same active components as the stack. Concerning the separator temperature and the cell performance, calculated results are compared with experimental data acquired during the operation of a 100-kW class stack. Good agreement is obtained. The applied numerical electric circuit model is modified to analyse the voltage distribution within each individual cell. The purpose of the model is to identify the cause of unexpected voltage differences within each cell during operation of a 100-kW class stack. Two causes are identified, namely, increase in the partial internal resistance (IR) and insufficient supply of fuel gas to the cell. The calculated cell voltage distribution and the observed voltage difference for a given cell exhibit similar behaviour.

  17. Prox1 Inhibits Proliferation and Is Required for Differentiation of the Oligodendrocyte Cell Lineage in the Mouse

    PubMed Central

    Kato, Kentaro; Konno, Daijiro; Berry, Martin; Matsuzaki, Fumio; Logan, Ann; Hidalgo, Alicia

    2015-01-01

    Central nervous system injury induces a regenerative response in ensheathing glial cells comprising cell proliferation, spontaneous axonal remyelination, and limited functional recovery, but the molecular mechanisms are not fully understood. In Drosophila, this involves the genes prospero and Notch controlling the balance between glial proliferation and differentiation, and manipulating their levels in glia can switch the response to injury from prevention to promotion of repair. In the mouse, Notch1 maintains NG2 oligodendrocyte progenitor cells (OPCs) in a progenitor state, but what factor may enable oligodendrocyte (OL) differentiation and functional remyelination is not understood. Here, we asked whether the mammalian homologue of prospero, Prox1, is involved. Our data show that Prox1 is distributed in NG2+ OPCs and in OLs in primary cultured cells, and in the mouse spinal cord in vivo. siRNA prox1 knockdown in primary OPCs increased cell proliferation, increased NG2+ OPC cell number and decreased CC1+ OL number. Prox1 conditional knockout in the OL cell lineage in mice increased NG2+ OPC cell number, and decreased CC1+ OL number. Lysolecithin-induced demyelination injury caused a reduction in CC1+ OLs in homozygous Prox1-/- conditional knockout mice compared to controls. Remarkably, Prox1-/- conditional knockout mice had smaller lesions than controls. Altogether, these data show that Prox1 is required to inhibit OPC proliferation and for OL differentiation, and could be a relevant component of the regenerative glial response. Therapeutic uses of glia and stem cells to promote regeneration and repair after central nervous system injury would benefit from manipulating Prox1. PMID:26709696

  18. Derivation of iPSCs after Culture of Human Dental Pulp Cells under Defined Conditions

    PubMed Central

    Takeda-Kawaguchi, Tomoko; Sugiyama, Ken; Chikusa, Shunji; Iida, Kazuki; Aoki, Hitomi; Tamaoki, Naritaka; Hatakeyama, Daijiro; Kunisada, Takahiro; Shibata, Toshiyuki; Fusaki, Noemi; Tezuka, Ken-ichi

    2014-01-01

    Human dental pulp cells (hDPCs) are a promising resource for regenerative medicine and tissue engineering and can be used for derivation of induced pluripotent stem cells (iPSCs). However, current protocols use reagents of animal origin (mainly fetal bovine serum, FBS) that carry the potential risk of infectious diseases and unwanted immunogenicity. Here, we report a chemically defined protocol to isolate and maintain the growth and differentiation potential of hDPCs. hDPCs cultured under these conditions showed significantly less primary colony formation than those with FBS. Cell culture under stringently defined conditions revealed a donor-dependent growth capacity; however, once established, the differentiation capabilities of the hDPCs were comparable to those observed with FBS. DNA array analyses indicated that the culture conditions robustly altered hDPC gene expression patterns but, more importantly, had little effect on neither pluripotent gene expression nor the efficiency of iPSC induction. The chemically defined culture conditions described herein are not perfect serum replacements, but can be used for the safe establishment of iPSCs and will find utility in applications for cell-based regenerative medicine. PMID:25521610

  19. Busulfan Conditioning Enhances Engraftment of Hematopoietic Donor-derived Cells in the Brain Compared With Irradiation

    PubMed Central

    Wilkinson, Fiona L; Sergijenko, Ana; Langford-Smith, Kia J; Malinowska, Marcela; Wynn, Rob F; Bigger, Brian W

    2013-01-01

    Hematopoietic stem cell gene therapy for neurological disorders relies on transmigration of donor-derived monocytes to the brain, where they can engraft as microglia and deliver therapeutic proteins. Many mouse studies use whole-body irradiation to investigate brain transmigration pathways, but chemotherapy is generally used clinically. The current evidence for transmigration to the brain after chemotherapy is conflicting. We compared hematopoietic donor cell brain engraftment after bone marrow (BM) transplants in busulfan- or irradiation-conditioned mice. Significantly more donor-derived microglial cells engrafted posttransplant in busulfan-conditioned brain compared with the irradiated, in both the short and long term. Although total Iba-1+ microglial content was increased in irradiated brain in the short term, it was similar between groups over long-term engraftment. MCP-1, a key regulator of monocyte transmigration, showed long-term elevation in busulfan-conditioned brain, whereas irradiated brains showed long-term elevation of the proinflammatory chemokine interleukin 1α (IL-1α), with increased in situ proliferation of resident microglia, and significant increases in the relative number of amoeboid activated microglia in the brain. This has implications for the choice of conditioning regimen to promote hematopoietic cell brain engraftment and the relevance of irradiation in mouse models of transplantation. PMID:23423338

  20. Three-dimensional Culture Conditions Lead to Decreased Radiation Induced Crytoxicity in Human Mammary Epithelial Cells

    SciTech Connect

    Sowa, Marianne B.; Chrisler, William B.; Zens, Kyra D.; Ashjian, Emily J.; Opresko, Lee K.

    2010-05-01

    For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extra cellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D vs. 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. ~4 fold increased survival at 5 Gy). The cell cycle delay induced following exposure to 2 and 5 Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures

  1. Helminth-conditioned dendritic cells prime CD4+ T cells to IL-4 production in vivo.

    PubMed

    Connor, Lisa M; Tang, Shiau-Choot; Camberis, Mali; Le Gros, Graham; Ronchese, Franca

    2014-09-15

    Dendritic cells (DC) are critical for the initiation of immune responses; however, their role in priming IL-4-producing Th2 cells in vivo is not fully understood. We used a model of intradermal injection with fluorescent-labeled, nonviable larvae from the helminth parasite nonviable Nippostrongylus brasiliensis L3 larvae (Nb), a strong inducer of Th2 responses, together with IL-4-GFP reporter mice that enable a sensitive detection of IL-4 production to examine the contribution of DC to the priming of IL-4-producing CD4(+) T cells in vivo. We found that parasite material is taken up by two distinct DC populations in draining lymph nodes: a mostly CD11c(int)MHC class II (MHCII)(hi)CD11b(+)Ly6C(-) dermal DC population and a CD11c(hi)MHCII(int)CD11b(+)Ly6C(+) monocyte-derived DC population. After Nb treatment, these two DC populations appeared in the draining lymph nodes in comparable numbers and with similar kinetics; however, treatment with pertussis toxin blocked the migration of dermal DC and the priming of IL-4-producing T cells, but only partially affected monocyte-derived DC numbers. In line with this observation, transfer of OVA-loaded CD11c(int)MHCII(hi) DC from Nb-treated mice into naive hosts could sensitize OVA-specific CD4(+) T cells to IL-4 production, whereas transfer of CD11c(int)MHCII(hi) DC from naive mice, or CD11c(hi)MHCII(int) DC from Nb-treated or naive mice, induced CD4(+) T cell expansion but no IL-4 production. Phenotypic analysis of Nb-loaded CD11c(int)MHCII(hi) DC revealed expression of programmed death ligand 2, CD301b, IFN regulatory factor 4, and moderate upregulation of OX40 ligand. However, thymic stromal lymphopoietin and OX40 ligand were not required for Th2 priming. Thus, our data suggest that appropriate stimuli can induce DC to express the unique signals sufficient to direct CD4(+) T cells to Th2 differentiation. PMID:25108019

  2. Investigation of degradation effects in polymer electrolyte fuel cells under automotive-related operating conditions

    NASA Astrophysics Data System (ADS)

    Enz, S.; Dao, T. A.; Messerschmidt, M.; Scholta, J.

    2015-01-01

    The influence of artificial starvation effects during automotive-related operating conditions is investigated within a polymer electrolyte fuel cell (PEFC) using non-dispersive infrared sensors and a current scan shunt. Driving cycles (DC) and single load change experiments are performed with specific fuel and oxidant starvation conditions. Within the DC experiments, a maximal CO2 amount of 4.67 μmol per cycle is detected in the cathode and 0.97 μmol per cycle in the anode exhaust without reaching fuel starvation conditions during the DC. Massive cell reversal conditions occur within the single load change experiments as a result of anodic fuel starvation. As soon as a fuel starvation appears, the emitted CO2 increases exponentially in the anode and cathode exhaust. A maximal CO2 amount of 143.8 μmol CO2 on the anode side and 5.8 μmol CO2 on the cathode side is detected in the exhaust gases. The critical cell reversal conditions only occur by using hydrogen reformate as anode reactant. The influence of the starvation effects on the PEFC performance is investigated via polarization curves, cyclic and linear sweep voltammetry as well as electrochemical impedance spectroscopy. The PEFC performance is reduced by 47% as a consequence of the dynamic operation.

  3. Studies on Culture and Osteogenic Induction of Human Mesenchymal Stem Cells under CO2-Independent Conditions

    PubMed Central

    Chen, Jian; Zhang, Cui; Feng, Yiding; Zong, Chen; Chen, Jiarong; Tang, Zihua; Jia, Bingbing; Tong, Xiangming; Zheng, Qiang

    2013-01-01

    Abstract Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce

  4. Effect of dendritic cell state and antigen-presentation conditions on resulting T-cell phenotypes and Th cytokine profiles.

    PubMed

    de Lastic, Anne-Lise; Rodi, Maria; Mouzaki, Athanasia

    2016-08-01

    T cells play a pivotal role in controlling the immune response and have been the focus of extensive research. We studied the process of in vitro generation of antigen-specific T effector cells (Teffs) to assess the dynamics of antigen presentation and determine the best conditions for cell therapy. We used a peptidic construct consisting of combined HLA class I and II epitopes of the tumor antigen MAGE-3 as an antigen. Monocytes were isolated from healthy donors and were differentiated to dendritic cells (DCs) in vitro. The peptide was added to the DC culture, the pulsed cells were transferred to a co-culture with lymphocytes from the same donor, either as irradiated feeders or untreated, and were cultured in the presence or absence of IL-2. Several rounds of restimulation followed. The cells were analyzed by Flow Cytometry, and cytokine levels were measured by ELISA and Cytometric Bead Array for Th1/Th2/Th17 profiling. The results showed that the lymphocytes in culture upregulated their activation markers and produced Th1 proinflammatory cytokines in response to the peptide, optimally when it was presented by non-irradiated dendritic cells in the presence of IL-2. In contrast, DC irradiation resulted in low activation potential and a shift toward a suppressive phenotype. After prolonged antigenic stimulation, the culture displayed Th17 polarization. In conclusion, the functional integrity of DCs is necessary for the development of antigen-specific Teffs, and culture conditions can be developed to create Teffs with specific properties for eventual use in cell therapy applications. PMID:27130240

  5. A Two-State Cell Damage Model Under Hyperthermic Conditions: Theory and In Vitro Experiments

    PubMed Central

    Feng, Yusheng; Oden, J. Tinsley; Rylander, Marissa Nichole

    2010-01-01

    The ultimate goal of cancer treatment utilizing thermotherapy is to eradicate tumors and minimize damage to surrounding host tissues. To achieve this goal, it is important to develop an accurate cell damage model to characterize the population of cell death under various thermal conditions. The traditional Arrhenius model is often used to characterize the damaged cell population under the assumption that the rate of cell damage is proportional to exp(−Ea/RT), where Ea is the activation energy, R is the universal gas constant, and T is the absolute temperature. However, this model is unable to capture transition phenomena over the entire hyperthermia and ablation temperature range, particularly during the initial stage of heating. Inspired by classical statistical thermodynamic principles, we propose a general two-state model to characterize the entire cell population with two distinct and measurable subpopulations of cells, in which each cell is in one of the two microstates, viable (live) and damaged (dead), respectively. The resulting cell viability can be expressed as C(τ, T) = exp(−Φ(τ, T)/kT)/(1+exp(−Φ(τ, T)/kT)), where k is a constant. The in vitro cell viability experiments revealed that the function Φ(τ, T) can be defined as a function that is linear in exposure time τ when the temperature T is fixed, and linear as well in terms of the reciprocal of temperature T when the variable τ is held as constant. To determine parameters in the function Φ(τ, T), we use in vitro cell viability data from the experiments conducted with human prostate cancerous (PC3) and normal (RWPE-1) cells exposed to thermotherapeutic protocols to correlate with the proposed cell damage model. Very good agreement between experimental data and the derived damage model is obtained. In addition, the new two-state model has the advantage that is less sensitive and more robust due to its well behaved model parameters. PMID:18601458

  6. The induction of human peripheral blood lymphoid colonies by conditioned media from human tumour cell lines.

    PubMed Central

    Vesole, D H; Moore, G E

    1980-01-01

    Conditioned medium (CM) from 29 human tumour cell lines and 3 malignant pleural fluids were tested for their ability to stimulate lymphoid colony formation in semi-solid agar; 9 of 14 malignant melanomas, 3 of 6 colonic carcinomas, 2 of 5 ovarian carcinomas, 3 of 4 breast carcinomas and 1 of 3 pleural fluids from breast cancer patients contained colony-stimulating activity (CSA) for human peripheral blood lymphoid cells (PBL) in semi-solid agar. Conditioned media also stimulated PBL proliferation in liquid medium; these effects were dose dependent. With the exception of one pleural fluid, extensive dialysis of CM did not significantly increase colony formation; CM from two tumour cell lines demonstrated a significant decrease in the induction of colony formation after dialysis. PMID:6970165

  7. Increased performance of hydrogen production in microbial electrolysis cells under alkaline conditions.

    PubMed

    Rago, Laura; Baeza, Juan A; Guisasola, Albert

    2016-06-01

    This work reports the first successful enrichment and operation of alkaline bioelectrochemical systems (microbial fuel cells, MFC, and microbial electrolysis cells, MEC). Alkaline (pH=9.3) bioelectrochemical hydrogen production presented better performance (+117%) compared to conventional neutral conditions (2.6 vs 1.2 litres of hydrogen gas per litre of reactor per day, LH2·L(-1)REACTOR·d(-1)). Pyrosequencing results of the anodic biofilm showed that while Geobacter was mainly detected under conventional neutral conditions, Geoalkalibacter sp. was highly detected in the alkaline MFC (21%) and MEC (48%). This is the first report of a high enrichment of Geoalkalibacter from an anaerobic mixed culture using alkaline conditions in an MEC. Moreover, Alkalibacter sp. was highly present in the anodic biofilm of the alkaline MFC (37%), which would indicate its potentiality as a new exoelectrogen. PMID:26855359

  8. In-Depth Proteomic Quantification of Cell Secretome in Serum-Containing Conditioned Medium.

    PubMed

    Weng, Yejing; Sui, Zhigang; Shan, Yichu; Jiang, Hao; Zhou, Yuan; Zhu, Xudong; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-05-01

    Secreted proteins play key roles during cellular communication, proliferation, and migration. The comprehensive profiling of secreted proteins in serum-containing culture media is technically challenging. Most studies have been performed under serum-free conditions. However, these conditions might alter the status of the cells. Herein, we describe an efficient strategy that avoids the disturbance of serum by combining metabolic labeling, protein "equalization," protein fractionation, and filter-aided sample preparation, called MLEFF, enabling the identification of 534 secreted proteins from HeLa conditioned media, including 31 cytokines, and growth factors. This MLEFF strategy was also successfully applied during a comparative secretome analysis of two human hepatocellular carcinoma cell lines with differentially metastatic potentials, enabling the quantification of 61 significantly changed proteins involved in tumor invasion and metastasis. PMID:27042867

  9. Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines.

    PubMed

    Rahman, Maryam; Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W; Stringer, Brett W; Boyd, Andrew W; Johns, Terrance G; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A

    2015-03-01

    Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture. PMID:25806119

  10. Regenerative and reparative effects of human chorion-derived stem cell conditioned medium on photo-aged epidermal cells.

    PubMed

    Li, Qiankun; Chen, Yan; Ma, Kui; Zhao, Along; Zhang, Cuiping; Fu, Xiaobing

    2016-04-17

    Epidermal cells are an important regenerative source for skin wound healing. Aged epidermal cells have a low ability to renew themselves and repair skin injury. Ultraviolet (UV) radiation, particularly UVB, can cause photo-aging of the skin by suppressing the viability of human epidermal cells. A chorion-derived stem cell conditioned medium (CDSC-CNM) is thought to have regenerative properties. This study aimed to determine the regenerative effects of CDSC-CNM on UVB-induced photo-aged epidermal cells. Epidermal cells were passaged four times and irradiated with quantitative UVB, and non-irradiated cells served as a control group. Cells were then treated with different concentrations of CDSC-CNM. Compared to the non-irradiated group, the proliferation rates and migration rates of UVB-induced photo-aged epidermal cells significantly decreased (p < 0.05) with increasing intracellular radical oxygen species (ROS) generation and DNA damage. After treatment with CDSC-CNM, photo-aged epidermal cells significantly improved their viability, and their ROS generation and DNA damage decreased. The secretory factors in CDSC-CNM, including epidermal growth factor (EGF), transforming growth factor-β (TGF-β), interleukin (IL)-6, and IL-8 and the related signaling pathway protein levels, increased compared to the control medium (CM). The potential regenerative and reparative effects of CDSC-CNM indicate that it may be a candidate material for the treatment of prematurely aged skin. The functions of the secretory factors and the mechanisms of CDSC-CNM therapy deserve further attention. PMID:27097375

  11. Extracellular Membrane Vesicles as Vehicles for Brain Cell-to-Cell Interactions in Physiological as well as Pathological Conditions

    PubMed Central

    Schiera, Gabriella; Di Liegro, Carlo Maria; Di Liegro, Italia

    2015-01-01

    Extracellular vesicles are involved in a great variety of physiological events occurring in the nervous system, such as cross talk among neurons and glial cells in synapse development and function, integrated neuronal plasticity, neuronal-glial metabolic exchanges, and synthesis and dynamic renewal of myelin. Many of these EV-mediated processes depend on the exchange of proteins, mRNAs, and noncoding RNAs, including miRNAs, which occurs among glial and neuronal cells. In addition, production and exchange of EVs can be modified under pathological conditions, such as brain cancer and neurodegeneration. Like other cancer cells, brain tumours can use EVs to secrete factors, which allow escaping from immune surveillance, and to transfer molecules into the surrounding cells, thus transforming their phenotype. Moreover, EVs can function as a way to discard material dangerous to cancer cells, such as differentiation-inducing proteins, and even drugs. Intriguingly, EVs seem to be also involved in spreading through the brain of aggregated proteins, such as prions and aggregated tau protein. Finally, EVs can carry useful biomarkers for the early diagnosis of diseases. Herein we summarize possible roles of EVs in brain physiological functions and discuss their involvement in the horizontal spreading, from cell to cell, of both cancer and neurodegenerative pathologies. PMID:26583089

  12. Characteristics of human CD34+ cells exposed to ionizing radiation under cytokine-free conditions

    PubMed Central

    Ishikawa, Junya; Hayashi, Naoki; Yamaguchi, Masaru; Monzen, Satoru; Kashiwakura, Ikuo

    2015-01-01

    To clarify the mechanisms underlying radiation-induced hematopoietic stem cell death, we investigated the effects of excessive ionizing radiation on the clonogenic potential of CD34+ cells obtained from human umbilical cord blood under cytokine-free conditions. The CD34+ cells were X-ray–irradiated (up to 2 Gy) and were cultured for 0–48 h under cytokine-free conditions. At various time-points, the CD34+ cells were investigated for survival, clonogenic potential and the generation of mitochondrial superoxide. At 12 h after X-ray irradiation, the number of viable cells had decreased to ∼70–80% compared with the 0-h non-irradiated control, whereas the clonogenic potential in the X-ray–irradiated cells had decreased to ∼50%–60% compared with the 0-h non-irradiated control. Furthermore, significant generation of mitochondrial superoxide was observed at 6 h, and reached a maximum value between 12 and 24 h after X-ray irradiation. However, no significant differences were observed between non-irradiated and X-ray–irradiated cells in terms of the generation of reactive oxygen species or in the intracellular mitochondrial contents. In addition, a cDNA microarray analysis showed that the majority of the altered genes in the CD34+ cells at 6 h after X-ray irradiation were apoptosis-related genes. These results suggest the possibility that the elimination of the clonogenic potentials of CD34+ cells involves the generation of mitochondrial superoxide induced by ionizing radiation. PMID:25877692

  13. The antioxidant effect of the Malaysian Gelam honey on pancreatic hamster cells cultured under hyperglycemic conditions.

    PubMed

    Batumalaie, Kalaivani; Qvist, Rajes; Yusof, Kamaruddin Mohd; Ismail, Ikram Shah; Sekaran, Shamala Devi

    2014-05-01

    Type 2 diabetes consists of progressive hyperglycemia, insulin resistance, and pancreatic β-cell failure which could result from glucose toxicity, inflammatory cytokines, and oxidative stress. In the present study, we investigate the effect of pretreatment with Gelam honey (Melaleuca spp.) and the individual flavonoid components chrysin, luteolin, and quercetin, on the production of reactive oxygen species (ROS), cell viability, lipid peroxidation, and insulin content in hamster pancreatic cells (HIT-T15 cells), cultured under normal and hyperglycemic conditions. Phenolic extracts from a local Malaysian species of Gelam honey (Melaleuca spp.) were prepared using the standard extraction methods. HIT-T15 cells were cultured in 5 % CO2 and then preincubated with Gelam honey extracts (20, 40, 60, and 80 μg/ml) as well as some of its flavonoid components chrysin, luteolin, and quercetin (20, 40, 60, and 80 μM), prior to stimulation by 20 and 50 mM of glucose. The antioxidative effects were measured in these cultured cells at different concentrations and time point by DCFH-DA assay. Pretreatment of cells with Gelam honey extract or the flavonoid components prior to culturing in 20 or 50 mM glucose showed a significant decrease in the production of ROS, glucose-induced lipid peroxidation, and a significant increase in insulin content and the viability of cells cultured under hyperglycemic condition. Our results show the in vitro antioxidative property of the Gelam honey and the flavonoids on the β-cells from hamsters and its cytoprotective effect against hyperglycemia. PMID:23584372

  14. Conditions for initiating Lake Victoria haplochromine (Oreochromis esculentus) primary cell cultures from caudal fin biopsies.

    PubMed

    Filice, Melissa; Lee, C; Mastromonaco, Gabriela F

    2014-10-01

    The global decline of freshwater fishes has created a need to cryopreserve biological materials from endangered species in an effort to conserve the biodiversity within this taxon. Since maternal gametes and embryos from fish are difficult to cryopreserve, somatic cells obtained from caudal fins have become an increasingly popular resource as they contain both maternal and paternal DNA ensuring valuable traits are not lost from the population. Somatic cells stored in cryobanks can be used to supplement endangered populations with genetically valuable offspring with the use of assisted reproductive technologies. However, initiating primary cell cultures from caudal fin biopsies of endangered species can be challenging as standardized protocols have not yet been developed. The objective of this study was to identify culture conditions, including antibiotic supplementation, biopsy size, and culture temperature, suitable for establishing primary cell cultures of ngege (Oreochromis esculentus), a critically endangered African cichlid. Six-millimeter caudal fin biopsies provided sufficient material to develop a primary cell culture when incubated at 25°C using standard fish cell culture medium containing 1× Primocin. Further investigation and application of these culture conditions for other endangered freshwater fishes is necessary. PMID:24985486

  15. Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells.

    PubMed

    Jiang, Nan-Wei; Wang, De-Juan; Xie, Ya-Jun; Zhou, Liang; Su, Li-Da; Li, Huashun; Wang, Qin-Wen; Shen, Ying

    2016-06-01

    Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor-mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells. PMID:26194056

  16. Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

    PubMed Central

    Rafiq, Qasim A.; Coopman, Karen; Nienow, Alvin W.

    2016-01-01

    Abstract Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost‐effective manufacturing processes. Microcarriers enable the culture of anchorage‐dependent cells in stirred‐tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow‐derived MSC (hBM‐MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM‐MSC1 underwent more cumulative population doublings over three passages in comparison to hBM‐MSC2 and hBM‐MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM‐MSC expansion. HBM‐MSCs were successfully harvested and characterised, demonstrating hBM‐MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier‐based allogeneic cell therapy manufacture. PMID:26632496

  17. Osteogenic priming of mesenchymal stem cells by chondrocyte-conditioned factors and mineralized matrix.

    PubMed

    Ro, Hyunuk; Park, Jungha; Yang, Kisuk; Kim, Jiyong; Yim, Hyun-Gu; Jung, Giyoung; Lee, Hyukjin; Cho, Seung-Woo; Hwang, Nathaniel S

    2015-10-01

    Transient cartilage and a mineralizing microenvironment play pivotal roles in mesenchymal cell ossification during bone formation. In order to recreate these microenvironmental cues, C3H10T1/2 murine mesenchymal stem cells (MSCs) were exposed to chondrocyte-conditioned medium (CM) and seeded onto three-dimensional mineralized scaffolds for bone regeneration. Expansion of C3H10T1/2 cells with CM resulted in enhanced expression levels of chondrogenic markers such as aggrecan, type II collagen, type X collagen, and Sox9, rather than of osteogenic genes. Interestingly, CM expansion led to reduced expression levels of osteogenic genes such as alkaline phosphatase (ALP), type I collagen, osteocalcin, and Runx2. However, CM-expanded C3H10T1/2 cells showed enhanced osteogenic differentiation as indicated by increased ALP and Alizarin Red S staining upon osteogenic factor exposure. In vivo, CM-expanded C3H10T1/2 mesenchymal cells were seeded onto mineralized scaffolds (fabricated with polydopamine and coated with simulated body fluids) and implanted into critical-sized calvarial-defect mouse models. After 8 weeks of implantation, mouse skulls were collected, and bone tissue regeneration was evaluated by micro-computed tumography and Masson's trichrome staining. In accordance with the in vitro analysis, CM-expanded C3H10T1/2 cells gave enhanced bone mineral deposition. Thus, chondrocyte-conditioned factors and a mineralized microenvironment stimulate the bone formation of MSCs. PMID:25956591

  18. Conditional Cripto overexpression in satellite cells promotes myogenic commitment and enhances early regeneration

    PubMed Central

    Prezioso, Carolina; Iaconis, Salvatore; Andolfi, Gennaro; Zentilin, Lorena; Iavarone, Francescopaolo; Guardiola, Ombretta; Minchiotti, Gabriella

    2015-01-01

    Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. Despite extensive studies, knowledge of the molecular mechanisms underlying the early events associated with satellite cell activation and myogenic commitment in muscle regeneration remains still incomplete. Cripto is a novel regulator of postnatal skeletal muscle regeneration and a promising target for future therapy. Indeed, Cripto is expressed both in myogenic and inflammatory cells in skeletal muscle after acute injury and it is required in the satellite cell compartment to achieve effective muscle regeneration. A critical requirement to further explore the in vivo cellular contribution of Cripto in regulating skeletal muscle regeneration is the possibility to overexpress Cripto in its endogenous configuration and in a cell and time-specific manner. Here we report the generation and the functional characterization of a novel mouse model for conditional expression of Cripto, i.e., the Tg:DsRedloxP/loxPCripto-eGFP mice. Moreover, by using a satellite cell specific Cre-driver line we investigated the biological effect of Cripto overexpression in vivo, and provided evidence that overexpression of Cripto in the adult satellite cell compartment promotes myogenic commitment and differentiation, and enhances early regeneration in a mouse model of acute injury. PMID:26052513

  19. Efficacious and Clinically Relevant Conditioned Medium of Human Adipose-derived Stem Cells for Therapeutic Angiogenesis

    PubMed Central

    Bhang, Suk Ho; Lee, Seahyoung; Shin, Jung-Youn; Lee, Tae-Jin; Jang, Hyeon-Ki; Kim, Byung-Soo

    2014-01-01

    Using stem cell–conditioned medium (CM) might be a viable alternative to stem cell transplantation, which is often hampered by low grafting efficiency and potential tumorigenesis, but the concentrations of angiogenic growth factors in CM are too low for therapeutic use and some components of the medium are not for human use. We used three-dimensional (3D) spheroid culture of human adipose-derived stem cells (ADSCs) with clinically relevant medium composed of amino acids, vitamins, glucose, and human serum to produce clinically relevant CM containing angiogenic and/or antiapoptotic factors such as vascular endothelial cell growth factor, fibroblast growth factor 2, hepatocyte growth factor, and chemokine (C-X-C motif) ligand 12. The concentrations of these factors were 23- to 27-fold higher than that in CM produced by conventional monolayer culture. Compared with injection of either monolayer culture CM or human ADSC, injection of spheroid culture CM to an ischemic region in mice significantly enhanced endothelial cell growth, CD34+/PTPRC− (endothelial progenitor) cell mobilization from bone marrow, and bone marrow cell homing to the ischemic region, resulting in improved blood vessel density, limb salvage, and blood perfusion in a mouse hindlimb ischemia model. The stem cell CM developed in this study will likely be an effective alternative to conventional stem cell transplantation therapy. PMID:24413377

  20. Optical examination of cell culture in bioreactors creating simulated in vivo conditions

    NASA Astrophysics Data System (ADS)

    Rolfe, Peter J.

    2005-01-01

    Cell culture using bioreactors is a vital part of Cellular and Tissue Engineering. Bioreactor design continues to advance, in order to allow control over physical and chemical parameters as well as continuous assessment of cell behaviour, gene expression, and tissue formation and growth. Measurement or monitoring of many such parameters or features can be achieved with optical techniques. The current aim of cell culture is to re-create in vivo conditions and in order to achieve this control of the chemical environment is required and some cell types must be subjected to shear stress and/or axial loads. For creating tissue engineered cartilage chondrocytes are cultured within a biodegradable scaffold. Influences of cyclic loading and of oxygen supply on phenotype are studied. Vascular endothelial cells are subjected to fluid shear stress and the influence on prostacyclin production is measured. Optical interrogation of culture fluid, attached cells, cells in suspension and tissue constructs is carried out using a combination of spectrophotometry, analysis of scattering, and chemical sensing. Insertion of sensing probes within the culture vessel presents problems of protein adsorption to sensing surfaces. Approaches based on cell membrane mimicry are being evaluated for their potential to overcome this problem. Sensors based on immobilised fluorophores and chromaphores within either wall-mounted membranes or within optical fibres are assessed. Culture fluid turbidity is evaluated with scattering determinations and circulating glucose concentration is measured spectrophotometrically. Formed tissue is interrogated with NIR radiation and in the future will include the use of OCT.

  1. T Cells Enhance Stem-Like Properties and Conditional Malignancy in Gliomas

    PubMed Central

    Irvin, Dwain K.; Jouanneau, Emmanuel; Duvall, Gretchen; Zhang, Xiao-xue; Zhai, Yuying; Sarayba, Danielle; Seksenyan, Akop; Panwar, Akanksha; Black, Keith L.; Wheeler, Christopher J.

    2010-01-01

    Background Small populations of highly tumorigenic stem-like cells (cancer stem cells; CSCs) can exist within, and uniquely regenerate cancers including malignant brain tumors (gliomas). Many aspects of glioma CSCs (GSCs), however, have been characterized in non-physiological settings. Methods We found gene expression similarity superiorly defined glioma “stemness”, and revealed that GSC similarity increased with lower tumor grade. Using this method, we examined stemness in human grade IV gliomas (GBM) before and after dendritic cell (DC) vaccine therapy. This was followed by gene expression, phenotypic and functional analysis of murine GL26 tumors recovered from nude, wild-type, or DC-vaccinated host brains. Results GSC similarity was specifically increased in post-vaccine GBMs, and correlated best to vaccine-altered gene expression and endogenous anti-tumor T cell activity. GL26 analysis confirmed immune alterations, specific acquisition of stem cell markers, specifically enhanced sensitivity to anti-stem drug (cyclopamine), and enhanced tumorigenicity in wild-type hosts, in tumors in proportion to anti-tumor T cell activity. Nevertheless, vaccine-exposed GL26 cells were no more tumorigenic than parental GL26 in T cell-deficient hosts, though they otherwise appeared similar to GSCs enriched by chemotherapy. Finally, vaccine-exposed GBM and GL26 exhibited relatively homogeneous expression of genes expressed in progenitor cells and/or differentiation. Conclusions T cell activity represents an inducible physiological process capable of proportionally enriching GSCs in human and mouse gliomas. Stem-like gliomas enriched by strong T cell activity, however, may differ from other GSCs in that their stem-like properties may be disassociated from increased tumor malignancy and heterogeneity under specific host immune conditions. PMID:20539758

  2. Altered differentiation and paracrine stimulation of mammary epithelial cell proliferation by conditionally activated Smoothened

    PubMed Central

    Visbal, Adriana P.; LaMarca, Heather L.; Villanueva, Hugo; Toneff, Michael J.; Li, Yi; Rosen, Jeffrey M.; Lewis, Michael T.

    2011-01-01

    The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation. PMID:21276786

  3. Human Wharton's jelly stem cells, its conditioned medium and cell-free lysate inhibit the growth of human lymphoma cells.

    PubMed

    Lin, Hao Daniel; Fong, Chui Yee; Biswas, Arijit; Choolani, Mahesh; Bongso, Ariff

    2014-08-01

    Several groups have reported that primitive mesenchymal stem cells from the gelatinous matrix of the Wharton's jelly of the human umbilical cord (hWJSCs) possess tumoricidal properties and inhibit the growth of solid tumours such as human mammary carcinoma, ovarian carcinoma and osteosarcoma. This unique characteristic led to the hypothesis that hWJSCs serve as a natural defence against migrating cancer cells from mother to fetus thus explaining why tumorigenesis in the fetus is rare. However, it is not known whether non-solid malignant hematopoietic cells are also inhibited by hWJSCs and what the exact tumoricidal mechanisms are. We therefore evaluated the influence of hWJSCs and its extracts on Burkitt's lymphoma cells. Cell proliferation (BrdU and Ki67+), viability (MTT) and cell death (Annexin V-Propidium iodide and live/dead) assays showed significant inhibition of lymphoma cell growth after 48 h exposure to hWJSCs or its extracts compared to controls. Increased cell death was observed at sub-G1 and S and decreased proliferation at G2/M phases of the mitotic cycle. Superoxide dismutase and hydrogen peroxide activity were significantly increased and glutathione peroxidase significantly decreased in treated lymphoma cells. Time lapse imaging and confocal z-stack images showed yellow fluorescent in situ hybridization (FISH) signals of lymphoma cell Y chromosomes within the cytoplasm of female red labelled hWJSCs. We hypothesize that the growth of lymphoma cells is inhibited by the molecules secreted by hWJSCs that use oxidative stress pathways to induce cell death followed by engulfment of the apoptotic remains of the lymphoma cells by the hWJSCs. PMID:24789672

  4. In vivo ectopic bone formation by devitalized mineralized stem cell carriers produced under mineralizing culture condition.

    PubMed

    Chai, Yoke Chin; Geris, Liesbet; Bolander, Johanna; Pyka, Grzegorz; Van Bael, Simon; Luyten, Frank P; Schrooten, Jan

    2014-12-01

    Functionalization of tissue engineering scaffolds with in vitro-generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca(2+)) and phosphate (PO4 (3-)) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography

  5. In Vivo Ectopic Bone Formation by Devitalized Mineralized Stem Cell Carriers Produced Under Mineralizing Culture Condition

    PubMed Central

    Chai, Yoke Chin; Geris, Liesbet; Bolander, Johanna; Pyka, Grzegorz; Van Bael, Simon; Luyten, Frank P.

    2014-01-01

    Abstract Functionalization of tissue engineering scaffolds with in vitro–generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca2+) and phosphate (PO43−) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano

  6. PPAR-delta promotes survival of breast cancer cells in harsh metabolic conditions

    PubMed Central

    Wang, X; Wang, G; Shi, Y; Sun, L; Gorczynski, R; Li, Y-J; Xu, Z; Spaner, D E

    2016-01-01

    Expression of the nuclear receptor peroxisome proliferator activated receptor delta (PPARδ) in breast cancer cells is negatively associated with patient survival, but the underlying mechanisms are not clear. High PPARδ protein levels in rat breast adenocarcinomas were found to be associated with increased growth in soft agar and mice. Transgenic expression of PPARδ increased the ability of human breast cancer cell lines to migrate in vitro and form lung metastases in mice. PPARδ also conferred the ability to grow in exhausted tissue culture media and survive in low-glucose and other endoplasmic reticulum stress conditions such as hypoxia. Upregulation of PPARδ by glucocorticoids or synthetic agonists also protected human breast cancer cells from low glucose. Survival in low glucose was related to increased antioxidant defenses mediated in part by catalase and also to late AKT phosphorylation, which is associated with the prolonged glucose-deprivation response. Synthetic antagonists reversed the survival benefits conferred by PPARδ in vitro. These findings suggest that PPARδ conditions breast cancer cells to survive in harsh microenvironmental conditions by reducing oxidative stress and enhancing survival signaling responses. Drugs that target PPARδ may have a role in the treatment of breast cancer. PMID:27270614

  7. Plasticity in variation of xylem and phloem cell characteristics of Norway spruce under different local conditions

    PubMed Central

    Gričar, Jožica; Prislan, Peter; de Luis, Martin; Gryc, Vladimír; Hacurová, Jana; Vavrčík, Hanuš; Čufar, Katarina

    2015-01-01

    There is limited information on intra-annual plasticity of secondary tissues of tree species growing under different environmental conditions. To increase the knowledge about the plasticity of secondary growth, which allows trees to adapt to specific local climatic regimes, we examined climate–radial growth relationships of Norway spruce [Picea abies (L.) H. Karst.] from three contrasting locations in the temperate climatic zone by analyzing tree-ring widths for the period 1932–2010, and cell characteristics in xylem and phloem increments formed in the years 2009–2011. Variation in the structure of xylem and phloem increments clearly shows that plasticity in seasonal dynamics of cambial cell production and cell differentiation exists on xylem and phloem sides. Anatomical characteristics of xylem and phloem cells are predominantly site-specific characteristics, because they varied among sites but were fairly uniform among years in trees from the same site. Xylem and phloem tissues formed in the first part of the growing season seemed to be more stable in structure, indicating their priority over latewood and late phloem for tree performance. Long-term climate and radial growth analyses revealed that growth was in general less dependent on precipitation than on temperature; however, growth sensitivity to local conditions differed among the sites. Only partial dependence of radial growth of spruce on climatic factors on the selected sites confirms its strategy to adapt the structure of wood and phloem increments to function optimally in local conditions. PMID:26442044

  8. PPAR-delta promotes survival of breast cancer cells in harsh metabolic conditions.

    PubMed

    Wang, X; Wang, G; Shi, Y; Sun, L; Gorczynski, R; Li, Y-J; Xu, Z; Spaner, D E

    2016-01-01

    Expression of the nuclear receptor peroxisome proliferator activated receptor delta (PPARδ) in breast cancer cells is negatively associated with patient survival, but the underlying mechanisms are not clear. High PPARδ protein levels in rat breast adenocarcinomas were found to be associated with increased growth in soft agar and mice. Transgenic expression of PPARδ increased the ability of human breast cancer cell lines to migrate in vitro and form lung metastases in mice. PPARδ also conferred the ability to grow in exhausted tissue culture media and survive in low-glucose and other endoplasmic reticulum stress conditions such as hypoxia. Upregulation of PPARδ by glucocorticoids or synthetic agonists also protected human breast cancer cells from low glucose. Survival in low glucose was related to increased antioxidant defenses mediated in part by catalase and also to late AKT phosphorylation, which is associated with the prolonged glucose-deprivation response. Synthetic antagonists reversed the survival benefits conferred by PPARδ in vitro. These findings suggest that PPARδ conditions breast cancer cells to survive in harsh microenvironmental conditions by reducing oxidative stress and enhancing survival signaling responses. Drugs that target PPARδ may have a role in the treatment of breast cancer. PMID:27270614

  9. Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

    SciTech Connect

    Kover, Karen; Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu; Tasch, James; Hager, Melissa; Clements, Mark; Moore, Wayne V.

    2015-06-19

    Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose

  10. Conditional Knockout of Telomerase Reverse Transcriptase in Mesenchymal Cells Impairs Mouse Pulmonary Fibrosis

    PubMed Central

    Liu, Tianju; Yu, Hongfeng; Ding, Lin; Wu, Zhe; Gonzalez De Los Santos, Francina; Liu, Jianhua; Ullenbruch, Matthew; Hu, Biao; Martins, Vanessa; Phan, Sem H.

    2015-01-01

    Telomerase is typically expressed in cellular populations capable of extended replication, such as germ cells, tumor cells, and stem cells, but is also induced in tissue injury, repair and fibrosis. Its catalytic component, telomerase reverse transcriptase (TERT) is induced in lung fibroblasts from patients with fibrotic interstitial lung disease and in rodents with bleomycin-induced pulmonary fibrosis. To evaluate the fibroblast specific role of TERT in pulmonary fibrosis, transgenic mice bearing a floxed TERT allele were generated, and then crossed with an inducible collagen α2(I)-Cre mouse line to generate fibroblast specific TERT conditional knockout mice. TERT-specific deficiency in mesenchymal cells caused attenuation of pulmonary fibrosis as manifested by reduced lung hydroxyproline content, type I collagen and α-smooth muscle actin mRNA levels. The TERT-deficient mouse lung fibroblasts displayed decreased cell proliferative capacity and higher susceptibility to induced apoptosis compared with control cells. Additionally TERT deficiency was associated with heightened α-smooth muscle actin expression indicative of myofibroblast differentiation. However the impairment of cell proliferation and increased susceptibility to apoptosis would cause a reduction in the myofibroblast progenitor population necessary to mount a successful myofibroblast-dependent fibrotic response. These findings identified a key role for TERT in fibroblast proliferation and survival essential for pulmonary fibrosis. PMID:26555817

  11. Effect of Conditioned Medium and Bone Marrow Stem Cell Lysate on the Course of Acetaminophen-Induced Liver Failure.

    PubMed

    Khubutiya, M Sh; Temnov, A A; Vagabov, V A; Sklifas, A N; Rogov, K A; Zhgutov, Yu A

    2015-05-01

    A composition containing culture medium conditioned by mesenchymal stem cells and mesenchymal stem cell lysate improves biochemical parameters, reduces inflammation, and stimulates regenerative processes in the liver. PMID:26033600

  12. Developing RCM Strategy for Hydrogen Fuel Cells Utilizing On Line E-Condition Monitoring

    NASA Astrophysics Data System (ADS)

    Baglee, D.; Knowles, M. J.

    2012-05-01

    Fuel cell vehicles are considered to be a viable solution to problems such as carbon emissions and fuel shortages for road transport. Proton Exchange Membrane (PEM) Fuel Cells are mainly used in this purpose because they can run at low temperatures and have a simple structure. Yet high maintenance costs and the inherent dangers of maintaining equipment using hydrogen are two main issues which need to be addressed. The development of appropriate and efficient strategies is currently lacking with regard to fuel cell maintenance. A Reliability Centered Maintenance (RCM) approach offers considerable benefit to the management of fuel cell maintenance since it includes an identification and consideration of the impact of critical components. Technological developments in e-maintenance systems, radio-frequency identification (RFID) and personal digital assistants (PDAs) have proven to satisfy the increasing demand for improved reliability, efficiency and safety. RFID technology is used to store and remotely retrieve electronic maintenance data in order to provide instant access to up-to-date, accurate and detailed information. The aim is to support fuel cell maintenance decisions by developing and applying a blend of leading-edge communications and sensor technology including RFID. The purpose of this paper is to review and present the state of the art in fuel cell condition monitoring and maintenance utilizing RCM and RFID technologies. Using an RCM analysis critical components and fault modes are identified. RFID tags are used to store the critical information, possible faults and their cause and effect. The relationship between causes, faults, symptoms and long term implications of fault conditions are summarized. Finally conclusions are drawn regarding suggested maintenance strategies and the optimal structure for an integrated, cost effective condition monitoring and maintenance management system.

  13. Transcriptomic changes in human renal proximal tubular cells revealed under hypoxic conditions by RNA sequencing.

    PubMed

    Yu, Wenmin; Li, Yiping; Wang, Zhi; Liu, Lei; Liu, Jing; Ding, Fengan; Zhang, Xiaoyi; Cheng, Zhengyuan; Chen, Pingsheng; Dou, Jun

    2016-09-01

    Chronic hypoxia often occurs among patients with chronic kidney disease (CKD). Renal proximal tubular cells may be the primary target of a hypoxic insult. However, the underlying transcriptional mechanisms remain undefined. In this study, we revealed the global changes in gene expression in HK‑2 human renal proximal tubular cells under hypoxic and normoxic conditions. We analyzed the transcriptome of HK‑2 cells exposed to hypoxia for 24 h using RNA sequencing. A total of 279 differentially expressed genes was examined, as these genes could potentially explain the differences in HK‑2 cells between hypoxic and normoxic conditions. Moreover, 17 genes were validated by qPCR, and the results were highly concordant with the RNA seqencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these differentially expressed genes. The upregulated genes appeared to be significantly enriched in the pathyway of extracellular matrix (ECM)-receptor interaction, and in paticular, the pathway of renal cell carcinoma was upregulated under hypoxic conditions. The downregulated genes were enriched in the signaling pathway related to antigen processing and presentation; however, the pathway of glutathione metabolism was downregulated. Our analysis revealed numerous novel transcripts and alternative splicing events. Simultaneously, we also identified a large number of single nucleotide polymorphisms, which will be a rich resource for future marker development. On the whole, our data indicate that transcriptome analysis provides valuable information for a more in depth understanding of the molecular mechanisms in CKD and renal cell carcinoma. PMID:27432315

  14. Conditional knockout of retinal determination genes in differentiating cells in Drosophila.

    PubMed

    Jin, Meng; Eblimit, Aiden; Pulikkathara, Merlyn; Corr, Stuart; Chen, Rui; Mardon, Graeme

    2016-08-01

    Conditional gene knockout in postmitotic cells is a valuable technique which allows the study of gene function with spatiotemporal control. Surprisingly, in contrast to its long-term and extensive use in mouse studies, this technology is lacking in Drosophila. Here, we use a novel method for generating complete loss of eyes absent (eya) or sine oculis (so) function in postmitotic cells posterior to the morphogenetic furrow (MF). Specifically, genomic rescue constructs with flippase recognition target (FRT) sequences flanking essential exons are used to generate conditional null alleles. By removing gene function in differentiating cells, we show that eya and so are dispensable for larval photoreceptor differentiation, but are required for differentiation during pupal development. Both eya and so are necessary for photoreceptor survival and the apoptosis caused by loss of eya or so function is likely a secondary consequence of inappropriate differentiation. We also confirm their requirement for cone cell development and reveal a novel role in interommatidial bristle (IOB) formation. In addition, so is required for normal eye disc morphology. This is the first report of a knockout method to study eya and so function in postmitotic cells. This technology will open the door to a large array of new functional studies in virtually any tissue and at any stage of development or in adults. PMID:27257739

  15. Nanostructured porous silicon micropatterns as a tool for substrate-conditioned cell research

    NASA Astrophysics Data System (ADS)

    Punzón-Quijorna, Esther; Sánchez-Vaquero, Vanessa; Muñoz-Noval, Álvaro; Pérez-Roldán, M. Jesus; Martín-Palma, Raúl J.; Rossi, Francois; Climent-Font, Aurelio; Manso-Silván, Miguel; Ruiz, J. Predestinacion García; Torres-Costa, Vicente

    2012-07-01

    The localized irradiation of Si allows a precise patterning at the microscale of nanostructured materials such as porous silicon (PS). PS patterns with precisely defined geometries can be fabricated using ion stopping masks. The nanoscale textured micropatterns were used to explore their influence as microenvironments for human mesenchymal stem cells (hMSCs). In fact, the change of photoluminescence emission from PS upon aging in physiological solution suggests the intense formation of silanol surface groups, which may play a relevant role in ulterior cell adhesion. The experimental results show that hMSCs are sensitive to the surface micropatterns. In this regard, preliminary β-catenin labeling studies reveal the formation of cell to cell interaction structures, while microtubule orientation is strongly influenced by the selective adhesion conditions. Relevantly, Ki-67 assays support a proliferative state of hMSCs on such nanostructured micropatterns comparable to that of standard cell culture platforms, which reinforce the candidature of porous silicon micropatterns to become a conditioning structure for in vitro culture of HMSCs.

  16. Emerging role of angiogenin in stress response and cell survival under adverse conditions

    PubMed Central

    Li, Shuping; Hu, Guo-Fu

    2011-01-01

    Angiogenin (ANG), also known as ribonuclease (RNASE) 5, is a member of the vertebrate-specific, secreted RNASE superfamily. ANG was originally identified as a tumor angiogenic factor, but its biological activity has been extended from inducing angiogenesis to stimulating cell proliferation and more recently, to promoting cell survival. Under growth conditions, ANG is translocated to nucleus where it accumulates in nucleolus and stimulates ribosomal RNA (rRNA) transcription, thus facilitating cell growth and proliferation. Under stress conditions, ANG is accumulated in cytoplasmic compartments and modulates the production of tiRNA, a novel class of small RNA that is derived from tRNA and is induced by stress. tiRNA suppress global protein translation by inhibiting both cap-dependent and -independent translation including that mediated by weak IRESes. However, strong IRES-mediated translation, a mechanism often used by genes involved in pro-survival and anti-apoptosis, is not affected. Thus, ANG-mediated tiRNA reprogram protein translation, save anabolic energy, and promote cell survival. This recently uncovered function of ANG presents a novel mechanism of action in regulating cell growth and survival. PMID:22021078

  17. Nanostructured porous silicon micropatterns as a tool for substrate-conditioned cell research

    PubMed Central

    2012-01-01

    The localized irradiation of Si allows a precise patterning at the microscale of nanostructured materials such as porous silicon (PS). PS patterns with precisely defined geometries can be fabricated using ion stopping masks. The nanoscale textured micropatterns were used to explore their influence as microenvironments for human mesenchymal stem cells (hMSCs). In fact, the change of photoluminescence emission from PS upon aging in physiological solution suggests the intense formation of silanol surface groups, which may play a relevant role in ulterior cell adhesion. The experimental results show that hMSCs are sensitive to the surface micropatterns. In this regard, preliminary β-catenin labeling studies reveal the formation of cell to cell interaction structures, while microtubule orientation is strongly influenced by the selective adhesion conditions. Relevantly, Ki-67 assays support a proliferative state of hMSCs on such nanostructured micropatterns comparable to that of standard cell culture platforms, which reinforce the candidature of porous silicon micropatterns to become a conditioning structure for in vitro culture of HMSCs. PMID:22799489

  18. System for measuring oxygen consumption rates of mammalian cells in static culture under hypoxic conditions.

    PubMed

    Kagawa, Yuki; Miyahara, Hirotaka; Ota, Yuri; Tsuneda, Satoshi

    2016-01-01

    Estimating the oxygen consumption rates (OCRs) of mammalian cells in hypoxic environments is essential for designing and developing a three-dimensional (3-D) cell culture system. However, OCR measurements under hypoxic conditions are infrequently reported in the literature. Here, we developed a system for measuring OCRs at low oxygen levels. The system injects nitrogen gas into the environment and measures the oxygen concentration by an optical oxygen microsensor that consumes no oxygen. The developed system was applied to HepG2 cells in static culture. Specifically, we measured the spatial profiles of the local dissolved oxygen concentration in the medium, then estimated the OCRs of the cells. The OCRs, and also the pericellular oxygen concentrations, decreased nonlinearly as the oxygen partial pressure in the environment decreased from 19% to 1%. The OCRs also depended on the culture period and the matrix used for coating the dish surface. Using this system, we can precisely estimate the OCRs of various cell types under environments that mimic 3-D culture conditions, contributing crucial data for an efficient 3-D culture system design. PMID:26558344

  19. Membrane resistance and current distribution measurements under various operating conditions in a polymer electrolyte fuel cell

    NASA Astrophysics Data System (ADS)

    Brett, D. J. L.; Atkins, S.; Brandon, N. P.; Vasileiadis, N.; Vesovic, V.; Kucernak, A. R.

    The ability to make spatially resolved measurements in a fuel cell provides one of the most useful ways in which to monitor and optimise their performance. Localised membrane resistance and current density measurements for a single channel polymer electrolyte fuel cell are presented for a range of operating conditions. The current density distribution results are compared with an analytical model that exhibited generally good agreement across a broad range of operating conditions. However, under conditions of high air flow rate, an increase in current is observed along the channel which is not predicted by the model. Under such circumstances, localised electrochemical impedance measurements show a decrease in membrane resistance along the channel. This phenomenon is attributed to drying of the electrolyte at the start of the channel and is more pronounced with increasing operating temperature. Under conditions of reactant depletion, an increase in electrolyte resistance with decreasing current is observed. This is due to the hydrating effect of product water and electro-osmotic drag through the membrane when ionic current is flowing. Localised conduction is shown to be an effective means of conditioning previously unused membrane electrode assemblies by forcing passage of ionic current through the electrolyte.

  20. Treosulfan-based conditioning regimen for allogeneic haematopoietic stem cell transplantation in children with sickle cell disease.

    PubMed

    Strocchio, Luisa; Zecca, Marco; Comoli, Patrizia; Mina, Tommaso; Giorgiani, Giovanna; Giraldi, Eugenia; Vinti, Luciana; Merli, Pietro; Regazzi, Mario; Locatelli, Franco

    2015-06-01

    Although allogeneic haematopoietic stem cell transplantation (HSCT) still represents the only consolidated possibility of cure for sickle cell disease (SCD) patients, its use has been limited by the risk of morbidity and mortality associated with conventional myeloablative therapy. The introduction of treosulfan to replace busulfan in conditioning regimens has recently been explored by virtue of its lower toxicity profile. We report our experience with a treosulfan/thiotepa/fludarabine conditioning for human leucocyte antigen (HLA)-matched sibling or unrelated donor-HSCT in 15 children with SCD, and compare patient outcomes with those of a historical cohort (15 patients) given a busulfan-based regimen. Engraftment was achieved in 28 out of 30 patients (93%), with one case of graft failure in either group. The conditioning regimen was well tolerated in both groups, with no cases of grade III-IV regimen-related toxicity. The 7-year overall survival (OS) and disease-free survival (DFS) for the whole cohort were 100% and 93%, respectively, with a 93% DFS in both busulfan and treosulfan groups. No SCD-related adverse events occurred after engraftment in patients with complete or mixed donor chimerism. This retrospective analysis suggests that a treosulfan-based conditioning regimen is able to ensure engraftment with excellent OS/DFS and low regimen-related toxicity in patients with SCD. PMID:25818248

  1. Subcritical crack velocities in titanium diboride under simulated hall-heroult cell conditions

    SciTech Connect

    Bumgartner

    1984-09-01

    Static fatigue crack velocities were measured in two types of titanium diboride under conditions existing in aluminum reduction cells. Double torsion specimens were heavily loaded (up to K/K /SUB lc/ = 0.85) for extended times at 960/sup 0/C while subjected to environments of aluminum, electrolyte, or electrolysis. Within the sensitivity limits of the method (about 10/sup -10/ m/s), one material did not exhibit slow crack growth. In the other material, crack velocity increased under cathodic conditions as compared to the rate in aluminum without electrolysis.

  2. PKC/MAPK signaling suppression by retinal pericyte conditioned medium prevents retinal endothelial cell proliferation.

    PubMed

    Kondo, Tetsu; Hosoya, Ken-Ichi; Hori, Satoko; Tomi, Masatoshi; Ohtsuki, Sumio; Terasaki, Tetsuya

    2005-05-01

    Little is known about the regulation mechanism of endothelial cell proliferation by retinal pericytes. The purpose of this study was to elucidate the suppression mechanism of retinal capillary endothelial cell growth by soluble factors derived from retinal pericytes. Conditioned medium of retinal pericytes (rPCT1-CM) suppressed ischemia-induced retinal neovascularization. The growth and DNA synthesis of TR-iBRB2 cells, a conditionally immortalized rat retinal capillary endothelial cell line, were suppressed in a concentration-dependent manner by concentrated rPCT1-CM. The number of human cultured endothelial cells was also reduced by rPCT1-CM. These results provide the first evidence that CM from the cultivation of pericytes alone can inhibit retinal neovascularization in vivo and in vitro. Although the growth reduction of TR-iBRB2 cells was only partly reversed by treatment of rPCT1-CM with antibodies to transforming growth factor-beta1, it was completely lost by heat-treatment of rPCT1-CM, suggesting that anti-angiogenic factors are soluble proteins. The levels of expression of G1/S-phase-related proteins, such as cyclin D1, cyclin-dependent kinase (cdk)4, cdk6, and proliferating cell nuclear antigen, were reduced and a cdk inhibitor, p21(Cip1), was induced in rPCT1-CM-treated TR-iBRB2 cells. Moreover, phosphorylated p44/42 mitogen-activated protein kinase (p44/42 MAPK) in TR-iBRB2 cells was reduced by rPCT1-CM treatment and phosphorylated protein kinase C (PKC)alpha/betaII, which is upstream of p44/42 MAPK, was also suppressed. In conclusion, CM from retinal pericytes suppresses PKC-p44/42 MAPK signaling, inhibits endothelial cell growth, and prevents retinal neovascularization. Anti-angiogenic factors derived from retinal pericytes are likely to play a critical role in the regulation of retinal endothelial cell growth. PMID:15499572

  3. Mast cells in human digestive tube in normal and pathological conditions.

    PubMed

    Rizzi, Anna; Crivellato, Enrico; Benagiano, Vincenzo; Ribatti, Domenico

    2016-09-01

    Mast cells (MCs) are localized in connective tissues and are more numerous near the boundaries between the external environment and the internal milieu including the skin, the respiratory tract, the gastrointestinal tract and the conjunctiva. In the gastrointestinal tract, MCs represent 1-5% of mononuclear cells in the lamina propria of the mucosa and in the submucosa, and they are also found inside the epithelium and deep in the muscle and serosal layers. The gastrointestinal MCs perform their biological functions, releasing mediators, as amines (histamine, serotonin), cytokines, proteases, lipid mediators (leukotrienes, prostaglandins), and heparin. MCs are involved in the pathogenesis of different inflammatory conditions and tumors of the gastrointestinal tract. The use of MCs' tryptase inhibitors or c-KitR tyrosine kinase inhibitors could represent a potential anti-MC therapeutic approach in all the inflammatory and tumor pathological conditions of the digestive tube in which MCs are involved. PMID:27393495

  4. Novel management of acute or secondary biliary liver conditions using hepatically differentiated human dental pulp cells.

    PubMed

    Ishkitiev, Nikolay; Yaegaki, Ken; Imai, Toshio; Tanaka, Tomoko; Fushimi, Naho; Mitev, Vanyo; Okada, Mio; Tominaga, Noriko; Ono, Sachie; Ishikawa, Hiroshi

    2015-02-01

    The current definitive treatment for acute or chronic liver condition, that is, cirrhosis, is liver transplantation from a limited number of donors, which might cause complications after donation. Hence, bone marrow stem cell transplantation has been developed, but the risk of carcinogenesis remains. We have recently developed a protocol for hepatic differentiation of CD117(+) stem cells from human exfoliated deciduous teeth (SHED). In the present study, we examine whether SHED hepatically differentiated (hd) in vitro could be used to treat acute liver injury (ALI) and secondary biliary cirrhosis. The CD117(+) cell fraction was magnetically separated from SHED and then differentiated into hepatocyte-like cells in vitro. The cells were transplanted into rats with either ALI or induced secondary biliary cirrhosis. Engraftment of human liver cells was determined immunohistochemically and by in situ hybridization. Recovery of liver function was examined by means of histochemical and serological tests. Livers of transplanted animals were strongly positive for human immunohistochemical factors, and in situ hybridization confirmed engraftment of human hepatocytes. The tests for recovery of liver function confirmed the presence of human hepatic markers in the animals' blood serum and lack of fibrosis and functional integration of transplanted human cells into livers. No evidence of malignancy was found. We show that in vitro hdSHED engraft morphologically and functionally into the livers of rats having acute injury or secondary biliary cirrhosis. SHED are readily accessible adult stem cells, capable of proliferating in large numbers before differentiating in vitro. This makes SHED an appropriate and safe stem cell source for regenerative medicine. PMID:25234861

  5. Visualization of Fuel Cell Water Transport and Performance Characterization under Freezing Conditions

    SciTech Connect

    Kandlikar, Satish G.; Lu, Zijie; Rao, Navalgund; Sergi, Jacqueline; Rath, Cody; McDade, Christopher; Trabold, Thomas; Owejan, Jon; Gagliardo, Jeffrey; Allen, Jeffrey; Yassar, Reza S.; Medici, Ezequiel; Herescu, Alexandru

    2010-05-30

    In this program, Rochester Institute of Technology (RIT), General Motors (GM) and Michigan Technological University (MTU) have focused on fundamental studies that address water transport, accumulation and mitigation processes in the gas diffusion layer and flow field channels of the bipolar plate. These studies have been conducted with a particular emphasis on understanding the key transport phenomena which control fuel cell operation under freezing conditions.

  6. Adropin reduces paracellular permeability of rat brain endothelial cells exposed to ischemia-like conditions.

    PubMed

    Yang, Changjun; DeMars, Kelly M; Hawkins, Kimberly E; Candelario-Jalil, Eduardo

    2016-07-01

    Adropin is a peptide encoded by the energy homeostasis associated gene (Enho) and plays a critical role in the regulation of lipid metabolism, insulin sensitivity, and endothelial function. Little is known of the effects of adropin in the brain and whether this peptide modulates ischemia-induced blood-brain barrier (BBB) injury. Here, we used an in vitro BBB model of rat brain microvascular endothelial cells (RBE4) and hypothesized that adropin would reduce endothelial permeability during ischemic conditions. To mimic ischemic conditions in vitro, RBE4 cell monolayers were subjected to 16h hypoxia/low glucose (HLG). This resulted in a significant increase in paracellular permeability to FITC-labeled dextran (40kDa), a dramatic upregulation of vascular endothelial growth factor (VEGF), and the loss of junction proteins occludin and VE-cadherin. Notably, HLG also significantly decreased Enho expression and adropin levels. Treatment of RBE4 cells with synthetic adropin (1, 10 and 100ng/ml) concentration-dependently reduced endothelial permeability after HLG, but this was not mediated through protection to junction proteins or through reduced levels of VEGF. We found that HLG dramatically increased myosin light chain 2 (MLC2) phosphorylation in RBE4 cells, which was significantly reduced by adropin treatment. We also found that HLG significantly increased Rho-associated kinase (ROCK) activity, a critical upstream effector of MLC2 phosphorylation, and that adropin treatment attenuated that effect. These data indicate that treatment with adropin reduces endothelial cell permeability after HLG insult by inhibition of the ROCK-MLC2 signaling pathway. These promising findings suggest that adropin protects against endothelial barrier dysfunction during ischemic conditions. PMID:27020249

  7. IRE1 prevents endoplasmic reticulum membrane permeabilization and cell death under pathological conditions

    PubMed Central

    Kanekura, Kohsuke; Ma, Xiucui; Murphy, John T.; Zhu, Lihua J.; Diwan, Abhinav; Urano, Fumihiko

    2015-01-01

    The endoplasmic reticulum (ER) has emerged as a critical regulator of cell fate. IRE1 is a transmembrane protein with kinase and RNase activities that is localized to the ER and that promotes resistance to ER stress. Here we showed a mechanism by which IRE1 conferred protection against ER stress-mediated cell death. IRE1 signaling prevented ER membrane permeabilization mediated by Bax and Bak and cell death under ER stress conditions. Suppression of IRE1 signaling led to the accumulation of the BH3 domain-containing protein Bnip3, which in turn triggered the oligomerization of Bax and Bak in the ER membrane and ER membrane permeabilization. As a result, cells deficient in IRE1 were susceptible to leakage of ER contents in response to ER stress, which was associated with the accumulation of calcium in mitochondria, oxidative stress in the cytosol, and cell death. Our results reveal a role for IRE1 in preventing an initial step of cell death emanating from the ER and provide a potential target for treating diseases characterized by ER stress, including diabetes and Wolfram syndrome. PMID:26106220

  8. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies

    PubMed Central

    Tang, Jonathan CY; Drokhlyansky, Eugene; Etemad, Behzad; Rudolph, Stephanie; Guo, Binggege; Wang, Sui; Ellis, Emily G; Li, Jonathan Z; Cepko, Constance L

    2016-01-01

    The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes. DOI: http://dx.doi.org/10.7554/eLife.15312.001 PMID:27205882

  9. Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress.

    PubMed

    Zaugg, Kathrin; Yao, Yi; Reilly, Patrick T; Kannan, Karuppiah; Kiarash, Reza; Mason, Jacqueline; Huang, Ping; Sawyer, Suzanne K; Fuerth, Benjamin; Faubert, Brandon; Kalliomäki, Tuula; Elia, Andrew; Luo, Xunyi; Nadeem, Vincent; Bungard, David; Yalavarthi, Sireesha; Growney, Joseph D; Wakeham, Andrew; Moolani, Yasmin; Silvester, Jennifer; Ten, Annick You; Bakker, Walbert; Tsuchihara, Katsuya; Berger, Shelley L; Hill, Richard P; Jones, Russell G; Tsao, Ming; Robinson, Murray O; Thompson, Craig B; Pan, Guohua; Mak, Tak W

    2011-05-15

    Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors. PMID:21576264

  10. Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress

    PubMed Central

    Zaugg, Kathrin; Yao, Yi; Reilly, Patrick T.; Kannan, Karuppiah; Kiarash, Reza; Mason, Jacqueline; Huang, Ping; Sawyer, Suzanne K.; Fuerth, Benjamin; Faubert, Brandon; Kalliomäki, Tuula; Elia, Andrew; Luo, Xunyi; Nadeem, Vincent; Bungard, David; Yalavarthi, Sireesha; Growney, Joseph D.; Wakeham, Andrew; Moolani, Yasmin; Silvester, Jennifer; Ten, Annick You; Bakker, Walbert; Tsuchihara, Katsuya; Berger, Shelley L.; Hill, Richard P.; Jones, Russell G.; Tsao, Ming; Robinson, Murray O.; Thompson, Craig B.; Pan, Guohua; Mak, Tak W.

    2011-01-01

    Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors. PMID:21576264

  11. Hypoxic Culture Conditions as a Solution for Mesenchymal Stem Cell Based Regenerative Therapy

    PubMed Central

    Haque, Nazmul; Rahman, Mohammad Tariqur; Abu Kasim, Noor Hayaty; Alabsi, Aied Mohammed

    2013-01-01

    Cell-based regenerative therapies, based on in vitro propagation of stem cells, offer tremendous hope to many individuals suffering from degenerative diseases that were previously deemed untreatable. Due to the self-renewal capacity, multilineage potential, and immunosuppressive property, mesenchymal stem cells (MSCs) are considered as an attractive source of stem cells for regenerative therapies. However, poor growth kinetics, early senescence, and genetic instability during in vitro expansion and poor engraftment after transplantation are considered to be among the major disadvantages of MSC-based regenerative therapies. A number of complex inter- and intracellular interactive signaling systems control growth, multiplication, and differentiation of MSCs in their niche. Common laboratory conditions for stem cell culture involve ambient O2 concentration (20%) in contrast to their niche where they usually reside in 2–9% O2. Notably, O2 plays an important role in maintaining stem cell fate in terms of proliferation and differentiation, by regulating hypoxia-inducible factor-1 (HIF-1) mediated expression of different genes. This paper aims to describe and compare the role of normoxia (20% O2) and hypoxia (2–9% O2) on the biology of MSCs. Finally it is concluded that a hypoxic environment can greatly improve growth kinetics, genetic stability, and expression of chemokine receptors during in vitro expansion and eventually can increase efficiency of MSC-based regenerative therapies. PMID:24068884

  12. Modifications of the cell wall of yeasts grown on hexadecane and under starvation conditions.

    PubMed

    Dmitriev, Vladimir V; Crowley, David E; Zvonarev, Anton N; Rusakova, Tatiana G; Negri, Maria C; Kolesnikova, Svetlana A

    2016-02-01

    Electron-microscopic examinations have demonstrated local modifications in the cell wall of the yeast Candida maltosa grown on hexadecane. In our earlier studies, these modified sites, observed in other yeasts grown on oil hydrocarbons, were conventionally called 'canals'. The biochemical and cytochemical studies of C. maltosa have revealed a correlation between the formation of 'canals' and decrease in the amount of cell wall polysaccharides, glucan and mannan. The ultrathin sections and surface replicas have shown that the 'canals' are destroyed by pronase, thus indicating that a significant proportion of their content is represented by proteins. This finding was compatible with our earlier data on the localization of oxidative enzymes in 'canals' and possible participation of the 'canals' in the primary oxidation of hydrocarbons. A completely unexpected and intriguing phenomenon has been the appearance of 'canals' in the yeast C. maltosa under starvation conditions. Unlike the yeasts grown on hexadecane, mannan almost disappears in starving cells, while the quantity of glucan first decreases and then is restored to its initial level. The role of 'canals' in starving cells is as yet unclear; it is assumed that they acquire exoenzymes involved in the utilization of products of cell lysis in the starving population. In the future, 'canals' of starving cells will be studied in connection with their possible participation in apoptosis. PMID:26833628

  13. Efficient conditional gene expression following transplantation of retrovirally transduced bone marrow stem cells.

    PubMed

    Chung, Jie-Yu; Mackay, Fabienne; Alderuccio, Frank

    2015-01-01

    Retroviral gene therapy combined with bone marrow stem cell transplantation can be used to generate mice with ectopic gene expression in the bone marrow compartment in a quick and cost effective manner when compared to generating and maintaining transgenic mouse lines. However a limitation of this procedure is the lack of cell specificity in gene expression that is associated with the use of endogenous retroviral promoters. Restricting gene expression to specific cell subsets utilising tissue-specific promoter driven retroviral vectors is a challenge. Here we describe the generation of conditional expression of retrovirally encoded genes in specific bone marrow derived cell lineages utilising a Cre-dependent retroviral vector. By utilising Lck and CD19 restricted Cre transgenic bone marrow stem cells, we generate chimeric animals with T or B lymphocyte restricted gene expression respectively. The design of the Cre-dependent retroviral vector enables expression of encoded MOG and GFP genes only in association with Cre mediated DNA inversion. Importantly this strategy does not significantly increase the size of the retroviral vector; as such we are able to generate bone marrow chimeric animals with significantly higher chimerism levels than previous studies utilising Cre-dependent retroviral vectors and Cre transgenic bone marrow stem cells. This demonstrates that the use of Cre-dependent retroviral vectors is able to yield high chimerism levels for experimental use and represent a viable alternative to generating transgenic animals. PMID:25445328

  14. Olfactory aversive conditioning alters olfactory bulb mitral/tufted cell glomerular odor responses

    PubMed Central

    Fletcher, Max L.

    2012-01-01

    The anatomical organization of receptor neuron input into the olfactory bulb (OB) allows odor information to be transformed into an odorant-specific spatial map of mitral/tufted (M/T) cell glomerular activity at the upper level of the OB. In other sensory systems, neuronal representations of stimuli can be reorganized or enhanced following learning. While the mammalian OB has been shown to undergo experience-dependent plasticity at the glomerular level, it is still unclear if similar representational change occurs within (M/T) cell glomerular odor representations following learning. To address this, odorant-evoked glomerular activity patterns were imaged in mice expressing a GFP-based calcium indicator (GCaMP2) in OB (M/T) cells. Glomerular odor responses were imaged before and after olfactory associative conditioning to aversive foot shock. Following conditioning, we found no overall reorganization of the glomerular representation. Training, however, did significantly alter the amplitudes of individual glomeruli within the representation in mice in which the odor was presented together with foot shock. Further, the specific pairing of foot shock with odor presentations lead to increased responses primarily in initially weakly activated glomeruli. Overall, these results suggest that associative conditioning can enhance the initial representation of odors within the OB by enhancing responses to the learned odor in some glomeruli. PMID:22461771

  15. Effects of Low-dose Triamcinolone Acetonide on Rat Retinal Progenitor Cells under Hypoxia Condition

    PubMed Central

    Xing, Yao; Cui, Li-Jun; Kang, Qian-Yan

    2016-01-01

    Background: Retinal degenerative diseases are the leading causes of blindness in developed world. Retinal progenitor cells (RPCs) play a key role in retina restoration. Triamcinolone acetonide (TA) is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition. Methods: RPCs were primary cultured and identified by immunofluorescence staining. Cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions. For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48–72 h. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression of cyclin D1, Akt, p-Akt, nuclear factor (NF)-κB p65, and caspase-3. Results: CCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group (P < 0.05). After 72 h, the cell viability was enhanced in both 0.01 mg/ml and 0.02 mg/ml TA groups compared with control group (all P < 0.05). Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group (P < 0.05). RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups (all P < 0.05). There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-κB p65 protein expression and downregulation of caspase-3 and cyclin D1 protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group (all P < 0.05). Conclusion: Low-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation. PMID:27364798

  16. Changes in functional activity of bone tissue cells under space flight conditions.

    NASA Astrophysics Data System (ADS)

    Rodionova, Natalia; Nesterenko, Olga; Kabitskaya, Olga

    The space flight conditions affect considerably the state of bone tissue, leading to the development of osteoporosis and osteopenia. Many aspects of reactions of bone tissue cells still remain unclear until now. With the use of electron microscopy we studied the samples gathered from the femoral bonеs metaphyses of rats flown on board the space laboratory (Spacelab - 2) during 2 weeks and samples from tibial bones of mice C57 Black ( Bion М-1). It was established, that under microgravity conditions there occur remodelling processes in a spongy bone related with a deficit of support load. In this work the main attention is focused on studying the ultrastructure of osteogenetic cells and osteoclasts. The degree of differentiation and functional state are evaluated according to the degree of development of organelles for specific biosynthesis: rough endoplasmic reticulum (RER), Golgy complex (GC), as well as the state of mitochondria and cell nucleus. As compared with a synchronous control, the population of osteogenetic cells from zones of bone reconstruction shows a decrease in the number of functionally active forms. We can judge of this from the reduction of a specific volume of RER, GC, mitochondria in osteoblasts. RER loses architectonics typical for osteoblasts and, as against the control, is represented by short narrow canaliculi distributed throughout the cytoplasm; some canals disintegrate. GC is slightly pronounced, mitochondria become smaller in size and acquire an optically dark matrix. These phenomena are supposed to be associated with the desorganization of microtubules and microfilaments in the cells under microgravity conditions. The population of osteogenetic cells shows a decrease in the number of differentiating osteoblasts and an increase in the number of little-differentiated stromal cells. In the population of osteoblasts, degrading and apoptotic cells are sometimes encountered. Such zones show a numerical increase of monocytic cells and

  17. Complexes of DNA with cationic peptides: conditions of formation and factors effecting internalization by mammalian cells.

    PubMed

    Dizhe, E B; Ignatovich, I A; Burov, S V; Pohvoscheva, A V; Akifiev, B N; Efremov, A M; Perevozchikov, A P; Orlov, S V

    2006-12-01

    This work was devoted to the study of conditions of the formation of DNA/K8 complex and analysis of factors effecting the entry of DNA/K8 complex into mammalian cells in comparison with DNA complexes with arginine-rich fragment (47-57) of human immunodeficiency virus (type 1) transcription factor Tat (Tat peptide). The stoichiometry of positively charged DNA/K8 complexes has been studied for the first time. Non-cooperative character of DNA-K8 interaction was revealed. It has been shown that along with the positive charge of such complexes, the presence of an excess of free K8 peptide in the culture medium is a necessary condition for maximal efficiency of cell transfection with DNA/K8 complexes. A stimulatory effect of free K8 peptide on the efficiency of mammalian cell transfection by DNA/K8 complexes is likely to be mediated by the interactions of cationic peptide K8 with negatively charged proteoglycans on the cell surface, which leads to protection of DNA/K8 complexes from disruption by cellular heparan sulfates. However, the protective role of free cationic peptides depends not only on their positive charge, but also on the primary structure of the peptide. In contrast with the results obtained for DNA complexes with molecular conjugates based on poly-L-lysine, the aggregation of DNA/K8 complexes leads to a significant increase in the expression of transferred gene. PMID:17223788

  18. Hypoxic conditions increases H₂S-induced ER stress in A2870 cells.

    PubMed

    Lencesova, Lubomira; Vlcek, Miroslav; Krizanova, Olga; Hudecova, Sona

    2016-03-01

    Hypoxia - a state of lower oxygen demand-is responsible for a higher aggressiveness of tumors and therefore a worse prognosis. During hypoxia, several metabolic pathways are re-organized, e.g., energetic metabolism, modulation of pH, and calcium transport. Calcium is an important second messenger that regulates variety of processes in the cell. Thus, aim of this work was to compare H2S modulation of the intracellular calcium transport systems in hypoxia and in cells grown in standard culture conditions. For all experiments, we used ovarian cancer cell line (A2780). H2S is a novel gasotransmitter, known to be involved in a modulation of several calcium transport systems, thus resulting in altered calcium signaling. Two models of hypoxia were used in our study-chemical (induced by dimethyloxallyl glycine) and 2 % O2 hypoxia, both combined with a treatment using a slow H2S donor GYY4137. In hypoxia, we observed rapid changes in cytosolic and reticular calcium levels compared to cells grown in standard culture conditions, and these changes were even more exagerrated when combined with the GYY4137. Changes in a calcium homeostasis result from IP3 receptor´s up-regulation and down-regulation of the SERCA 2, which leads to a development of the endoplasmic reticulum stress. Based on our results, we propose a higher vulnerability of calcium transport systems to H2S regulation under hypoxia. PMID:26868821

  19. Final Report - Durable Catalysts for Fuel Cell Protection during Transient Conditions

    SciTech Connect

    Atanasoski, Radoslav; van der Vliet, Dennis; Cullen, David; Atanasoska, Ljiljana

    2015-01-26

    The objective of this project was to develop catalysts that will enable proton exchange membranes (PEM) fuel cell systems to weather the damaging conditions in the fuel cell at voltages beyond the thermodynamic stability of water during the transient periods of start-up/shut-down and fuel starvation. Such catalysts are required to make it possible for the fuel cell to satisfy the 2015 DOE targets for performance and durability. The project addressed a key issue of importance for successful transition of PEM fuel cell technology from development to pre-commercial phase. This issue is the failure of the catalyst and the other thermodynamically unstable membrane electrode assembly (MEA) components during start-up/shut-down and local fuel starvation at the anode, commonly referred to as transient conditions. During these periods the electrodes can reach potentials higher than the usual 1.23V upper limit during normal operation. The most logical way to minimize the damage from such transient events is to minimize the potential seen by the electrodes. At lower positive potentials, increased stability of the catalysts themselves and reduced degradation of the other MEA components is expected.

  20. Chemosensitivity measurements of human tumour cells by soft agar assays are influenced by the culture conditions.

    PubMed Central

    Endresen, L.; Tveit, K. M.; Rugstad, H. E.; Pihl, A.

    1985-01-01

    To investigate the influence of culture conditions on the in vitro responses of tumour cells to anticancer drugs, the sensitivities observed with the soft agar methods of Hamburger & Salmon (1977) (H-S) and of Courtenay & Mills (1978) (C-M) were compared. In all cases the ID50 values were determined from dose-response curves. Six human tumour cell lines exposed to 10 different agents, and 9 patients' melanomas exposed to 5 different agents, were examined. In the studies of cell lines the H-S method gave higher sensitivity values than the C-M method in 38 out of 52 cases, whereas in 14 cases the results were the same. In the patients' tumours the H-S method gave higher sensitivity in 21 of 35 cases, equal sensitivity in 11, and lower sensitivity in 3 cases. In many instances the ID50 values obtained with the two test systems differed by factors of 10 or more, both in the case of cell lines and tumour specimens. Systematic alterations in the culture conditions indicated that the presence or absence of rat erythrocytes is the most important factor responsible for the differences observed. Also, other factors, such as supplements (in the H-S method) and the use of different serum types, appeared to influence both colony growth and chemosensitivity. PMID:4005141

  1. Nanoparticle growth and surface chemistry changes in cell-conditioned culture medium

    PubMed Central

    Kendall, Michaela; Hodges, Nikolas J.; Whitwell, Harry; Tyrrell, Jess; Cangul, Hakan

    2015-01-01

    When biomolecules attach to engineered nanoparticle (ENP) surfaces, they confer the particles with a new biological identity. Physical format may also radically alter, changing ENP stability and agglomeration state within seconds. In order to measure which biomolecules are associated with early ENP growth, we studied ENPs in conditioned medium from A549 cell culture, using dynamic light scattering (DLS) and linear trap quadrupole electron transfer dissociation mass spectrometry. Two types of 100 nm polystyrene particles (one uncoated and one with an amine functionalized surface) were used to measure the influence of surface type. In identically prepared conditioned medium, agglomeration was visible in all samples after 1 h, but was variable, indicating inter-sample variability in secretion rates and extracellular medium conditions. In samples conditioned for 1 h or more, ENP agglomeration rates varied significantly. Agglomerate size measured by DLS was well correlated with surface sequestered peptide number for uncoated but not for amine coated polystyrene ENPs. Amine-coated ENPs grew much faster and into larger agglomerates associated with fewer sequestered peptides, but including significant sequestered lactose dehydrogenase. We conclude that interference with extracellular peptide balance and oxidoreductase activity via sequestration is worthy of further study, as increased oxidative stress via this new mechanism may be important for cell toxicity. PMID:25533102

  2. Nanoparticle growth and surface chemistry changes in cell-conditioned culture medium.

    PubMed

    Kendall, Michaela; Hodges, Nikolas J; Whitwell, Harry; Tyrrell, Jess; Cangul, Hakan

    2015-02-01

    When biomolecules attach to engineered nanoparticle (ENP) surfaces, they confer the particles with a new biological identity. Physical format may also radically alter, changing ENP stability and agglomeration state within seconds. In order to measure which biomolecules are associated with early ENP growth, we studied ENPs in conditioned medium from A549 cell culture, using dynamic light scattering (DLS) and linear trap quadrupole electron transfer dissociation mass spectrometry. Two types of 100 nm polystyrene particles (one uncoated and one with an amine functionalized surface) were used to measure the influence of surface type. In identically prepared conditioned medium, agglomeration was visible in all samples after 1 h, but was variable, indicating inter-sample variability in secretion rates and extracellular medium conditions. In samples conditioned for 1 h or more, ENP agglomeration rates varied significantly. Agglomerate size measured by DLS was well correlated with surface sequestered peptide number for uncoated but not for amine coated polystyrene ENPs. Amine-coated ENPs grew much faster and into larger agglomerates associated with fewer sequestered peptides, but including significant sequestered lactose dehydrogenase. We conclude that interference with extracellular peptide balance and oxidoreductase activity via sequestration is worthy of further study, as increased oxidative stress via this new mechanism may be important for cell toxicity. PMID:25533102

  3. Provision of antifungal immunity and concomitant alloantigen tolerization by conditioned dendritic cells in experimental hematopoietic transplantation.

    PubMed

    Montagnoli, Claudia; Perruccio, Katia; Bozza, Silvia; Bonifazi, Pierluigi; Zelante, Teresa; De Luca, Antonella; Moretti, Silvia; D'Angelo, Carmen; Bistoni, Francesco; Martelli, Massimo; Aversa, Franco; Velardi, Andrea; Romani, Luigina

    2008-01-01

    FoxP3(+) regulatory T (Treg) cells are important mediators of peripheral tolerance, and deficiency of this population is associated with autoimmune inflammation and onset of acute lethal graft-vs.-host disease in transplantation. Type I IFN-producing plasmacytoid dendritic cells (pDC) are implicated in the induction and maintenance of tolerance and contribute to engraftment facilitation and prevention of graft-vs.-host disease after allogeneic hematopoietic stem cells transplantation (HSCT). Because host DC function is impaired during the immediate period post-transplant, the administration of donor DC may be useful for the educational program of recovering T cells. Distinct DC subsets could be derived from bone marrow (murine) or peripheral CD14(+) cell (human) cultures in the presence of either GM-CSF/IL-4 (myeloid DC) or FLT3-ligand (mainly pDC). The ability of either DC subset to induce Th1/Treg cell priming against Aspergillus fumigatus as well as the relative contribution of murine DC subsets to antifungal priming upon adoptive transfer in hematopoietic transplanted mice with aspergillosis is not known. We found specialization and complementarity in priming and tolerization by the different DC subsets, with FL-DC fulfilling the requirement for (i) Th1/Treg antifungal priming; ii) tolerization toward alloantigens and (iii) diversion from alloantigen-specific to antigen-specific T cell responses in the presence of donor T lymphocytes. Interestingly, thymosin alpha1 (Talpha1), known to modulate human pDC functions trough TLR9, affects mobilization and tolerization of pDC by activating the indoleamine 2,3-dioxygenase-dependent pathway, and this resulted in Treg development and tolerization. Thus, transplantation tolerance and concomitant pathogen clearance could be achieved through the therapeutic induction of antigen-specific Treg cells via instructive immunotherapy with pathogen- or TLR-conditioned donor DC. PMID:17827038

  4. Effect of ionizing radiation on acinar morphogenesis of human prostatic epithelial cells under three-dimensional culture conditions.

    PubMed

    Wang, T; X, S Ma; Kong, D; Yi, H; Wang, X; Liang, B; Xu, H; He, M; Jia, L; Qased, A B; Yang, Y; Liu, X

    2012-01-01

    Homeostasis is maintained by the interplay of multiple factors that directly or indirectly regulate cell proliferation and cell death. Complex multiple interactions between cells and the extracellular matrix occur during acinar morphogenesis and changes in these might indicate carcinogenesis of cells from a normal to a malignant, invasive phenotype. In this study, the human prostatic epithelial cell line RWPE-1 was cultured under three-dimensional (3-D) culture conditions, and the effect of ionizing radiation on acinar morphogenesis and its association with autophagy were discussed. The results illustrated that formation of specific spheroid (acinar) structures was detectable under 3-D culture conditions. Radiation induced the disruption of acini in different cell models using either gene overexpression (Akt) or gene knock-down (Beclin 1 and ATG7). Introduction of Akt not only accelerated the growth of cells (i.e., caused the cells to manifest elongating and microspike-like structures that are obviously different from structures seen in wild-type RWPE-1 cells under two-dimensional conditions), but also changed their morphological characteristics under 3-D culture conditions. Knock-down of autophagy-related genes (Beclin 1 and ATG7) increased the radiosensitivity of cells under 3-D culture conditions, and cells died of non-apoptotic death after radiation. The results suggested that ionizing radiation may change the cell phenotype and the formation of acini. Additionally even the autophagy mechanism may play a role in these processes. PMID:22296497

  5. Albumin leak across human pulmonary microvascular vs. umbilical vein endothelial cells under septic conditions.

    PubMed

    Shelton, Jennifer L; Wang, Lefeng; Cepinskas, Gediminas; Sandig, Martin; Inculet, Richard; McCormack, David G; Mehta, Sanjay

    2006-01-01

    Human pulmonary microvascular endothelial cell (HPMVEC) injury is central to the pathophysiology of human lung injury. However, septic HPMVEC barrier dysfunction and the contribution of neutrophils have not been directly addressed in vitro. Instead, human EC responses are often extrapolated from studies of human umbilical vein EC (HUVEC). We hypothesized that HUVEC was not a good model for investigating HPMVEC barrier function under septic conditions. HPMVEC was isolated from lung tissue resected from lung cancer patients using magnetic bead-bound anti-PECAM-1 antibody. In confluent monolayers in 3-mum cell-culture inserts, we assessed trans-EC Evans-Blue (EB)-conjugated albumin leak under basal, unstimulated conditions and following stimulation with either lipopolysaccharide or a mixture of equal concentrations of TNF-alpha, IL-1beta and IFN-gamma (cytomix). Basal EB-albumin leak was significantly lower across HPMVEC than HUVEC (0.64 +/- 0.06% vs. 1.13 +/- 0.10%, respectively, P < 0.001). Lipopolysaccharide and cytomix increased leak across both HPMVEC and HUVEC in a dose-dependent manner, with a similar increase relative to basal leak in both cell types. The presence of neutrophils markedly and dose-dependently enhanced cytomix-induced EB-albumin leak across HPMVEC (P < 0.01), but had no effect on EB-albumin leak across HUVEC. Both cytomix and lipopolysaccharide-induced albumin leak was not associated with a loss of cell viability. In conclusion, HPMVEC barrier dysfunction under septic conditions is dramatically enhanced by neutrophil presence, and HUVEC is not a suitable model for studying HPMVEC septic barrier responses. The direct study of HPMVEC septic responses will lead to a better understanding of human lung injury. PMID:16376951

  6. Phenotype overlap in glial cell populations: astroglia, oligodendroglia and NG-2(+) cells

    PubMed Central

    Alghamdi, Badrah; Fern, Robert

    2015-01-01

    The extent to which NG-2(+) cells form a distinct population separate from astrocytes is central to understanding whether this important cell class is wholly an oligodendrocyte precursor cell (OPC) or has additional functions akin to those classically ascribed to astrocytes. Early immuno-staining studies indicate that NG-2(+) cells do not express the astrocyte marker GFAP, but orthogonal reconstructions of double-labeled confocal image stacks here reveal a significant degree of co-expression in individual cells within post-natal day 10 (P10) and adult rat optic nerve (RON) and rat cortex. Extensive scanning of various antibody/fixation/embedding approaches identified a protocol for selective post-embedded immuno-gold labeling. This first ultrastructural characterization of identified NG-2(+) cells revealed populations of both OPCs and astrocytes in P10 RON. NG-2(+) astrocytes had classic features including the presence of glial filaments but low levels of glial filament expression were also found in OPCs and myelinating oligodendrocytes. P0 RONs contained few OPCs but positively identified astrocytes were observed to ensheath pre-myelinated axons in a fashion previously described as a definitive marker of the oligodendrocyte lineage. Astrocyte ensheathment was also apparent in P10 RONs, was absent from developing nodes of Ranvier and was never associated with compact myelin. Astrocyte processes were also shown to encapsulate some oligodendrocyte somata. The data indicate that common criteria for delineating astrocytes and oligodendroglia are insufficiently robust and that astrocyte features ascribed to OPCs may arise from misidentification. PMID:26106302

  7. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

    SciTech Connect

    Iyer, Soumya C; Kannan, Anbarasu; Gopal, Ashidha; Devaraj, Niranjali; Halagowder, Devaraj

    2015-08-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target. - Highlights: • HIF1α increases NICD, induces TRPC6 in lx2 cells. • TRPC6 a novel regulator in the activation of HSC. • HSCs as target for HCC therapy.

  8. Conditional inactivation of PDCD2 induces p53 activation and cell cycle arrest

    PubMed Central

    Granier, Celine J.; Wang, Wei; Tsang, Tiffany; Steward, Ruth; Sabaawy, Hatem E.; Bhaumik, Mantu; Rabson, Arnold B.

    2014-01-01

    ABSTRACT PDCD2 (programmed cell death domain 2) is a highly conserved, zinc finger MYND domain-containing protein essential for normal development in the fly, zebrafish and mouse. The molecular functions and cellular activities of PDCD2 remain unclear. In order to better understand the functions of PDCD2 in mammalian development, we have examined PDCD2 activity in mouse blastocyst embryos, as well as in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs). We have studied mice bearing a targeted PDCD2 locus functioning as a null allele through a splicing gene trap, or as a conditional knockout, by deletion of exon2 containing the MYND domain. Tamoxifen-induced knockout of PDCD2 in MEFs, as well as in ESCs, leads to defects in progression from the G1 to the S phase of cell cycle, associated with increased levels of p53 protein and p53 target genes. G1 prolongation in ESCs was not associated with induction of differentiation. Loss of entry into S phase of the cell cycle and marked induction of nuclear p53 were also observed in PDCD2 knockout blastocysts. These results demonstrate a unique role for PDCD2 in regulating the cell cycle and p53 activation during early embryonic development of the mouse. PMID:25150276

  9. Evaluation of the antineoplastic activity of gallic acid in oral squamous cell carcinoma under hypoxic conditions.

    PubMed

    Guimaraes, Talita A; Farias, Lucyana C; Fraga, Carlos A; Feltenberger, John D; Melo, Geraldo A; Coletta, Ricardo D; Souza Santos, Sergio H; de Paula, Alfredo M B; Guimaraes, Andre L

    2016-06-01

    The purpose of the current study was to develop and test a theoretical model that could explain the mechanism of action of gallic acid (GA) in the oral squamous cell carcinoma context for the first time. The theoretical model was developed using bioinformatics and interaction network analysis to evaluate the effect of GA on oral squamous cell carcinoma. In a second step to confirm theoretical results, migration, invasion, proliferation, and gene expression (Col1A1, E-cadherin, HIF-1α, and caspase-3) were performed under normoxic and hypoxic conditions. Our study indicated that treatment with GA resulted in the inhibition of cell proliferation, migration, and invasion in neoplastic cells. Observation of the molecular mechanism showed that GA upregulates E-cadherin expression and downregulates Col1A1 and HIF-1α expression, suggesting that GA might be a potential anticancer compound. In conclusion, the present study demonstrated that GA significantly reduces cell proliferation, invasion, and migration by increasing E-cadherin and repressing Col1A1. PMID:26849170

  10. Conditional inactivation of PDCD2 induces p53 activation and cell cycle arrest.

    PubMed

    Granier, Celine J; Wang, Wei; Tsang, Tiffany; Steward, Ruth; Sabaawy, Hatem E; Bhaumik, Mantu; Rabson, Arnold B

    2014-01-01

    PDCD2 (programmed cell death domain 2) is a highly conserved, zinc finger MYND domain-containing protein essential for normal development in the fly, zebrafish and mouse. The molecular functions and cellular activities of PDCD2 remain unclear. In order to better understand the functions of PDCD2 in mammalian development, we have examined PDCD2 activity in mouse blastocyst embryos, as well as in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs). We have studied mice bearing a targeted PDCD2 locus functioning as a null allele through a splicing gene trap, or as a conditional knockout, by deletion of exon2 containing the MYND domain. Tamoxifen-induced knockout of PDCD2 in MEFs, as well as in ESCs, leads to defects in progression from the G1 to the S phase of cell cycle, associated with increased levels of p53 protein and p53 target genes. G1 prolongation in ESCs was not associated with induction of differentiation. Loss of entry into S phase of the cell cycle and marked induction of nuclear p53 were also observed in PDCD2 knockout blastocysts. These results demonstrate a unique role for PDCD2 in regulating the cell cycle and p53 activation during early embryonic development of the mouse. PMID:25150276

  11. Promotion of glioma cell survival by acyl-CoA synthetase 5 under extracellular acidosis conditions.

    PubMed

    Mashima, T; Sato, S; Sugimoto, Y; Tsuruo, T; Seimiya, H

    2009-01-01

    Extracellular acidosis (low pH) is a tumor microenvironmental stressor that has a critical function in the malignant progression and metastatic dissemination of tumors. To survive under stress conditions, tumor cells must evolve resistance to stress-induced toxicity. Acyl-CoA synthetase 5 (ACSL5) is a member of the ACS family, which converts fatty acid to acyl-CoA. ACSL5 is frequently overexpressed in malignant glioma, whereas its functional significance is still unknown. Using retrovirus-mediated stable gene transfer (gain of function) and small interfering RNA-mediated gene silencing (loss of function), we show here that ACSL5 selectively promotes human glioma cell survival under extracellular acidosis. ACSL5 enhanced cell survival through its ACS catalytic activity. To clarify the genome-wide changes in cell signaling pathways by ACSL5, we performed cDNA microarray analysis and identified an ACSL5-dependent gene expression signature. The analysis revealed that ACSL5 was critical to the expression of tumor-related factors including midkine (MDK), a heparin-binding growth factor frequently overexpressed in cancer. Knockdown of MDK expression significantly attenuated ACSL5-mediated survival under acidic state. These results indicate that ACSL5 is a critical factor for survival of glioma cells under acidic tumor microenvironment, thus providing novel molecular basis for cancer therapy. PMID:18806831

  12. Conception on the cell mechanisms of bone tissue loss under spase flight conditions

    NASA Astrophysics Data System (ADS)

    Rodionova, Natalia; Oganov, Victor; Kabitskaya, Olga

    Basing on the analysis of available literature and the results of our own electron microscopic and radioautographic researches the data are presented about the morpho-functional peculiarities and succession of cellular interactions in adaptive remodeling of bone structures under normal conditions and after exposure of animals (rats, monkeys, mice) to microgravity (SLS-2, Bion-11, BionM-1). The probable cellular mechanisms of the development of osteopenia and osteoporosis are considered. Our conception on remodeling proposes the following sequence in the development of cellular interactions after decrease of the mechanical loading: a primary response of osteocytes (mechanosensory cells) to the mechanical stimulus; osteocytic remodeling (osteolysis); transmission of the mechanical signals through a system of canals and processes to functionally active osteoblasts and surface osteocytes as well as to the bone-marrow stromal cells and to those lying on bone surfaces. As a response to the mechanical stimulus (microgravity) the system of stromal cell-preosteoblast-osteoblast shows a delay in proliferation, differentiation and specific functioning of the osteogenetic cells, some of the osteoblasts undergo apoptosis. Then the osteoclastic reaction occurs (attraction of monocytes and formation of osteoclasts and bone matrix resorption in the loci of apoptosis of osteoblasts and osteocytes). The macrophagal reaction is followed by osteoblastogenesis, which appears to be a rehabilitating process. However, during prolonged absence of mechanical stimuli (microgravity, long-time immobilization) the adaptive activization of osteoblastogenesis doesn’t occur (as it is the case during the physiological remodeling of bone tissue) or it occurs to a smaller degree. The loading deficit leads to an adaptive differentiation of stromal cells to fibroblastic cells and adipocytes in these remodeling loci. These cell reactions are considered as adaptive-compensatory, but they don’t result

  13. Metabolic analysis of antibody producing Chinese hamster ovary cell culture under different stresses conditions.

    PubMed

    Badsha, Md Bahadur; Kurata, Hiroyuki; Onitsuka, Masayoshi; Oga, Takushi; Omasa, Takeshi

    2016-07-01

    Chinese hamster ovary (CHO) cells are commonly used as the host cell lines concerning their ability to produce therapeutic proteins with complex post-translational modifications. In this study, we have investigated the time course extra- and intracellular metabolome data of the CHO-K1 cell line, under a control and stress conditions. The addition of NaCl and trehalose greatly suppressed cell growth, where the maximum viable cell density of NaCl and trehalose cultures were 2.2-fold and 2.8-fold less than that of a control culture. Contrariwise, the antibody production of both the NaCl and trehalose cultures was sustained for a longer time to surpass that of the control culture. The NaCl and trehalose cultures showed relatively similar dynamics of cell growth, antibody production, and substrate/product concentrations, while they indicated different dynamics from the control culture. The principal component analysis of extra- and intracellular metabolome dynamics indicated that their dynamic behaviors were consistent with biological functions. The qualitative pattern matching classification and hierarchical clustering analyses for the intracellular metabolome identified the metabolite clusters whose dynamic behaviors depend on NaCl and trehalose. The volcano plot revealed several reporter metabolites whose dynamics greatly change between in the NaCl and trehalose cultures. The elastic net identified some critical, intracellular metabolites that are distinct between the NaCl and trehalose. While a relatively small number of intracellular metabolites related to the cell growth, glucose, glutamine, lactate and ammonium ion concentrations, the mechanism of antibody production was suggested to be very complicated or not to be explained by elastic net regression analysis. PMID:26803706

  14. Role of nesprin-1 in nuclear deformation in endothelial cells under static and uniaxial stretching conditions

    SciTech Connect

    Anno, Toshiro; Sakamoto, Naoya; Sato, Masaaki

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Nesprin-1 knockdown decreases widths of nuclei in ECs under static condition. Black-Right-Pointing-Pointer Nuclear strain caused by stretching is increased by nesprin-1 knockdown in ECs. Black-Right-Pointing-Pointer We model mechanical interactions of F-actin with the nucleus in stretched cells. Black-Right-Pointing-Pointer F-actin bound to nesprin-1 may cause sustainable force transmission to the nucleus. -- Abstract: The linker of nucleus and cytoskeleton (LINC) complex, including nesprin-1, has been suggested to be crucial for many biological processes. Previous studies have shown that mutations in nesprin-1 cause abnormal cellular functions and diseases, possibly because of insufficient force transmission to the nucleus through actin filaments (F-actin) bound to nesprin-1. However, little is known regarding the mechanical interaction between the nucleus and F-actin through nesprin-1. In this study, we examined nuclear deformation behavior in nesprin-1 knocked-down endothelial cells (ECs) subjected to uniaxial stretching by evaluating nuclear strain from lateral cross-sectional images. The widths of nuclei in nesprin-1 knocked-down ECs were smaller than those in wild-type cells. In addition, nuclear strain in nesprin-1 knocked-down cells, which is considered to be compressed by the actin cortical layer, increased compared with that in wild-type cells under stretching condition. These results indicate that nesprin-1 knockdown releases the nucleus from the tension of F-actin bound to the nucleus, thereby increasing allowance for deformation before stretching, and that F-actin bound to the nucleus through nesprin-1 causes sustainable force transmission to the nucleus.

  15. The Capacity of Red Blood Cells to Reduce Nitrite Determines Nitric Oxide Generation under Hypoxic Conditions

    PubMed Central

    Fens, Marcel H.; Larkin, Sandra K.; Oronsky, Bryan; Scicinski, Jan; Morris, Claudia R.; Kuypers, Frans A.

    2014-01-01

    Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release. PMID:25007272

  16. Mathematical model of a plate fin heat exchanger operating under solid oxide fuel cell working conditions

    NASA Astrophysics Data System (ADS)

    Kaniowski, Robert; Poniewski, Mieczysław

    2013-12-01

    Heat exchangers of different types find application in power systems based on solid oxide fuel cells (SOFC). Compact plate fin heat exchangers are typically found to perfectly fit systems with power output under 5 kWel. Micro-combined heat and power (micro-CHP) units with solid oxide fuel cells can exhibit high electrical and overall efficiencies, exceeding 85%, respectively. These values can be achieved only when high thermal integration of a system is assured. Selection and sizing of heat exchangers play a crucial role and should be done with caution. Moreover, performance of heat exchangers under variable operating conditions can strongly influence efficiency of the complete system. For that reason, it becomes important to develop high fidelity mathematical models allowing evaluation of heat exchangers under modified operating conditions, in high temperature regimes. Prediction of pressure and temperatures drops at the exit of cold and hot sides are important for system-level studies. Paper presents dedicated mathematical model used for evaluation of a plate fin heat exchanger, operating as a part of micro-CHP unit with solid oxide fuel cells.

  17. Modification of decellularized vascular scaffold with conditioned medium to enhance cell reseeding.

    PubMed

    Zhao, Liang; Chen, Hong-Li; Xie, Li-Qin; Wang, Mian; Li, Xia-Fei; Feng, Zhi-Wei; Li, Min

    2016-08-01

    Repopulation of decellularized vascular scaffolds (DVS) is limited because of change in the repertoire and ratios of the remaining extracellular matrix (ECM) proteins, for example, loss of glycoproteins and the retention of type I collagen. Pre-treatment of DVS with defined ECM proteins, which match the repertoire of integrin receptors expressed by the embryonic stem cells (mESCs) to be seeded, can increase the reseeding efficacy. mESCs mainly express high levels of functional receptors for LM and FN. Reseeding efficiency of DVS with mESCs was very low, but was sigficantly increased (2.5 ± 0.1 fold) by pre-treating the DVS with A549-conditioned media. In addition, pre-treatment with A549-conditioned media led to a more homogeneous distribution of the seeded mESCs throughout the engineered blood vessel as compared to untreated DVS. This paper may promote blood vessel engineering by stressing the importance of matching the cell binding motifs of DVS and the integrin receptor repertoire of seeded cells. PMID:27193380

  18. Reduced-intensity conditioning allogeneic hematopoietic-cell transplantation for older patients with acute myeloid leukemia

    PubMed Central

    Goyal, Gaurav; Gundabolu, Krishna; Vallabhajosyula, Saraschandra; Silberstein, Peter T.; Bhatt, Vijaya Raj

    2016-01-01

    Elderly patients (>60 years) with acute myeloid leukemia have a poor prognosis with a chemotherapy-alone approach. Allogeneic hematopoietic-cell transplantation (HCT) can improve overall survival (OS). However, myeloablative regimens can have unacceptably high transplant-related mortality (TRM) in an unselected group of older patients. Reduced-intensity conditioning (RIC) or nonmyeloablative (NMA) conditioning regimens preserve the graft-versus-leukemia effects but reduce TRM. NMA regimens result in minimal cytopenia and may not require stem cell support for restoring hematopoiesis. RIC regimens, intermediate in intensity between NMA and myeloablative regimens, can cause prolonged myelosuppresion and usually require stem cell support. A few retrospective and prospective studies suggest a possibility of lower risk of relapse with myeloablative HCT in fit older patients with lower HCT comorbidity index; however, RIC and NMA HCTs have an important role in less-fit patients and those with significant comorbidities because of lower TRM. Whether early tapering of immunosuppression, monitoring of minimal residual disease, and post-transplant maintenance therapy can improve the outcomes of RIC and NMA HCT in elderly patients will require prospective trials. PMID:27247754

  19. Effects of Operating Conditions on Internal Resistances in Enzyme Fuel Cells Studied via Electrochemical Impedance Spectroscopy

    SciTech Connect

    Aaron, D; Borole, Abhijeet P; Yiacoumi, Sotira; Tsouris, Costas

    2012-01-01

    Enzyme fuel cells (EFCs) offer some advantages over traditional precious-metal-catalyzed fuel cells, such as polymer electrolyte membrane fuel cells (PEMFCs). However, EFCs exhibit far less power output than PEMFCs and have relatively short life spans before materials must be replaced. In this work, electrochemical impedance spectroscopy (EIS) is used to analyze the internal resistances throughout the EFC at a variety of operating conditions. EIS analysis is focused primarily on the resistances of the anode, solution/membrane, and cathode. Increased enzyme loading results in improved power output and reductions in internal resistance. Conditions are identified for which enzyme loading does not limit the EFC performance. EIS experiments are also reported for EFCs operated continuously for 2 days; power output declines sharply over time, while all internal resistances increase. Drying of the cathode and enzyme/mediator degradation are believed to have contributed to this behavior. Finally, experiments are performed at varying air-humidification temperatures. Little effect on internal resistances or power output is observed. However, it is anticipated that increased air humidification can improve longevity by delivering more water to the cathode. Improvements to the enzymatic cathode are needed for EFC development. These improvements need to focus on improving transport rather than increasing enzyme loading.

  20. Reduced-intensity conditioning allogeneic hematopoietic-cell transplantation for older patients with acute myeloid leukemia.

    PubMed

    Goyal, Gaurav; Gundabolu, Krishna; Vallabhajosyula, Saraschandra; Silberstein, Peter T; Bhatt, Vijaya Raj

    2016-06-01

    Elderly patients (>60 years) with acute myeloid leukemia have a poor prognosis with a chemotherapy-alone approach. Allogeneic hematopoietic-cell transplantation (HCT) can improve overall survival (OS). However, myeloablative regimens can have unacceptably high transplant-related mortality (TRM) in an unselected group of older patients. Reduced-intensity conditioning (RIC) or nonmyeloablative (NMA) conditioning regimens preserve the graft-versus-leukemia effects but reduce TRM. NMA regimens result in minimal cytopenia and may not require stem cell support for restoring hematopoiesis. RIC regimens, intermediate in intensity between NMA and myeloablative regimens, can cause prolonged myelosuppresion and usually require stem cell support. A few retrospective and prospective studies suggest a possibility of lower risk of relapse with myeloablative HCT in fit older patients with lower HCT comorbidity index; however, RIC and NMA HCTs have an important role in less-fit patients and those with significant comorbidities because of lower TRM. Whether early tapering of immunosuppression, monitoring of minimal residual disease, and post-transplant maintenance therapy can improve the outcomes of RIC and NMA HCT in elderly patients will require prospective trials. PMID:27247754

  1. Proliferation and responsiveness to estrogen of human endometrial cancer cells under serum-free culture conditions.

    PubMed

    Holinka, C F; Anzai, Y; Hata, H; Kimmel, N; Kuramoto, H; Gurpide, E

    1989-06-15

    Studies of hormonal growth regulation in cultured human endometrial cancer cells are limited by the requirement of exogenous growth factors, usually supplied by addition of serum. The present report provides evidence that estradiol can stimulate proliferation of endometrial cancer cells of the Ishikawa line in the absence of serum or added growth factors. Mitogenic effects of estrogen were demonstrated in two different experimental systems, in cells attached to the substratum of mammalian tissue culture dishes, and in cells forming colonies in soft agar under anchorage-independent conditions. Addition of estradiol to a mixture of serum-free, phenol red-free Dulbecco's minimal essential medium and Ham's F-12 medium, supplemented with L-glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [basal medium: (BM)] significantly increased the proliferation of cells attached to culture dishes. Dose-response experiments revealed maximal estradiol stimulation at 10 nM; significant responses were also observed at 1 nM and at 100 nM concentrations. The mitogenic effect of 10 nM estradiol was comparable to that of 1% charcoal-treated fetal bovine serum and the two effects were additive. The presence of estradiol in serum-free BM resulted in a shortening of the doubling time of exponentially proliferating cells from 38 to 29 h. From the labeling index, measured after exposure to a pulse of [3H]thymidine, and from the mitotic index, both determined in exponentially proliferating cells, the lengths of the S and M phases were calculated to be 11 and 1 h, respectively. From these data it was estimated that estradiol shortened the G1 phase by approximately 40%, from 22 to 13 h. Estradiol doubled the colony formation efficiency of cells plated in BM containing 0.3% agar in the absence of serum as well as in the presence of 1% charcoal-treated fetal bovine serum. The stimulation of colony formation by estradiol was influenced by medium components, since no effects were

  2. Differentiations and Functional State of Osteogenic Cells in Conditions of Microgravity

    NASA Astrophysics Data System (ADS)

    Onishchenko, Ganna; Rodionova, Natalia; Markevich, Ganna; Markevich, Ganna

    The space flight factors (space radiation, magnetic fields etc.) affect considerably the state of bone tissue, leading to the development of osteoporosis and osteopenia in the bone skeleton. Many aspects of reactions of bone tissue cells still remain unclear until now. With the use of electron microscopy and autoradiography with 3H-thymidine we studied the samples gathered from the femoral bone epiphyses and metaphyses of rats flown on board American Spacelab -2 and in experiments with modeling of microgravity ("tail suspension" method). In our work the main attention is focused on studying the ultrastructure and metabolism of osteogenetic cells. The degree of differentiation and functional state are evaluated according to the degree of development of organelles for specific biosynthesis: rough endoplasmic reticulum (RER), Golgy complex (GC), as well as the state of mitochondria and cell nucleus. As compared with a control, the population of osteogenetic cells from zones of bone reconstruction shows a decrease in the number of functionally active forms. We can judge of this from the reduction volume of RER, GC, mitochondria in osteoblasts. RER loses architectonics typical for osteoblasts and, as against the control, is represented by short narrow canaliculi distributed throughout the cy-toplasm; some canals disintegrate. GC is slightly pronounced, mitochondria become smaller in size and acquire an optically dark matrix. These phenomena are supposed to be associated with the desorganization of microtubules and microfilaments in the cells under microgravity condi-tions. The number of degrading and apoptotic cells increases in the population of osteoblasts. The dynamics of labeled cells following various intervals after 3H-thymidine injection testifies to a delay in the rates of osteoblasts' differentiation and their transformation to osteocytes in the experiment animals. A lower 3H-glycine uptake by the osteogenic cells and bone matrix as compared with a control is

  3. Differentiation of cones in cultured rabbit retina: effects of retinal pigment epithelial cell-conditioned medium.

    PubMed

    Mack, Andreas F; Uhlmann, Daniela; Germer, Angela; Szél, Agoston; Enzmann, Volker; Reichenbach, Andreas

    2003-04-24

    This study was aimed at investigating the postnatal differentiation of cone photoreceptors in the rabbit retina in an organotypic explant culture system. Both short wavelength (S) and middle wavelength (M) cone opsins were expressed in culture but M cones appeared only in retinal explants from the dorsal half of the eye. Stimulating the explants with retinal pigment epithelial cell (RPE) conditioned medium resulted in a suppression of opsin expression despite of an increase of the number of presumptive peanut agglutinin-labeled cones. These results suggest that at birth the immature cones are largely undetermined in terms of their final cone identity although some positional information ('dorsal' vs. 'ventral' retina) is present. Furthermore, factors from RPE may inhibit as well as stimulate different steps of cone cell differentiation. PMID:12676342

  4. Conditioned Media From Adipose-Derived Stromal Cells Accelerates Healing in 3-Dimensional Skin Cultures.

    PubMed

    Collawn, Sherry S; Mobley, James A; Banerjee, N Sanjib; Chow, Louise T

    2016-04-01

    Wound healing involves a number of factors that results in the production of a "closed" wound. Studies have shown, in animal models, acceleration of wound healing with the addition of adipose-derived stromal cells (ADSC). The cause for the positive effect which these cells have on wound healing has not been elucidated. We have previously shown that addition of ADSC to the dermal equivalent in 3-dimensional skin cultures accelerates reepithelialization. We now demonstrate that conditioned media (CM) from cultured ADSC produced a similar rate of healing. This result suggests that a feedback from the 3-dimensional epithelial cultures to ADSC was not necessary to effect the accelerated reepithelialization. Mass spectrometry of CM from ADSC and primary human fibroblasts revealed differences in secretomes, some of which might have roles in the accelerating wound healing. Thus, the use of CM has provided some preliminary information on a possible mode of action. PMID:26954733

  5. Real-time thermal imaging of solid oxide fuel cell cathode activity in working condition.

    PubMed

    Montanini, Roberto; Quattrocchi, Antonino; Piccolo, Sebastiano A; Amato, Alessandra; Trocino, Stefano; Zignani, Sabrina C; Faro, Massimiliano Lo; Squadrito, Gaetano

    2016-09-01

    Electrochemical methods such as voltammetry and electrochemical impedance spectroscopy are effective for quantifying solid oxide fuel cell (SOFC) operational performance, but not for identifying and monitoring the chemical processes that occur on the electrodes' surface, which are thought to be strictly related to the SOFCs' efficiency. Because of their high operating temperature, mechanical failure or cathode delamination is a common shortcoming of SOFCs that severely affects their reliability. Infrared thermography may provide a powerful tool for probing in situ SOFC electrode processes and the materials' structural integrity, but, due to the typical design of pellet-type cells, a complete optical access to the electrode surface is usually prevented. In this paper, a specially designed SOFC is introduced, which allows temperature distribution to be measured over all the cathode area while still preserving the electrochemical performance of the device. Infrared images recorded under different working conditions are then processed by means of a dedicated image processing algorithm for quantitative data analysis. Results reported in the paper highlight the effectiveness of infrared thermal imaging in detecting the onset of cell failure during normal operation and in monitoring cathode activity when the cell is fed with different types of fuels. PMID:27607294

  6. Conditioned Medium from the Stem Cells of Human Exfoliated Deciduous Teeth Ameliorates Experimental Autoimmune Encephalomyelitis.

    PubMed

    Shimojima, Chiaki; Takeuchi, Hideyuki; Jin, Shijie; Parajuli, Bijay; Hattori, Hisashi; Suzumura, Akio; Hibi, Hideharu; Ueda, Minoru; Yamamoto, Akihito

    2016-05-15

    Multiple sclerosis (MS) is a major neuroinflammatory demyelinating disease of the CNS. Current MS treatments, including immunomodulators and immunosuppressants, do not result in complete remission. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells derived from dental pulp. Both SHED and SHED-conditioned medium (SHED-CM) exhibit immunomodulatory and regenerative activities and have the potential to treat various diseases. In this study, we investigated the efficacy of SHED-CM in treating experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. EAE mice treated with a single injection of SHED-CM exhibited significantly improved disease scores, reduced demyelination and axonal injury, and reduced inflammatory cell infiltration and proinflammatory cytokine expression in the spinal cord, which was associated with a shift in the microglia/macrophage phenotype from M1 to M2. SHED-CM also inhibited the proliferation of myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, as well as their production of proinflammatory cytokines in vitro. Treatment of EAE mice with the secreted ectodomain of sialic acid-binding Ig-like lectin-9, a major component of SHED-CM, recapitulated the effects of SHED-CM treatment. Our data suggest that SHED-CM and secreted ectodomain of sialic acid-binding Ig-like lectin-9 may be novel therapeutic treatments for autoimmune diseases, such as MS. PMID:27053763

  7. Human umbilical mesenchymal stem cells conditioned medium promote primary wound healing regeneration

    PubMed Central

    Kusindarta, Dwi Liliek; Wihadmadyatami, Hevi; Fibrianto, Yuda Heru; Nugroho, Widagdo Sri; Susetya, Heru; Musana, Dewi Kania; Wijayanto, Hery; Prihatna, Surya Agus; Wahyuni, A. E. T. H.

    2016-01-01

    Aim: This research was conducted to clarify the capability of human umbilical mesenchymal stem cells conditioned medium (HU-MSCM) to promote regenerations of primary wound healing on the incision skin injury. Materials and Methods: In this study, two approaches in vitro and in vivo already done. On in vitro analysis, tube formation was performed using HU vein endothelial cells in the presence of HU-MSCM, in some experiments cells line was incubated prior the presence of lipopolysaccharide and HU-MSCM then apoptosis assay was performed. Furthermore, in vivo experiments 12 female rats (Rattus norvegicus) were used after rats anesthetized, 7 mm wound was made by incision on the left side of the body. The wound was treated with HU-MSCM containing cream, povidone iodine was run as a control. Wound healing regenerations on the skin samples were visualized by hematoxylin-eosin staining. Results: In vitro models elucidate HU-MSCM may decreasing inflammation at the beginning of wound healing, promote cell migration and angiogenesis. In addition in vivo models show that the incision length on the skin is decreasing and more smaller, HE staining describe decreasing of inflammation phase, increasing of angiogenesis, accelerate fibroplasia, and maturation phase. Conclusions: Taken together our observation indicates that HU-MSCM could promote the acceleration of skin tissue regenerations in primary wound healing process. PMID:27397984

  8. Novel electrical energy storage system based on reversible solid oxide cells: System design and operating conditions

    NASA Astrophysics Data System (ADS)

    Wendel, C. H.; Kazempoor, P.; Braun, R. J.

    2015-02-01

    Electrical energy storage (EES) is an important component of the future electric grid. Given that no other widely available technology meets all the EES requirements, reversible (or regenerative) solid oxide cells (ReSOCs) working in both fuel cell (power producing) and electrolysis (fuel producing) modes are envisioned as a technology capable of providing highly efficient and cost-effective EES. However, there are still many challenges and questions from cell materials development to system level operation of ReSOCs that should be addressed before widespread application. This paper presents a novel system based on ReSOCs that employ a thermal management strategy of promoting exothermic methanation within the ReSOC cell-stack to provide thermal energy for the endothermic steam/CO2 electrolysis reactions during charging mode (fuel producing). This approach also serves to enhance the energy density of the stored gases. Modeling and parametric analysis of an energy storage concept is performed using a physically based ReSOC stack model coupled with thermodynamic system component models. Results indicate that roundtrip efficiencies greater than 70% can be achieved at intermediate stack temperature (680 °C) and elevated stack pressure (20 bar). The optimal operating condition arises from a tradeoff between stack efficiency and auxiliary power requirements from balance of plant hardware.

  9. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.

    PubMed

    Aiuti, Alessandro; Slavin, Shimon; Aker, Memet; Ficara, Francesca; Deola, Sara; Mortellaro, Alessandra; Morecki, Shoshana; Andolfi, Grazia; Tabucchi, Antonella; Carlucci, Filippo; Marinello, Enrico; Cattaneo, Federica; Vai, Sergio; Servida, Paolo; Miniero, Roberto; Roncarolo, Maria Grazia; Bordignon, Claudio

    2002-06-28

    Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID. PMID:12089448

  10. Testing the Paracrine Properties of Human Mesenchymal Stem Cells Using Conditioned Medium.

    PubMed

    Danieli, Patrizia; Malpasso, Giuseppe; Ciuffreda, Maria Chiara; Gnecchi, Massimiliano

    2016-01-01

    Mesenchymal stem cells (MSC) produce and secrete a great variety of cytokines and chemokines that play beneficial paracrine actions when MSC are used for tissue repair. The conditioned medium (CM) derived from MSC can be used both in vitro and in vivo to test specific paracrine effects or to screen putative paracrine/autocrine mediators by proteomics.In this chapter, we describe a straightforward method to prepare MSC-derived CM. Furthermore, we summarize some in vitro assays useful for testing the cytoprotective, angiogenic, and regenerative activity of CM. These assays are very helpful when studying the role of MSC in cardiac repair and regeneration. PMID:27236688

  11. Generation of ES cells for conditional expression of nuclear receptors and coregulators in vivo.

    PubMed

    Wu, San-Pin; Lee, Dong-Kee; Demayo, Francesco J; Tsai, Sophia Y; Tsai, Ming-Jer

    2010-06-01

    Nuclear receptors and coregulators orchestrate diverse aspects of biological functions and inappropriate expression of these factors often associates with human diseases. The present study describes a conditional overexpression system consisting of a minigene located at the Rosa26 locus in the genome of mouse embryonic stem (ES) cells. Before activation, the minigene is silent due to a floxed STOP cassette inserted between the promoter and the transgene. Upon cre-mediated excision of the STOP cassette, the minigene constitutively expresses the tagged transgene driven by the ubiquitous CAGGS promoter. Thus, this system can be used to express target gene in any tissue in a spatial and/or temporal manner if respective cre mouse lines are available. Serving as proof of principle, the CAG-S-hCOUP-TFI allele was generated in ES cells and subsequently in mice. This allele was capable of conditionally overexpressing human chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) in all tissues tested upon activation by cre drivers. This allele was further subjected to address functionality of expressed COUP-TFI and the functional similarity between COUP-TFI and COUP-TFII. Expression of COUP-TFI in COUP-TFII-ablated uterus suppressed aberrant estrogen receptor-alpha activities and rescued implantation and decidualization defects of COUP-TFII mutants, suggesting that COUP-TFI and COUP-TFII are able to functionally compensate for each other in the uterus. A toolbox currently under construction will contain ES cell lines for overexpressing all 48 nuclear receptors and selected 10 coregulators. Upon completion, it will be a very valuable resource for the scientific community. Several ES cells are currently available for distribution. PMID:20382891

  12. Transcriptome analysis and physiology of Bifidobacterium longum NCC2705 cells under continuous culture conditions.

    PubMed

    Mozzetti, V; Grattepanche, F; Moine, D; Berger, B; Rezzonico, E; Arigoni, F; Lacroix, C

    2012-12-01

    A central issue in the use of probiotics in food and food supplements is their sensitivity to many environmental stress factors. The resistance of probiotic cells to lethal stress can be improved by application of homologous or heterologous sub-lethal stress during culture. This screening procedure is generally performed using batch cultures. Continuous cultures could be a suitable and more efficient method to test different stress factors on one culture instead of repeating several batch cultures. However, before testing stresses using continuous cultures, the physiological stability of continuously produced cells over a considered time period must be first evaluated. A continuous culture of Bifidobacterium longum NCC2705 was maintained for 211 h at a dilution rate of 0.1 per h, mimicking a deceleration growth phase culture. Stable viable cell counts were measured over the culture period, decreasing only moderately from 8.8 to 8.6 log10 cfu/ml. A slight shift in metabolite production, characterized by increased lactate and decreased acetate, formate and ethanol concentrations was observed. Susceptibilities to antibiotics and stress conditions were stable (cefotaxim, ampicillin, ceftazidime) or moderately affected (simulated gastric juices, heat, bile salts, tetracycline, chloramphenicol, penicillin, vancomycin and neomycin) over culturing time. Comparison of gene transcription profiles between samples collected after 31 h of continuous culture and samples collected after 134 and 211 h revealed only limited changes in expression of 1.0 and 3.8% of total genes, respectively. Based on these results, we propose that continuous culture can be used to produce bacterial cells with stable physiological properties suitable for fast and efficient screening of sub-lethal stress conditions. PMID:23234728

  13. Identifying the microbial communities and operational conditions for optimized wastewater treatment in microbial fuel cells.

    PubMed

    Ishii, Shun'ichi; Suzuki, Shino; Norden-Krichmar, Trina M; Wu, Angela; Yamanaka, Yuko; Nealson, Kenneth H; Bretschger, Orianna

    2013-12-01

    Microbial fuel cells (MFCs) are devices that exploit microorganisms as "biocatalysts" to recover energy from organic matter in the form of electricity. MFCs have been explored as possible energy neutral wastewater treatment systems; however, fundamental knowledge is still required about how MFC-associated microbial communities are affected by different operational conditions and can be optimized for accelerated wastewater treatment rates. In this study, we explored how electricity-generating microbial biofilms were established at MFC anodes and responded to three different operational conditions during wastewater treatment: 1) MFC operation using a 750 Ω external resistor (0.3 mA current production); 2) set-potential (SP) operation with the anode electrode potentiostatically controlled to +100 mV vs SHE (4.0 mA current production); and 3) open circuit (OC) operation (zero current generation). For all reactors, primary clarifier effluent collected from a municipal wastewater plant was used as the sole carbon and microbial source. Batch operation demonstrated nearly complete organic matter consumption after a residence time of 8-12 days for the MFC condition, 4-6 days for the SP condition, and 15-20 days for the OC condition. These results indicate that higher current generation accelerates organic matter degradation during MFC wastewater treatment. The microbial community analysis was conducted for the three reactors using 16S rRNA gene sequencing. Although the inoculated wastewater was dominated by members of Epsilonproteobacteria, Gammaproteobacteria, and Bacteroidetes species, the electricity-generating biofilms in MFC and SP reactors were dominated by Deltaproteobacteria and Bacteroidetes. Within Deltaproteobacteria, phylotypes classified to family Desulfobulbaceae and Geobacteraceae increased significantly under the SP condition with higher current generation; however those phylotypes were not found in the OC reactor. These analyses suggest that species

  14. Food matrices and cell conditions influence survival of Lactobacillus rhamnosus GG under heat stresses and during storage.

    PubMed

    Endo, Akihito; Teräsjärvi, Johanna; Salminen, Seppo

    2014-03-17

    The present study evaluated impact of moisture content and cell conditions on survival of probiotic strain, Lactobacillus rhamnosus GG, under lethal heat stresses and during long-term storage using freeze-dried cells and oils as matrices. Viable cell counts of freeze-dried L. rhamnosus GG cells suspended in oils had only 1-log-reduction after 5min at 80°C and approximately 3-log-reduction after 20min, while no or very few viable cells were recorded for freeze dried cells suspended in buffer and cultured cells in oils. Surprisingly, freeze-dried cells suspended in oils still contained 4.3 to 6.7logCFU/ml after 5min at 95°C. Long-term storage study indicated that freeze-dried cells suspended in oils kept viable conditions for 4months, and a loss of the viability was only 0.3 to 0.6logCFU/ml. Viable cell counts of cultured cells suspended in oils were not present after 3days to 3months. These results clearly indicate that moisture and cell conditions have a great impact on survival of probiotics under severe heat stress in processing and during long-term storage. Combination of freeze-dried cells and oils as carrier provides beneficial options to preserve viability of probiotics in food processes and storage. PMID:24480189

  15. Feasibility of raw glycerol conversion into single cell oil by zygomycetes under non-aseptic conditions.

    PubMed

    Moustogianni, Anna; Bellou, Stamatia; Triantaphyllidou, Irene-Eva; Aggelis, George

    2015-04-01

    The use of plant oils as feedstock for the biodiesel manufacture has many drawbacks, thus, the interest has turned to single cell oil (SCO) as an alternative. However, the production of SCO is still too expensive, mainly due to the low oil productivity and the high cost of medium sterilization required. In this work raw glycerol was converted into SCO by oleaginous Zygomycetes under non-aseptic conditions on selective (i.e., containing essential oils and/or antibiotics) nitrogen limited media. The obtained data showed that although bacterial populations inhibited the fungal growth, lipid accumulation remained unaffected by the presence of bacteria in the growth medium compared to control experiments (conducted under aseptic conditions). Therefore, a two-stage process was developed in which growth was performed under aseptic conditions (1st stage) followed by lipid accumulation performed under non-aseptic conditions (2nd stage) in the presence of thyme essential oil as an antibacterial agent. Large amounts of lipids were accumulated inside the mycelia, yielding around 13% wt/wt of oil per glycerol consumed. PMID:25335774

  16. Variations of secretome profiles according to conditioned medium preparation: The example of human mesenchymal stem cell-derived adipocytes.

    PubMed

    Clabaut, Aline; Grare, Céline; Léger, Thibaut; Hardouin, Pierre; Broux, Odile

    2015-10-01

    One challenging point in analyzing cellular secretome collected as conditioned medium is cross-contamination by cell culture media components, especially bovine serum proteins. A common approach for serum removal is to wash the cells, an alternative is to grow cells using serum-free conditions. Given that the sample processing may influence the phenotype of cells and thus the secretome, it is important to establish the optimal protocol for each cell type. In this study, we compared two methods for preparing conditioned medium from human adipocytes derived from mesenchymal stem cells. Cells were either washed twice with PBS or cultured the last four days of differentiation in serum-free adipogenic medium. Gene expression of the cells was evaluated by using real-time PCR and 1D LC-MS/MS was used to compare secreted proteins present in the culture supernatants. Surprisingly, results showed significant differences in gene expression patterns of the cells and in protein content of the conditioned media and suggested that PBS washes induced severe modifications of the phenotype of cells and thus changes in protein secretion profiles. These data emphasize the significant variations in protein species related to cell manipulations and underline the importance of procedure optimization prior to any proteomic investigation. PMID:26105977

  17. Testicular cell-conditioned medium supports embryonic stem cell differentiation toward germ lineage and to spermatocyte- and oocyte-like cells.

    PubMed

    Shah, Syed M; Saini, Neha; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan S

    2016-08-01

    Testicular cells are believed to secrete various growth factors that activate signaling pathways finally leading to gametogenesis. In vitro gametogenesis is an obscure but paramountly important task primarily because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these limitations for development of in vitro gametes, the present study was designed to induce differentiation of buffalo embryonic stem (ES) cells into germ lineage cells on stimulation by testicular cell-conditioned medium (TCM), on the basis of the assumption that ES cells have the intrinsic property to differentiate into any cell type and TCM would provide the necessary growth factors for differentiation toward germ cell lineage. For this purpose, buffalo ES cells were differentiated as embryoid bodies (EB) in floating cultures and as monolayer adherent cultures in different doses (10%, 20%, and 40%) of TCM for different culture intervals (4, 8, and 14 days), to identify the optimum dose-and-time period. We observed that 40% TCM dose induces highest expression of primordial germ cell-specific (DAZL, VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, and PRM2), spermatocyte-specific (BOULE and TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) for a culture period of 14 days under both floating and adherent differentiation. Immunocytochemical analysis of EBs and adherent cultures revealed presence of primordial germ cell markers (c-KIT, DAZL, and VASA), meiotic markers (SYCP3, MLH1 and PROTAMINE1), spermatocyte markers (ACROSIN and HAPRIN), and oocyte markers (GDF9 and ZP4), indicating progression into post-meiotic gametogenesis. The detection of germ cell-specific proteins in Day 14 EBs like VASA, GDF9, and ZP4 by Western blotting further confirmed germ lineage differentiation. The significantly lower (P < 0.05) concentration of 5-methyl-2-deoxycytidine in optimally differentiated EBs is suggestive of the process of methylation erasure. Oocyte-like structures

  18. Interface engineering of hybrid perovskite solar cells with poly(3-thiophene acetic acid) under ambient conditions.

    PubMed

    Shit, Arnab; Nandi, Arun K

    2016-04-21

    The properties of methyl ammonium lead iodide (MAPbI3) perovskite solar cells with poly(3-thiophene acetic acid) (P3TAA) as a hole transporting material (HTM) and a dense layer of ZnO nanoparticle film as an electron transporting material (ETM) are described using the conventional ZnO (n)/perovskite (i)/P3TAA (p) (n-i-p) architecture. The FT-IR spectra of a MAPbI3/P3TAA mixture indicate a shift of the N-H stretching and the abolition of the N-H bending peak indicating the interaction between the components. UV-Vis spectra of the mixture exhibit a large red shift of the π-π* transition peak of the conjugated chain arising from the interaction causing an increase of the conjugation length. The cross-sectional SEM image of the device shows the sequence of the individual layers of ZnO, MAPbI3, P3TAA and Ag, respectively. The current density (J)-voltage (V) curves obtained upon illumination with a light of 100 mW cm(-2) indicate the average PCE to be 7.38 ± 0.59% under ambient conditions. The IPCE values of these cells reach about 63% across a broad range of wavelength (300-800 nm). The HOMO and the LUMO of P3TAA are measured using cyclic voltammetry and the optical band gap and the relative energy level of the components explain the operation of photocurrent in the cell. For comparison purposes a device using poly(3-hexyl thiophene) (P3HT) as the HTM is fabricated under similar conditions and it exhibits a lower PCE (5.85 ± 0.51%) than that of the P3TAA based device. The longevity of the P3TAA based cell is also found to be better than that of the P3HT based cell for storing in air. The UV-Vis and impedance spectral results clearly explain the above results, signifying the influence of the interface on the performance of hybrid solar cells. PMID:27020145

  19. Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

    PubMed Central

    Rao, Wei; Pan, Ning; Yang, Zhibo

    2016-01-01

    Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 µm) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (< 10 µm), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies. PMID:27341402

  20. Lethal cutaneous disease in transgenic mice conditionally expressing type I human T cell leukemia virus Tax.

    PubMed

    Kwon, Hakju; Ogle, Louise; Benitez, Bobby; Bohuslav, Jan; Montano, Mauricio; Felsher, Dean W; Greene, Warner C

    2005-10-21

    Type I human T cell leukemia virus (HTLV-I) is etiologically linked with adult T cell leukemia, an aggressive and usually fatal expansion of activated CD4+ T lymphocytes that frequently traffic to skin. T cell transformation induced by HTLV-I involves the action of the 40-kDa viral Tax transactivator protein. Tax both stimulates the HTLV-I long terminal repeat and deregulates the expression of select cellular genes by altering the activity of specific host transcription factors, including cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor, NF-kappaB/Rel, and serum response factor. To study initiating events involved in HTLV-I Tax-induced T cell transformation, we generated "Tet-off" transgenic mice conditionally expressing in a lymphocyte-restricted manner (EmuSR alpha promoter-enhancer) either wild-type Tax or mutant forms of Tax that selectively compromise the NF-kappaB (M22) or CREB/activating transcription factor (M47) activation pathways. Wild-type Tax and M47 Tax-expressing mice, but not M22-Tax expressing mice, developed progressive alopecia, hyperkeratosis, and skin lesions containing profuse activated CD4 T cell infiltrates with evidence of deregulated inflammatory cytokine production. In addition, these animals displayed systemic lymphadenopathy and splenomegaly. These findings suggest that Tax-mediated activation of NF-kappaB plays a key role in the development of this aggressive skin disease that shares several features in common with the skin disease occurring during the preleukemic stage in HTLV-I-infected patients. Of note, this skin disease completely resolved when Tax transgene expression was suppressed by administration of doxycycline, emphasizing the key role played by this viral oncoprotein in the observed pathology. PMID:16105841

  1. Effects of corneal stromal cell- and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells

    PubMed Central

    Zhu, Meng-Yu; Yao, Qin-Ke; Chen, Jun-Zhao; Shao, Chun-Yi; Yan, Chen-Xi; Ni, Ni; Fan, Xian-Qun; Gu, Ping; Fu, Yao

    2016-01-01

    AIM To explore the effects of conditioned media on the proliferation of corneal endothelial cells (CECs) and to compare the efficiency of different conditioned media (CM). METHODS Rat CECs, corneal stromal cells (CSCs), bone marrow-derived endothelial progenitor cells (BEPCs), and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and cultured in vitro. CM was collected from CSCs, BEPCs, and BMSCs. CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed. RESULTS After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone-like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na+/K+-ATP, aquaporin 1 (AQP1), and zonula occludens 1 (ZO-1). Based on quantitative polymerase chain reaction (qPCR) analysis, Na+/K+-ATP expression in CSC-CM was notably upregulated by 1.3-fold (±0.036) (P<0.05, n=3). The expression levels of ZO-1, neuron specific enolase (NSE), Vimentin, paired homebox 6 (PAX6), and procollagen type VIII (COL8A1) were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation. CONCLUSION CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation. PMID:27158599

  2. Photoinduced Interfacial Electron Injection Dynamics in Dye-Sensitized Solar Cells under Photovoltaic Operating Conditions.

    PubMed

    Teuscher, Joël; Décoppet, Jean-David; Punzi, Angela; Zakeeruddin, Shaik M; Moser, Jacques-E; Grätzel, Michael

    2012-12-20

    We report a pump-probe spectroscopy study of electron injection rates in dye-sensitized solar cell (DSSC) devices. We examine the case of working devices employing an N719 ruthenium sensitizer and an iodide electrolyte. Electron injection is found to occur mainly on a sub-100 fs time scale, followed by a slower component with a lifetime of 26.9 ps, in accordance with previous reports on model samples. The amplitude of this latter component varies with electrolyte composition from 25 to 9%. The appearance of slower components in the electron injection dynamics may be attributed to an aggregated or weakly bound state of the surface-adsorbed N719 sensitizer. Further measurements are reported varying the cell light bias and load conditions, revealing no influence on electron injection dynamics. No other electron injection event is found to occur up to 1 ns. These results show no evidence for a slowdown of electron injection under working conditions compared to model systems for the electrolytes examined in this study. PMID:26291112

  3. Protective effect of carbon monoxide pre-conditioning on LPS-induced endothelial cell stress

    PubMed Central

    Zannoni, Augusta; Bacci, Maria Laura; Forni, Monica

    2009-01-01

    Increasing evidence indicates that carbon monoxide (CO) may protect against several diseases including sepsis. The ability of CO pre-treatment to provide good pre-conditioning against lipopolysaccharide (LPS)-induced injury was tested using an in vitro model of primary culture of porcine aortic endothelial cells (pAEC). pAEC were exposed to CO (250 ppm) or air for 1 h prior to the addition of LPS (10 μg/ml). Hsp70, HO-1, and Egr-1 protein levels were determined as well as vascular endothelial growth factor (VEGF) secretion after 4, 7, and 15 h. The effect of CO on LPS-induced apoptosis was also detected at 15 h. CO pre-treatment before the addition of LPS, significantly reduced LPS-induced apoptosis. LPS induced an increase in the level of VEGF in culture media after 7 and 15 h, and a larger increase was detected in CO pre-treated cells. In addition, CO pre-treatment reduced LPS-induced Hsp70, HO-1, and Egr-1 protein expression. In conclusion, CO treatment seems to provide a good pre-conditioning for the prevention of LPS-induced endothelial injury. PMID:19693705

  4. Optimization of pre-transplantation conditions to enhance the efficacy of mesenchymal stem cells.

    PubMed

    Haque, Nazmul; Kasim, Noor Hayaty Abu; Rahman, Mohammad Tariqur

    2015-01-01

    Mesenchymal stem cells (MSCs) are considered a potential tool for cell based regenerative therapy due to their immunomodulatory property, differentiation potentials, trophic activity as well as large donor pool. Poor engraftment and short term survival of transplanted MSCs are recognized as major limitations which were linked to early cellular ageing, loss of chemokine markers during ex vivo expansion, and hyper-immunogenicity to xeno-contaminated MSCs. These problems can be minimized by ex vivo expansion of MSCs in hypoxic culture condition using well defined or xeno-free media i.e., media supplemented with growth factors, human serum or platelet lysate. In addition to ex vivo expansion in hypoxic culture condition using well defined media, this review article describes the potentials of transient adaptation of expanded MSCs in autologous serum supplemented medium prior to transplantation for long term regenerative benefits. Such transient adaptation in autologous serum supplemented medium may help to increase chemokine receptor expression and tissue specific differentiation of ex vivo expanded MSCs, thus would provide long term regenerative benefits. PMID:25678851

  5. Optimization of Pre-transplantation Conditions to Enhance the Efficacy of Mesenchymal Stem Cells

    PubMed Central

    Haque, Nazmul; Kasim, Noor Hayaty Abu; Rahman, Mohammad Tariqur

    2015-01-01

    Mesenchymal stem cells (MSCs) are considered a potential tool for cell based regenerative therapy due to their immunomodulatory property, differentiation potentials, trophic activity as well as large donor pool. Poor engraftment and short term survival of transplanted MSCs are recognized as major limitations which were linked to early cellular ageing, loss of chemokine markers during ex vivo expansion, and hyper-immunogenicity to xeno-contaminated MSCs. These problems can be minimized by ex vivo expansion of MSCs in hypoxic culture condition using well defined or xeno-free media i.e., media supplemented with growth factors, human serum or platelet lysate. In addition to ex vivo expansion in hypoxic culture condition using well defined media, this review article describes the potentials of transient adaptation of expanded MSCs in autologous serum supplemented medium prior to transplantation for long term regenerative benefits. Such transient adaptation in autologous serum supplemented medium may help to increase chemokine receptor expression and tissue specific differentiation of ex vivo expanded MSCs, thus would provide long term regenerative benefits. PMID:25678851

  6. Performance of diffusion-barrier scintillation cells under a variety of controlled environmental conditions

    SciTech Connect

    Spangler, R.R.; Langner, G.H., Jr.

    1989-02-01

    The US Department of Energy (DOE) Office of Remedial Action and Waste Technology established the Technical Measurements Center (TMC) at the Grand Junction, Colorado, Projects Office (GJPO), in part, to develop and evaluate new devices for the DOE remedial action projects. The TMC charged the GJPO Radon Laboratory, under the management of UNC Geotech (UNC), with developing and testing a passive scintillation-type, time-averaging radon monitor. Two types of monitors were developed--a diffusion-barrier scintillation cell (DBSC) and a diffusion-barrier liquid scintillation cell (DBLSC). The performance of the DBSCs was tested under different relative humidities, temperatures, and wind speeds. The test results of the DBSCs showed no statistically significant change in accuracy due to the environmental test conditions. Radon-concentration measurement results for diffusion-barrier charcoal canisters (DBCC), exposed along with the DBSCs, did show significant effects due to wind and temperature, but no effects due to relative humidity. The performance of the DBLSCs under a variety of environmental conditions was not tested because a sufficiently sensitive device could not be developed using the existing GJPO liquid-scintillation counting system and a nontoxic counting medium. 10 refs., 10 figs., 10 tabs.

  7. Effects of operating conditions on durability of polymer electrolyte membrane fuel cell Pt cathode catalyst layer

    NASA Astrophysics Data System (ADS)

    Ohyagi, Shinsuke; Matsuda, Toshihiko; Iseki, Yohei; Sasaki, Tatsuyoshi; Kaito, Chihiro

    In this study, we investigated the effects of humidity and oxygen reduction on the degradation of the catalyst of a polymer electrolyte membrane fuel cell (PEMFC) in a voltage cycling test. To elucidate the effect of humidity on the voltage cycling corrosion of a carbon-supported Pt catalyst with 3 nm Pt particles, voltage cycling tests based on 10,000 cycles were conducted using 100% relative humidity (RH) hydrogen as anode gas and nitrogen of varying humidities as cathode gas. The degradation rate of an electrochemical surface area (ECSA) was almost 50% under 189% RH nitrogen atmosphere and the Pt average particle diameter after 10,000 cycles under these conditions was about 2.3 times that of a particle of fresh catalyst because of the agglomeration of Pt particles. The oxygen reduction reaction (ORR) that facilitated Pt catalyst agglomeration when oxygen was employed as the cathode gas also demonstrated that Pt agglomeration was prominent in higher concentrations of oxygen. The ECSA degradation figure in 100% RH oxygen was similar to that in 189% RH nitrogen. It was concluded that liquid water, which was dropped under a supersaturated condition or generated by ORR, accelerated Pt agglomeration. In this paper, we suggest that the Pt agglomeration degradation occurs in a flooding area in a cell plane.

  8. Development and Maturation of the Neuromuscular Junciton in Cell Culture Under Conditions of Simulated Zero-gravity

    NASA Technical Reports Server (NTRS)

    Gruener, R.

    1985-01-01

    Alterations in gravitational conditions which alter the normal development and interactions of nerve and muscle cells grown in culture is examined. Clinostat conditions, similating Og, which produce changes in cell morphology and growth patterns is studied. Data show that rotation of cocultures of nerve and muscle cells results in morphologic changes which are predicted to significantly alter the functional interactions between the elements of a prototypic synapse. It is further predicted that similar alterations may occur in central synapses which may therefore affect the development of the central nervous system when subjected to altered gravitational conditions.

  9. Stabilization of Pt monolayer catalysts under harsh conditions of fuel cells

    SciTech Connect

    Zhang, Xiaoming; Liu, Ping; Yu, Shansheng; Qiao, Liang; Zheng, Weitao

    2015-05-21

    We employed density functional theory (DFT) to explore the stability of core (M = Cu, Ru, Rh, Pd, Ag, Os, Ir, Au)-shell (Pt) catalysts under harsh conditions, including solutions and reaction intermediates involved in the oxygen reduction reaction (ORR) in fuel cells. A pseudomorphic surface alloy (PSA) with a Pt monolayer (Pt1ML) supported on an M surface, Pt1ML/M(111) or (001), was considered as a model system. Different sets of candidate M cores were identified to achieve a stable Pt1ML shell depending on the conditions. In vacuum conditions, the Pt1ML shell can be stabilized on the most of M cores except Cu, Ag, and Au. The situation varies under various electrochemical conditions. Depending on the solutions and the operating reaction pathways of the ORR, different M should be considered. Pd and Ir are the only core metals studied, being able to keep the PtML shell intact in perchloric acid, sulfuric acid, phosphoric acid, and alkaline solutions as well as under the ORR conditions via different pathways. Ru and Os cores should also be paid attention, which only fall during the ORR via the *OOH intermediate. Rh core works well as long as the ORR does not undergo the pathway via *O intermediate. Our results show that PSAs can behave differently from the near surface alloy, Pt1ML/M1ML/Pt(111), highlighting the importance of considering both chemical environments and the atomic structures in rational design of highly stable core-shell nanocatalysts. Finally, the roles that d-band center of a core M played in determining the stability of supported Pt1ML shell were also discussed.

  10. Stabilization of Pt monolayer catalysts under harsh conditions of fuel cells

    SciTech Connect

    Zhang, Xiaoming; Yu, Shansheng; Zheng, Weitao E-mail: pingliu3@bnl.gov; Qiao, Liang; Liu, Ping E-mail: pingliu3@bnl.gov

    2015-05-21

    We employed density functional theory to explore the stability of core (M = Cu, Ru, Rh, Pd, Ag, Os, Ir, Au)-shell (Pt) catalysts under harsh conditions, including solutions and reaction intermediates involved in the oxygen reduction reaction (ORR) in fuel cells. A pseudomorphic surface alloy (PSA) with a Pt monolayer (Pt{sub 1ML}) supported on an M surface, Pt{sub 1ML}/M(111) or (001), was considered as a model system. Different sets of candidate M cores were identified to achieve a stable Pt{sub 1ML} shell depending on the conditions. In vacuum conditions, the Pt{sub 1ML} shell can be stabilized on the most of M cores except Cu, Ag, and Au. The situation varies under various electrochemical conditions. Depending on the solutions and the operating reaction pathways of the ORR, different M should be considered. Pd and Ir are the only core metals studied, being able to keep the Pt{sub ML} shell intact in perchloric acid, sulfuric acid, phosphoric acid, and alkaline solutions as well as under the ORR conditions via different pathways. Ru and Os cores should also be paid attention, which only fall during the ORR via the *OOH intermediate. Rh core works well as long as the ORR does not undergo the pathway via *O intermediate. Our results show that PSAs can behave differently from the near surface alloy, Pt{sub 1ML}/M{sub 1ML}/Pt(111), highlighting the importance of considering both chemical environments and the atomic structures in rational design of highly stable core-shell nanocatalysts. Finally, the roles that d-band center of a core M played in determining the stability of supported Pt{sub 1ML} shell were also discussed.

  11. Stabilization of Pt monolayer catalysts under harsh conditions of fuel cells

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaoming; Yu, Shansheng; Qiao, Liang; Zheng, Weitao; Liu, Ping

    2015-05-01

    We employed density functional theory to explore the stability of core (M = Cu, Ru, Rh, Pd, Ag, Os, Ir, Au)-shell (Pt) catalysts under harsh conditions, including solutions and reaction intermediates involved in the oxygen reduction reaction (ORR) in fuel cells. A pseudomorphic surface alloy (PSA) with a Pt monolayer (Pt1ML) supported on an M surface, Pt1ML/M(111) or (001), was considered as a model system. Different sets of candidate M cores were identified to achieve a stable Pt1ML shell depending on the conditions. In vacuum conditions, the Pt1ML shell can be stabilized on the most of M cores except Cu, Ag, and Au. The situation varies under various electrochemical conditions. Depending on the solutions and the operating reaction pathways of the ORR, different M should be considered. Pd and Ir are the only core metals studied, being able to keep the PtML shell intact in perchloric acid, sulfuric acid, phosphoric acid, and alkaline solutions as well as under the ORR conditions via different pathways. Ru and Os cores should also be paid attention, which only fall during the ORR via the *OOH intermediate. Rh core works well as long as the ORR does not undergo the pathway via *O intermediate. Our results show that PSAs can behave differently from the near surface alloy, Pt1ML/M1ML/Pt(111), highlighting the importance of considering both chemical environments and the atomic structures in rational design of highly stable core-shell nanocatalysts. Finally, the roles that d-band center of a core M played in determining the stability of supported Pt1ML shell were also discussed.

  12. Stabilization of Pt monolayer catalysts under harsh conditions of fuel cells

    DOE PAGESBeta

    Zhang, Xiaoming; Liu, Ping; Yu, Shansheng; Qiao, Liang; Zheng, Weitao

    2015-05-21

    We employed density functional theory (DFT) to explore the stability of core (M = Cu, Ru, Rh, Pd, Ag, Os, Ir, Au)-shell (Pt) catalysts under harsh conditions, including solutions and reaction intermediates involved in the oxygen reduction reaction (ORR) in fuel cells. A pseudomorphic surface alloy (PSA) with a Pt monolayer (Pt1ML) supported on an M surface, Pt1ML/M(111) or (001), was considered as a model system. Different sets of candidate M cores were identified to achieve a stable Pt1ML shell depending on the conditions. In vacuum conditions, the Pt1ML shell can be stabilized on the most of M cores exceptmore » Cu, Ag, and Au. The situation varies under various electrochemical conditions. Depending on the solutions and the operating reaction pathways of the ORR, different M should be considered. Pd and Ir are the only core metals studied, being able to keep the PtML shell intact in perchloric acid, sulfuric acid, phosphoric acid, and alkaline solutions as well as under the ORR conditions via different pathways. Ru and Os cores should also be paid attention, which only fall during the ORR via the *OOH intermediate. Rh core works well as long as the ORR does not undergo the pathway via *O intermediate. Our results show that PSAs can behave differently from the near surface alloy, Pt1ML/M1ML/Pt(111), highlighting the importance of considering both chemical environments and the atomic structures in rational design of highly stable core-shell nanocatalysts. Finally, the roles that d-band center of a core M played in determining the stability of supported Pt1ML shell were also discussed.« less

  13. Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency

    DOE PAGESBeta

    Barnhoorn, Sander; Uittenboogaard, Lieneke M.; Jaarsma, Dick; Vermeij, Wilbert P.; Tresini, Maria; Weymaere, Michael; Menoni, Hervé; Brandt, Renata M. C.; de Waard, Monique C.; Botter, Sander M.; et al

    2014-10-09

    As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which—in a C57BL6/FVB F1 hybrid genetic background—displays manymore » progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.« less

  14. Cell-Autonomous Progeroid Changes in Conditional Mouse Models for Repair Endonuclease XPG Deficiency

    PubMed Central

    Vermeij, Wilbert P.; Tresini, Maria; Weymaere, Michael; Menoni, Hervé; Brandt, Renata M. C.; de Waard, Monique C.; Botter, Sander M.; Sarker, Altaf H.; Jaspers, Nicolaas G. J.; van der Horst, Gijsbertus T. J.; Cooper, Priscilla K.; Hoeijmakers, Jan H. J.; van der Pluijm, Ingrid

    2014-01-01

    As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg−/− mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg−/− mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging. PMID:25299392

  15. Repression of retinal microvascular endothelial cells by transthyretin under simulated diabetic retinopathy conditions

    PubMed Central

    Shao, Jun; Yao, Yong

    2016-01-01

    AIM To investigate biological effects of transthyretin (TTR) on the development of neovascularization under simulated diabetic retinopathy (DR) condition associated with high glucose and hypoxia. METHODS Human retinal microvascular endothelial cells (hRECs) were cultured in normal and simulated DR environments with high glucose and hypoxia. The normal serum glucose concentration is approximately 5.5 mmol/L; thus, hyperglycemia was simulated with 25 mmol/L glucose, while hypoxia was induced using 200 µmol/L CoCl2. The influence of TTR on hRECs and human retinal pigment epithelial cells (hRPECs) was determined by incubating the cells with 4 µmol/L TTR in normal and abnormal media. A co-culture system was then employed to evaluate the effects of hRPECs on hRECs. RESULTS Decreased hRECs and hRPECs were observed under abnormal conditions, including high-glucose and hypoxic media. In addition, hRECs were significantly inhibited by 4 µmol/L exogenous TTR during hyperglycemic culture. During co-culture, hRPECs inhibited hRECs in both the normal and abnormal environments. CONCLUSION hREC growth is inhibited by exogenous TTR under simulated DR environments with high-glucose and hypoxic, particularly in the medium containing 25 mmol/L glucose. hRPECs, which manufacture TTR in the eye, also represses hRECs in the same environment. TTR is predicted to inhibit the proliferation of hRECs and neovascularization. PMID:27366679

  16. Cytokine-Conditioned Dendritic Cells Induce Humoral Tolerance to Protein Therapy in Mice

    PubMed Central

    Sule, Gautam; Suzuki, Masataka; Guse, Kilian; Cela, Racel; Rodgers, John R.

    2012-01-01

    Abstract A major obstacle in the genetic therapy of inherited metabolic disease is host immune responses to the therapeutic protein. This is best exemplified by inhibitor formation in the protein therapy for hemophilia A. An approach to overcoming this is induction of immunological tolerance to the therapeutic protein. Tolerogenic dendritic cells (DCtols) have been reported to induce tolerance. In addition, cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β1 are known to induce tolerance. To model protein therapy, we used ovalbumin (OVA) as antigen in BALB/c mice and their transgenic derivative, DO11.10 mice. In this study we show that adoptive transfer of antigen-pulsed dendritic cells (DCs) treated with a combination of IL-10 and TGF-β1 can suppress the antibody response in mice. Adoptive transfer of cytokine-conditioned DCs in preimmunized mice results in reduction of antibody response in the mice. Furthermore, the effect is antigen specific, as the recipient mice were able to mount a potent antibody response to the control antigen. Last, we show that TGF-β1 and IL-10-conditioned DCs are able to inhibit anti-FVIII antibody responses in FVIII knockout (KO) mice. Analysis of the contribution of IL-10 and TGF-β1 to the DCtol phenotype shows that IL-10 treatment of DCs is sufficient for inducing OVA-specific tolerance in BALB/c mice, but we observed a requirement for treatment with both human TGF-β1 and human IL-10 to significantly inhibit anti-FVIII antibody responses in FVIII KO mice. This paper demonstrates that autologous cell therapy for antigen-targeted immune suppression may be developed to facilitate long-term therapy. PMID:22468961

  17. Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency

    SciTech Connect

    Barnhoorn, Sander; Uittenboogaard, Lieneke M.; Jaarsma, Dick; Vermeij, Wilbert P.; Tresini, Maria; Weymaere, Michael; Menoni, Hervé; Brandt, Renata M. C.; de Waard, Monique C.; Botter, Sander M.; Sarker, Altaf H.; Jaspers, Nicolaas G. J.; van der Horst, Gijsbertus T. J.; Cooper, Priscilla K.; Hoeijmakers, Jan H. J.; van der Pluijm, Ingrid; Niedernhofer, Laura J.

    2014-10-09

    As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which—in a C57BL6/FVB F1 hybrid genetic background—displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.

  18. Studies on penetration of antibiotic in bacterial cells in space conditions (7-IML-1)

    NASA Technical Reports Server (NTRS)

    Tixador, R.

    1992-01-01

    The Cytos 2 experiment was performed aboard Salyut 7 in order to test the antibiotic sensitivity of bacteria cultivated in vitro in space. An increase of the Minimal Inhibitory Concentration (MIC) in the inflight cultures (i.e., an increase of the antibiotic resistance) was observed. Complementary studies of the ultrastructure showed a thickening of the cell envelope. In order to confirm the results of the Cytos 2 experiment, we performed the ANTIBIO experiment during the D1 mission to try to differentiate, by means of the 1 g centrifuge in the Biorack, between the biological effects of cosmic rays and those caused by microgravity conditions. The originality of this experiment was in the fact that it was designed to test the antibiotic sensitivity of bacteria cultivated in vitro during the orbital phase of the flight. The results show an increase in resistance to Colistin in in-flight bacteria. The MIC is practically double in the in-flight cultures. A cell count of living bacteria in the cultures containing the different Colistin concentrations showed a significant difference between the cultures developed during space flight and the ground based cultures. The comparison between the 1 g and 0 g in-flight cultures show similar behavior for the two sets. Nevertheless, a small difference between the two sets of ground based control cultures was noted. The cultures developed on the ground centrifuge (1.4 g) present a slight decrease in comparison with the cultures developed in the static rack (1 g). In order to approach the mechanisms of the increase of antibiotic resistance on bacteria cultivated in vitro in space, we have proposed the study on penetration of antibiotics in bacterial cells in space conditions. This experiment was selected for the International Microgravity Laboratory 1 (IML-1) mission.

  19. Roles of Mitochondrial Dynamics under Stressful and Normal Conditions in Yeast Cells

    PubMed Central

    Knorre, Dmitry A.; Popadin, Konstantin Y.; Sokolov, Svyatoslav S.; Severin, Fedor F.

    2013-01-01

    Eukaryotic cells contain dynamic mitochondrial filaments: they fuse and divide. Here we summarize data on the protein machinery driving mitochondrial dynamics in yeast and also discuss the factors that affect the fusion-fission balance. Fission is a general stress response of cells, and in the case of yeast this response appears to be prosurvival. At the same time, even under normal conditions yeast mitochondria undergo continuous cycles of fusion and fission. This seems to be a futile cycle and also expensive from the energy point of view. Why does it exist? Benefits might be the same as in the case of sexual reproduction. Indeed, mixing and separating of mitochondrial content allows mitochondrial DNA to segregate and recombine randomly, leading to high variation in the numbers of mutations per individual mitochondrion. This opens a possibility for effective purifying selection-elimination of mitochondria highly contaminated by deleterious mutations. The beneficial action presumes a mechanism for removal of defective mitochondria. We argue that selective mitochondrial autophagy or asymmetrical distribution of mitochondria during cell division could be at the core of such mechanism. PMID:23956814

  20. [Is it possible to "cancel" aging process of cell cultures under optimal conditions for cultivation?].

    PubMed

    Bozhkov, A I; Kovaleva, M K; Menzianova, N G

    2011-01-01

    The characteristics of the cells epigenotypes Dunaliella viridis Teod. in the process of chronological and replicative aging were investigated. By 40th day of accumulative cultivation (which coincided with the stationary growth phase) DNA content in the cells of Dunaliella viridis increased 2 times, triacylglycerides 3 times, beta-carotene and carbonyl proteins 2 times, RNA content decreased in comparison with cells in exponential growth phase, i. e., the 40th day of growth of culture forms the age-related epigenotype. 4 received subcultures were being transplanted during 2 years in mid-logarithmic growth phase (subculture-10), early stationary phase of growth (subculture-20), in the mid-stationary growth phase (subculture-30), and late stationary growth phase (subculture-40). It is shown that epigenotype of subculture-10 remained unchanged over 2 years of cultivation, i. e., it does not manifest replicative aging. At the same time, the subculture-20, although long enough (at least 40 passages), maintained epigenotype characteristic of young cultures, and showed age-related changes. Pronounced age-dependent changes of epigenotype in the course of cultivation were identified for subculture-30, and subculture-40 was characterized by unstable epigenotype. Thus, cultivation conditions determine the intensity of replicative aging in Dunaliella viridis. PMID:21809617

  1. Modeling and short circuit detection of 18650 Li-ion cells under mechanical abuse conditions

    NASA Astrophysics Data System (ADS)

    Sahraei, Elham; Campbell, John; Wierzbicki, Tomasz

    2012-12-01

    In this research a simple, yet accurate model of a single cell, needed for safety assessment of batteries under mechanical abuse conditions, was developed. Extensive testing was performed on a 18650 lithium ion cell, including indentation by a hemispherical punch, lateral indentation by a cylindrical rod, compression between two flat plates, and three-point bending. The batteries were tested in an environmental chamber at a 10% SOC. A finite element model was developed, composed of shell elements representing outside casing, and solid elements for the active material with a binder lumped together with the current collectors and the separator. The jelly roll is modeled as a homogenized and isotropic material. The homogenous model assumes different properties in tension and compression, but does not account for the effect of structural anisotropy caused by the layered nature of the jelly roll. Very good correlation was obtained between LS Dyna numerical simulation and test results in terms of load-displacement relations, deformed shape of the battery, and initiation and propagation of a crack in the shell casing. The FE model was also capable of predicting the onset of short circuit of the cell.

  2. Beneficial Effects of Adiponectin on Periodontal Ligament Cells under Normal and Regenerative Conditions

    PubMed Central

    Nokhbehsaim, Marjan; Keser, Sema; Cirelli, Joni Augusto; Jepsen, Søren; Jäger, Andreas

    2014-01-01

    Type 2 diabetes and obesity are increasing worldwide and linked to periodontitis, a chronic disease which is characterized by the irreversible destruction of the tooth-supporting tissues, that is, periodontium. The mechanisms underlying the association of diabetes mellitus and obesity with periodontal destruction and compromised periodontal healing are not well understood, but decreased plasma levels of adiponectin, as found in diabetic and obese individuals, might be a critical mechanistic link. The aim of this in vitro study was to examine the effects of adiponectin on periodontal ligament (PDL) cells under normal and regenerative conditions, and to study the regulation of adiponectin and its receptors in these cells. Adiponectin stimulated significantly the expression of growth factors and extracellular matrix, proliferation, and in vitro wound healing, reduced significantly the constitutive tumor necrosis factor-α expression, and caused a significant upregulation of its own expression. The beneficial actions of enamel matrix derivative on a number of PDL cell functions critical for periodontal regeneration were partially enhanced by adiponectin. The periodontopathogen Porphyromonas gingivalis inhibited the adiponectin expression and stimulated the expression of its receptors. In conclusion, reduced levels of adiponectin, as found in type 2 diabetes and obesity, may compromise periodontal health and healing. PMID:25121107

  3. Efficient method for slow cryopreservation of human embryonic stem cells in xeno-free conditions.

    PubMed

    Valbuena, Diana; Sánchez-Luengo, Silvia; Galán, Amparo; Sánchez, Eva; Gómez, Eva; Poo, M Eugenia; Ruiz, Verónica; Genbacev, Olga; Krtolica, Ana; Pellicer, Antonio; Moreno, Rubén; Simón, Carlos

    2008-07-01

    An effective, consistent and xeno-free cryopreservation technique is crucial for any human embryonic stem cell (hESC) laboratory with future perspectives for clinical application. This study presents a new slow freezing-rapid thawing method in serum-free conditions that allows the cryopreservation of a large number of colonies without the use of a programmable freezer. To test its efficacy, this method has been compared with two established vitrification methods and applied to three different hESC lines (H9, VAL-3 and VAL-5). The method is based on an increasing concentration of dimethylsulphoxide (1.0, 1.2, 1.5 and 2.0 mol/l) with a slow or a rapid cooling system. Using this method, approximately 60 colonies per cryovial could be cryopreserved, the survival rate ranged between 15 and 68% depending on the cell line used, and the majority of the surviving colonies were grade A. Post-cryopreserved hESC have been cultured for 20 passages, re-cryopreserved and re-thawed with consistent results. After thawing, cells retained the inherent undifferentiated characteristics of hESC and growth rate curve, with a stable karyotype, telomerase activity and teratoma formation when injected into severe combined immunodeficient animals, which was comparable with the fresh lines. This method has been tested for 3 years in two different laboratories. PMID:18616900

  4. Secondary solid cancers after allogeneic hematopoietic cell transplantation using busulfan-cyclophosphamide conditioning

    PubMed Central

    Brazauskas, Ruta; Rizzo, J. Douglas; Sobecks, Ronald M.; Wang, Zhiwei; Horowitz, Mary M.; Bolwell, Brian; Wingard, John R.; Socie, Gerard

    2011-01-01

    Risks of secondary solid cancers among allogeneic hematopoietic cell transplant (HCT) recipients who receive conditioning without total body irradiation are not well known. We evaluated the incidence and risk factors for solid cancers after HCT using high-dose busulfan-cyclophosphamide conditioning in 4318 recipients of first allogeneic HCT for acute myeloid leukemia in first complete remission (N = 1742) and chronic myeloid leukemia in first chronic phase (N = 2576). Our cohort represented 22 041 person-years at risk. Sixty-six solid cancers were reported at a median of 6 years after HCT. The cumulative-incidence of solid cancers at 5 and 10 years after HCT was 0.6% and 1.2% among acute myeloid leukemia and 0.9% and 2.4% among chronic myeloid leukemia patients. In comparison to general population incidence rates, HCT recipients had 1.4× higher than expected rate of invasive solid cancers (95% confidence interval, 1.08-1.79, P = .01). Significantly elevated risks were observed for tumors of the oral cavity, esophagus, lung, soft tissue, and brain. Chronic graft-versus-host disease was an independent risk factor for all solid cancers, and especially cancers of the oral cavity. Recipients of allogeneic HCT using busulfan-cyclophosphamide conditioning are at risk for developing solid cancers. Their incidence continues to increase with time, and lifelong cancer surveillance is warranted in this population. PMID:20926773

  5. Chimerism analysis following allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning.

    PubMed

    Valcárcel, D; Martino, R; Caballero, D; Mateos, M V; Pérez-Simón, J A; Canals, C; Fernández, F; Bargay, J; Muñiz-Díaz, E; Gonzalez, M; San Miguel, J F; Sierra, J

    2003-03-01

    We have performed a prospective study to evaluate early chimerism and its kinetics after allogeneic peripheral blood stem cell transplantation among 68 patients who received a reduced-intensity conditioning (RIC) regimen with fludarabine plus melphalan (n=40) or busulphan (n=28). Chimerism was analyzed by polymerase chain reaction amplification of short tandem repeats in unfractionated (UF) and/or fractionated nucleated cells from bone marrow and peripheral blood (PB). All of the patients showed initial donor engraftment and no patient presented primary or secondary graft failure. In UF samples, the probability of achieving stable complete donor chimerism (CDC) in PB within the first 6 months was 70% on day +30, 85% on day +100 and 95% on day +180. CDC in granulocytes was observed in nearly all cases from day +30 onwards. CDC in T cells, however, differed among melphalan and busulphan recipients during the first 3 months (100 vs 0% on day +30 and 93 vs 20% on day +90, respectively). In multivariate analysis, the only significant variable associated with the achievement of early CDC was having received more than two lines of chemotherapy pretransplant (P<0.02). No correlation was found between the rate of achieving early CDC and the occurrence of acute graft-versus-host disease (GVHD) or disease progression post-transplant. In multivariate analysis, the only variable that influenced the incidence of disease progression post-transplant was the development of chronic extensive GVHD (P<0.05). In conclusion, a state of CDC is readily obtained within the first 6 months after our RIC protocols. Donor myeloid engraftment occurs rapidly in all cases, while early T-cell CDC is more common in more immunosuppressed hosts and, perhaps, in melphalan recipients. PMID:12634730

  6. Impact of KIR and HLA Genotypes on Outcomes after Reduced-Intensity Conditioning Hematopoietic Cell Transplantation.

    PubMed

    Sobecks, Ronald M; Wang, Tao; Askar, Medhat; Gallagher, Meighan M; Haagenson, Michael; Spellman, Stephen; Fernandez-Vina, Marcelo; Malmberg, Karl-Johan; Müller, Carlheinz; Battiwalla, Minoo; Gajewski, James; Verneris, Michael R; Ringdén, Olle; Marino, Susana; Davies, Stella; Dehn, Jason; Bornhäuser, Martin; Inamoto, Yoshihiro; Woolfrey, Ann; Shaw, Peter; Pollack, Marilyn; Weisdorf, Daniel; Milller, Jeffrey; Hurley, Carolyn; Lee, Stephanie J; Hsu, Katharine

    2015-09-01

    Natural killer cells are regulated by killer cell immunoglobulin-like receptor (KIR) interactions with HLA class I ligands. Several models of natural killer cell reactivity have been associated with improved outcomes after myeloablative allogeneic hematopoietic cell transplantation (HCT), but this issue has not been rigorously addressed in reduced-intensity conditioning (RIC) unrelated donor (URD) HCT. We studied 909 patients undergoing RIC-URD HCT. Patients with acute myeloid leukemia (AML, n = 612) lacking ≥ 1 KIR ligands experienced higher grade III to IV acute graft-versus-host disease (GVHD) (HR, 1.6; 95% CI, 1.16 to 2.28; P = .005) compared to those with all ligands present. Absence of HLA-C2 for donor KIR2DL1 was associated with higher grade II to IV (HR, 1.4; P = .002) and III to IV acute GVHD (HR, 1.5; P = .01) compared with HLA-C2(+) patients. AML patients with KIR2DS1(+), HLA-C2 homozygous donors had greater treatment-related mortality compared with others (HR, 2.4; 95% CI, 1.4 to 4.2; P = .002) but did not experience lower relapse. There were no significant associations with outcomes for AML when assessing donor-activating KIRs or centromeric KIR content or for any donor-recipient KIR-HLA assessments in patients with myelodysplastic syndrome (n = 297). KIR-HLA combinations in RIC-URD HCT recapitulate some but not all KIR-HLA effects observed in myeloablative HCT. PMID:25960307

  7. Melatonin promotes development of haploid germ cells from early developing spermatogenic cells of Suffolk sheep under in vitro condition.

    PubMed

    Deng, Shou-Long; Chen, Su-Ren; Wang, Zhi-Peng; Zhang, Yan; Tang, Ji-Xin; Li, Jian; Wang, Xiu-Xia; Cheng, Jin-Mei; Jin, Cheng; Li, Xiao-Yu; Zhang, Bao-Lu; Yu, Kun; Lian, Zheng-Xing; Liu, Guo-Shi; Liu, Yi-Xun

    2016-05-01

    Promotion of spermatogonial stem cell (SSC) differentiation into functional sperms under in vitro conditions is a great challenge for reproductive physiologists. In this study, we observed that melatonin (10(-7) m) supplementation significantly enhanced the cultured SSCs differentiation into haploid germ cells. This was confirmed by the expression of sperm special protein, acrosin. The rate of SSCs differentiation into sperm with melatonin supplementation was 11.85 ± 0.93% which was twofold higher than that in the control. The level of testosterone, the transcriptions of luteinizing hormone receptor (LHR), and the steroidogenic acute regulatory protein (StAR) were upregulated with melatonin treatment. At the early stage of SSCs culture, melatonin suppressed the level of cAMP, while at the later stage, it promoted cAMP production. The similar pattern was observed in testosterone content. Expressions for marker genes of meiosis anaphase, Dnmt3a, and Bcl-2 were upregulated by melatonin. In contrast, Bax expression was downregulated. Importantly, the in vitro-generated sperms were functional and they were capable to fertilize oocytes. These fertilized oocytes have successfully developed to the blastula stage. PMID:26993286

  8. N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions

    PubMed Central

    Hwang, Priscilla Y; Jing, Liufang; Chen, Jun; Lim, Foon-Lian; Tang, Ruhang; Choi, Hyowon; Cheung, Kenneth M; Risbud, Makarand V; Gersbach, Charles A; Guilak, Farshid; Leung, Victor Y; Setton, Lori A

    2016-01-01

    Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a laminin-functionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with β-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues. PMID:27292569

  9. N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions.

    PubMed

    Hwang, Priscilla Y; Jing, Liufang; Chen, Jun; Lim, Foon-Lian; Tang, Ruhang; Choi, Hyowon; Cheung, Kenneth M; Risbud, Makarand V; Gersbach, Charles A; Guilak, Farshid; Leung, Victor Y; Setton, Lori A

    2016-01-01

    Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a laminin-functionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with β-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues. PMID:27292569

  10. Role of reduced intensity conditioning in T-cell and B-cell immune reconstitution after HLA-identical bone marrow transplantation in ADA-SCID

    PubMed Central

    Cancrini, Caterina; Ferrua, Francesca; Scarselli, Alessia; Brigida, Immacolata; Romiti, Maria Luisa; Barera, Graziano; Finocchi, Andrea; Roncarolo, Maria Grazia; Caniglia, Maurizio; Aiuti, Alessandro

    2010-01-01

    The treatment of choice for severe combined immunodeficiency is bone marrow transplantation from an HLA-identical donor sibling without conditioning. However, this may result in low donor stem cell chimerism, leading to reduced long-term immune reconstitution. We compared engraftment, metabolic, and T-cell and B-cell immune reconstitution of HLA-identical sibling bone marrow transplantation performed in 2 severe combined immunodeficiency infants with adenosine deaminase deficiency from the same family treated with or without a reduced intensity conditioning regimen (busulfan/fludarabine). Only the patient who received conditioning showed a stable mixed chimerism in all lineages, including bone marrow myeloid and B cells. The use of conditioning resulted in higher thymus-derived naïve T cells and T-cell receptor excision circles, normalization of the T-cell repertoire, and faster and complete B-cell and metabolic reconstitution. These results suggest the utility of exploring the use of reduced intensity conditioning in bone marrow transplantation from HLA-identical donor in severe combined immunodeficiency to improve long-term immune reconstitution. PMID:20460637

  11. Cell culture condition-dependent impact of AGE-rich food extracts on kinase activation and cell survival on human fibroblasts.

    PubMed

    Nass, Norbert; Weissenberg, Kristian; Somoza, Veronika; Ruhs, Stefanie; Silber, Rolf-Edgar; Simm, Andreas

    2014-03-01

    Advanced glycation end products (AGEs) are stable end products of the Maillard reaction. Effects of food extracts are often initially analysed in cellular test systems and it is not clear how different cell culture conditions might influence the results. Therefore, we compared the effects of two models for AGE-rich food, bread crust and coffee extract (CE) on WI-38 human lung fibroblasts under different cell culture conditions (sub-confluent versus confluent cells, with and without serum). WI-38 cells responded to coffee and bread crust extract (BCE) with a rapid phosphorylation of PKB (AKT), p42/44 MAPK (ERK 1/2) and p38 MAPK, strongly depending on culture conditions. BCE resulted in increased cell numbers, whereas CE appeared to be cytotoxic. When cell numbers under all culture conditions and treatments were correlated with kinase phosphorylation, the relation between phospho-p38 MAPK and phospho-AKT represented a good, cell culture condition-independent predictor of cell survival. PMID:24111510

  12. Local Area Water Removal Analysis of a Proton Exchange Membrane Fuel Cell under Gas Purge Conditions

    PubMed Central

    Lee, Chi-Yuan; Lee, Yu-Ming; Lee, Shuo-Jen

    2012-01-01

    In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold. PMID:22368495

  13. Reduced-intensity conditioning for hematopoietic cell transplantation of chronic granulomatous disease.

    PubMed

    Oshrine, Benjamin; Morsheimer, Megan; Heimall, Jennifer; Bunin, Nancy

    2014-08-30

    Hematopoietic cell transplantation (HCT) is the only available curative therapy for chronic granulomatous disease (CGD), but its use is limited by transplant-related mortality (TRM) in patients who often come to transplant with existing infections or organ dysfunction. Reduction in the intensity of the preparative regimen mitigates these risks, but increases the potential for mixed donor-recipient chimerism (MC) that may progress to graft loss. Recently a busulfan-based reduced-intensity conditioning (RIC) regimen has been described with excellent survival and little MC. We report our experience with a similar RIC regimen at our institution, demonstrating problems with donor chimerism and graft loss. Pediatr Blood Cancer © 2014 Wiley Periodicals, Inc. PMID:25175046

  14. Local area water removal analysis of a proton exchange membrane fuel cell under gas purge conditions.

    PubMed

    Lee, Chi-Yuan; Lee, Yu-Ming; Lee, Shuo-Jen

    2012-01-01

    In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold. PMID:22368495

  15. Engineering aptazyme switches for conditional gene expression in mammalian cells utilizing an in vivo screening approach.

    PubMed

    Rehm, Charlotte; Klauser, Benedikt; Hartig, Jörg S

    2015-01-01

    Artificial RNA switches are an emerging class of genetic controllers suitable for synthetic biology applications. Aptazymes are fusions composed of an aptamer domain and a self-cleaving ribozyme. The utilization of aptazymes for conditional gene expression displays several advantages over employing conventional transcription factor-based techniques as aptazymes require minimal genomic space, fulfill their function without the need of protein cofactors, and most importantly are reprogrammable with respect to ligand selectivity and the RNA function to be regulated. Technologies that enable the generation of aptazymes to defined input ligands are of interest for the construction of biocomputing devices and biosensing applications. In this chapter we present a method that facilitates the in vivo screening of randomized pools of aptazymes in mammalian cells. PMID:25967058

  16. Silicification-induced cell aggregation for the sustainable production of H2 under aerobic conditions.

    PubMed

    Xiong, Wei; Zhao, Xiaohong; Zhu, Genxing; Shao, Changyu; Li, Yaling; Ma, Weimin; Xu, Xurong; Tang, Ruikang

    2015-10-01

    Photobiological hydrogen production is of great importance because of its promise for generating clean renewable energy. In nature, green algae cannot produce hydrogen as a result of the extreme sensitivity of hydrogenase to oxygen. However, we find that silicification-induced green algae aggregates can achieve sustainable photobiological hydrogen production even under natural aerobic conditions. The core-shell structure of the green algae aggregates creates a balance between photosynthetic electron generation and hydrogenase activity, thus allowing the production of hydrogen. This finding provides a viable pathway for the solar-driven splitting of water into hydrogen and oxygen to develop green energy alternatives by using rationally designed cell-material complexes. PMID:26302695

  17. Early administration of recombinant erythropoietin improves hemoglobin recovery after reduced intensity conditioned allogeneic stem cell transplantation.

    PubMed

    Ivanov, V; Faucher, C; Mohty, M; Bilger, K; Ladaique, P; Sainty, D; Arnoulet, C; Chabannon, C; Vey, N; Camerlo, J; Bouabdallah, R; Viens, P; Maraninchi, D; Bardou, V J; Esterni, B; Blaise, D

    2005-11-01

    The use of recombinant human erythropoietin (rHuEPO) has been controversial after myeloablative allogeneic Stem cell transplantation (allo-SCT). Reduced intensity conditioning regimens (RIC) offer a novel approach that might translate into a different profile of erythropoietic recovery. We treated 20 consecutive patients with rHuEPO early after matched sibling RIC allo-SCT. Conditioning included fludarabine, busulfan and antithymocyte globulin. EPO treatment was analyzed in terms of toxicity, impact on the frequency of Red blood cell transfusions (RBCT) and kinetics of Hemoglobin recovery within the 60 days post-allo-SCT. Results were compared with 27 matched patients who did not receive rHuEPO. In the first 2 months after allo-SCT all patients receiving rHuEPO (100%) achieved an Hb level > 11 g/dl at a median of 30 (15-35) days post-allo-SCT, as compared to only 63% of the patients not receiving rHuEPO (P = 0.007) at a median of 35 (20-55) days (P = 0.03). A total of 70% (95% CI, 50-90) of rHuEPO patients maintained an Hb over 11 g/dl in the second month as compared to only 19% (95% CI, 4-34) in the other group (P = 0.0004). For patients receiving RBCT, the use of rHuEPO was associated with a trend towards reduced RBCT requirements. This pilot study suggests a potential benefit of early administration of rHuEPO after RIC allo-SCT on early erythropoietic recovery. PMID:16151421

  18. The biofilm environment offers a possible condition for inducing the competency of DNA recipient cells through nutritional starvation.

    PubMed

    Nishioka, Motomu; Mashayekhan, Shohreh; Onishi, Kyoko; Taya, Masahito

    2007-09-01

    Transformation phenomena occurring under conditions mimicking the biofilm environment were investigated using Escherichia coli IM302 (as DNA recipient cells) and Providencia sp. WW2 (as surrounding cells in the biofilm model). In the case of planktonic IM302 cells kept at 25 degrees C, the transformation took place exclusively in the absence of organic nutrients (COD = 0), and was not substantially observed in the range of COD = 30-1500 mg O2/L. On the other hand, in the case of biofilm IM302 cells, the transformation occurred at relatively high levels under the examined conditions (temperature = 5 or 25 degrees C and COD = 0-1500 mg O2/L). These results indicated that the competency of biofilm IM302 cells was induced even in the presence of organic nutrients owing to nutritional starvation caused by WW2 cells. PMID:17927052

  19. Anti-inflammatory effect of conditioned medium from human uterine cervical stem cells in uveitis.

    PubMed

    Bermudez, Maria A; Sendon-Lago, Juan; Seoane, Samuel; Eiro, Noemi; Gonzalez, Francisco; Saa, Jorge; Vizoso, Francisco; Perez-Fernandez, Roman

    2016-08-01

    The aim of the present study was to evaluate the effect of conditioned medium from human uterine cervical stem cells (CM-hUCESCs) in uveitis. To do that, uveitis was induced in rats after footpad injection of Escherichia coli lipopolysaccaride (LPS). Human retinal pigment epithelial (ARPE-19) cells after LPS challenge were used to test anti-inflammatory effect of CM-hUCESCs 'ìn vitro'. Real-time PCR was used to evaluate mRNA expression levels of the pro-inflammatory cytokines interkeukin-6, interkeukin-8, macrophage inflammatory protein-1 alpha, tumor necrosis factor alpha, and the anti-inflammatory interkeukin-10. Leucocytes from aqueous humor (AqH) were quantified in a Neubauer chamber, and eye histopathological analysis was done with hematoxylin-eosin staining. Additionally, using a human cytokine antibody array we evaluated CM-hUCESCs to determine mediating proteins. Results showed that administration of CM-hUCESCs significantly reduced LPS-induced pro-inflammatory cytokines both 'in vitro' and 'in vivo', and decreased leucocytes in AqH and ocular tissues. High levels of cytokines with anti-inflammatory effects were found in CM-hUCESCs, suggesting a possible role of these factors in reducing intraocular inflammation. In summary, treatment with CM-hUCESCs significantly reduces inflammation in uveitis. Our data indicate that CM-hUCESCs could be regarded as a potential therapeutic agent for patients suffering from ocular inflammation. PMID:27381329

  20. Re-evaluation of the culture condition of polymorphonuclear cells for the study of apoptosis induction.

    PubMed

    Hiroi, M; Tajima, M; Shimojima, T; Kashimata, M; Miyata, T; Sakagami, H

    1998-01-01

    The culture conditions of human peripheral blood polymorphonuclear leukocytes (PMN) in the study of apoptosis induction were re-evaluated. The changes in the relative viable cell number of PMNs after tumor necrosis factor (TNF) treatment were colorimetrically investigated using a cell counting kit. The relative potency of PMNs to produce the superoxide anion (O2-) was measured as the reduction of color intensity by addition of superoxide dismutase (SOD). When the PMNs were cultured in conventional RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), the stimulation effect of TNF on O2- generation by PMNs was observed only for the first 6 hours. When FBS was replaced with human serum, the effect of TNF was maintained for longer incubation periods. Prolonged incubation of PMNs spontaneously produced large DNA fragments, and the extent of DNA fragmentation was relatively smaller in human serum-containing medium. TNF, LPS, hyperthermia or potassium thiocyanate slightly accelerated the production of large DNA fragments, as well as the induction of trace amounts of internucleosomal DNA cleavage in PMNs, which became detectable only after concentration by fractional isopropanol precipitation. The present study suggests the importance of the use of human serum rather than conventional FBS for the study of apoptosis induction in PMNs. PMID:9673409

  1. IFNα/βR Signaling Promotes Regulatory T Cell Development and Function Under Stress Conditions

    PubMed Central

    Metidji, Amina; Rieder, Sadiye Amcaoglu; Glass, Deborah Dacek; Cremer, Isabelle; Punkosdy, George A.; Shevach, Ethan M.

    2015-01-01

    Type I IFNs are a family of cytokines with antiviral and immunomodulatory properties. While the antiviral effects of IFNs are well characterized, their immunomodulatory properties are less clear. To specifically address the effects of type I IFNs on Treg, we studied mixed bone morrow (BM) chimeras between wild-type (WT) and IFNα/βR (IFNAR) knockout (KO) mice, and heterozygous female mice expressing a Treg-specific deletion of the IFNAR. In these two models, IFNAR signaling promotes the development of the Treg lineage in the thymus and their survival in the periphery. IFNAR KO Treg had a higher expression of the pro-apoptotic gene Bim and higher frequency of active caspase positive cells. IFNAR KO Treg from chimeric mice displayed a more naïve phenotype, accompanied by lower levels of CD25 and phosphorylated STAT5. Therefore, in Treg IFNAR signaling may directly or indirectly affect phosphorylation of STAT5. In mixed chimeras with Scurfy fetal liver, Treg derived from IFNAR KO BM were unable to control T effector cell activation and tissue inflammation. Under stress conditions or in a competitive environment, IFNAR signaling may be required to maintain Treg homeostasis and function. PMID:25795758

  2. Classification of yeast cells from image features to evaluate pathogen conditions

    NASA Astrophysics Data System (ADS)

    van der Putten, Peter; Bertens, Laura; Liu, Jinshuo; Hagen, Ferry; Boekhout, Teun; Verbeek, Fons J.

    2007-01-01

    Morphometrics from images, image analysis, may reveal differences between classes of objects present in the images. We have performed an image-features-based classification for the pathogenic yeast Cryptococcus neoformans. Building and analyzing image collections from the yeast under different environmental or genetic conditions may help to diagnose a new "unseen" situation. Diagnosis here means that retrieval of the relevant information from the image collection is at hand each time a new "sample" is presented. The basidiomycetous yeast Cryptococcus neoformans can cause infections such as meningitis or pneumonia. The presence of an extra-cellular capsule is known to be related to virulence. This paper reports on the approach towards developing classifiers for detecting potentially more or less virulent cells in a sample, i.e. an image, by using a range of features derived from the shape or density distribution. The classifier can henceforth be used for automating screening and annotating existing image collections. In addition we will present our methods for creating samples, collecting images, image preprocessing, identifying "yeast cells" and creating feature extraction from the images. We compare various expertise based and fully automated methods of feature selection and benchmark a range of classification algorithms and illustrate successful application to this particular domain.

  3. Conditions for charge transport without recombination in low mobility organic solar cells and photodiodes (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Stolterfoht, Martin; Armin, Ardalan; Philippa, Bronson; White, Ronald D.; Burn, Paul L.; Meredith, Paul; Juška, Gytis; Pivrikas, Almantas

    2015-10-01

    Organic semiconductors typically possess low charge carrier mobilities and Langevin-type recombination dynamics, which both negatively impact the performance of organic solar cells and photodetectors. Charge transport in organic solar cells is usually characterized by the mobility-lifetime product. Using newly developed transient and steady state photocurrent measurement techniques we show that the onset of efficiency limiting photocarrier recombination is determined by the charge that can be stored on the electrodes of the device. It is shown that significant photocarrier recombination can be avoided when the total charge inside the device, defined by the trapped, doping-induced and mobile charge carriers, is less than the electrode charge. Based upon this physics we propose the mobility-recombination coefficient product as an alternative and more convenient figure of merit to minimize the recombination losses. We validate the results in 3 different organic semiconductor-based light harvesting systems with very different charge transport properties. The findings allow the determination of the charge collection efficiency in fully operational devices. In turn, knowing the conditions under which non-geminate recombination is eliminated enables one to quantify the generation efficiency of free charge carriers. The results are relevant to a wide range of light harvesting systems, particularly those based upon disordered semiconductors, and require a rethink of the critical parameters for charge transport.

  4. Characterization and optimization of cathodic conditions for H2O2 synthesis in microbial electrochemical cells.

    PubMed

    Sim, Junyoung; An, Junyeong; Elbeshbishy, Elsayed; Ryu, Hodon; Lee, Hyung-Sool

    2015-11-01

    Cathode potential and O2 supply methods were investigated to improve H2O2 synthesis in an electrochemical cell, and optimal cathode conditions were applied for microbial electrochemical cells (MECs). Using aqueous O2 for the cathode significantly improved current density, but H2O2 conversion efficiency was negligible at 0.3-12%. Current density decreased for passive O2 diffusion to the cathode, but H2O2 conversion efficiency increased by 65%. An MEC equipped with a gas diffusion cathode was operated with acetate medium and domestic wastewater, which presented relatively high H2O2 conversion efficiency from 36% to 47%, although cathode overpotential was fluctuated. Due to different current densities, the maximum H2O2 production rate was 141 mg H2O2/L-h in the MEC fed with acetate medium, but it became low at 6 mg H2O2/L-h in the MEC fed with the wastewater. Our study clearly indicates that improving anodic current density and mitigating membrane fouling would be key parameters for large-scale H2O2-MECs. PMID:26141667

  5. Achieving stringent CR is essential before reduced-intensity conditioning allogeneic hematopoietic cell transplantation in AML.

    PubMed

    Ustun, C; Wiseman, A C; Defor, T E; Yohe, S; Linden, M A; Oran, B; Burke, M; Warlick, E; Miller, J S; Weisdorf, D

    2013-11-01

    Reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (allo-HCT) can cure patients with AML in CR. However, relapse after RIC allo-HCT may indicate heterogeneity in the stringency of CR. Strict definition of CR requires no evidence of leukemia by both morphologic and flow cytometric criteria. We re-evaluated 85 AML patients receiving RIC allo-HCT in CR to test if a strict definition of CR had direct implications for the outcome. These patients had leukemia immunophenotype documented at diagnosis and analyzed at allo-HCT. Eight (9.4%) had persistent leukemia by flow cytometric criteria at allo-HCT. The patients with immunophenotypic persistent leukemia had a significantly increased relapse (hazard ratio (HR): 3.7; 95% confidence interval (CI): 1.3-10.3, P=0.01) and decreased survival (HR: 2.9; 95% CI: 1.3-6.4, P<0.01) versus 77 patients in CR by both morphology and flow cytometry. However, the pre-allo-HCT bone marrow (BM) blast count (that is, 0-4%) was not significantly associated with risks of relapse or survival. These data indicate the presence of leukemic cells, but not the BM blast count affects survival. A strict morphologic and clinical lab flow cytometric definition of CR predicts outcomes after RIC allo-HCT, and therefore is critical to achieve at transplantation. PMID:23933764

  6. Human Dermal Stem/Progenitor Cell-Derived Conditioned Medium Improves Senescent Human Dermal Fibroblasts

    PubMed Central

    Jung, Ji-Yong; Shim, Joong Hyun; Choi, Hyun; Lee, Tae Ryong; Shin, Dong Wook

    2015-01-01

    Adult skin stem cells are recognized as potential therapeutics to rejuvenate aged skin. We previously demonstrated that human dermal stem/progenitor cells (hDSPCs) with multipotent capacity could be enriched from human dermal fibroblasts using collagen type IV. However, the effects of hDSPCs on cellular senescence remain to be elucidated. In the present study, we investigated whether conditioned medium (CM) collected from hDSPC cultures (hDSPC-CM) exhibits beneficial effects on senescent fibroblasts. We found that hDSPC-CM promoted proliferation and decreased the expression level of senescence-associated β-galactosidase in senescent fibroblasts. In addition, p53 phosphorylation and p21 expression were significantly reduced in senescent fibroblasts treated with hDSPC-CM. hDSPC-CM restored the expression levels of collagen type I, collagen type III, and tissue inhibitor of metalloproteinase, and antagonized the increase of matrix metalloproteinase 1 expression. Finally, we demonstrated that hDSPC-CM significantly reduced reactive oxygen species levels by specifically up-regulating the expression level of superoxide dismutase 2. Taken together, these data suggest that hDSPC-CM can be applied as a potential therapeutic agent for improving human aged skin. PMID:26287165

  7. Risks and benefits of sex-mismatched hematopoietic cell transplantation differ according to conditioning strategy

    PubMed Central

    Nakasone, Hideki; Remberger, Mats; Tian, Lu; Brodin, Petter; Sahaf, Bita; Wu, Fang; Mattsson, Jonas; Lowsky, Robert; Negrin, Robert; Miklos, David B.; Meyer, Everett

    2015-01-01

    Sex-mismatched hematopoietic cell transplantation is linked to increased graft-versus-host disease and mortality in myeloablative conditioning. Here we evaluated outcomes of 1,041 adult transplant recipients at two centers between 2006 and 2013 and investigated how the effect of sex-mismatching differed in myeloablative, reduced-intensity, and non-myeloablative total lymphoid irradiation with anti-thymocyte globulin conditioning. Among patients who underwent myeloablative conditioning, male recipients with female donors had increased chronic graft-versus-host disease (hazard ratio 1.83, P<0.01), increased non-relapse mortality (hazard ratio 1.84, P=0.022) and inferior overall survival (hazard ratio 1.59, P=0.018). In contrast, among patients who received reduced-intensity conditioning, male recipients with female donors had increased acute graft-versus-host disease (hazard ratio 1.96, P<0.01) but no difference in non-relapse mortality or overall survival. Among the patients who underwent total lymphoid irradiation with anti-thymocyte globulin, male recipients with female donors showed no increase in graft-versus-host disease or non-relapse mortality. Notably, only in the cohort receiving total lymphoid irradiation with anti-thymocyte globulin were male recipients with female donors significantly associated with reduced relapse (hazard ratio 0.64, P<0.01), and allo-antibody responses against H-Y antigens were predictive of reduced relapse. In the cohort given total lymphoid irradiation with anti-thymocyte globulin, the graft-versus-leukemia effect resulted in superior overall survival in recipients of sex-mismatched grafts (HR 0.69, P=0.037). In addition, only in the cohort treated with total lymphoid irradiation with anti-thymocyte globulin were female recipients with male donors associated with reduced relapse (hazard ratio 0.59, P<0.01) and superior survival (hazard ratio 0.61, P=0.014) compared with sex-matched pairs. We conclude that the risks and benefits of

  8. Proteomic analysis of Staphylococcus aureus biofilm cells grown under physiologically relevant fluid shear stress conditions

    PubMed Central

    2014-01-01

    Background The biofilm forming bacterium Staphylococcus aureus is responsible for maladies ranging from severe skin infection to major diseases such as bacteremia, endocarditis and osteomyelitis. A flow displacement system was used to grow S. aureus biofilms in four physiologically relevant fluid shear rates (50, 100, 500 and 1000 s-1) to identify proteins that are associated with biofilm. Results Global protein expressions from the membrane and cytosolic fractions of S. aureus biofilm cells grown under the above shear rate conditions are reported. Sixteen proteins in the membrane-enriched fraction and eight proteins in the cytosolic fraction showed significantly altered expression (p < 0.05) under increasing fluid shear. These 24 proteins were identified using nano-LC-ESI-MS/MS. They were found to be associated with various metabolic functions such as glycolysis / TCA pathways, protein synthesis and stress tolerance. Increased fluid shear stress did not influence the expression of two important surface binding proteins: fibronectin-binding and collagen-binding proteins. Conclusions The reported data suggest that while the general metabolic function of the sessile bacteria is minimal under high fluid shear stress conditions, they seem to retain the binding capacity to initiate new infections. PMID:24855455

  9. Dental pulp-derived stem cell conditioned medium reduces cardiac injury following ischemia-reperfusion

    PubMed Central

    Yamaguchi, Satoshi; Shibata, Rei; Yamamoto, Noriyuki; Nishikawa, Masaya; Hibi, Hideharu; Tanigawa, Tohru; Ueda, Minoru; Murohara, Toyoaki; Yamamoto, Akihito

    2015-01-01

    Stem cells from human exfoliated deciduous teeth (SHEDs) can regenerate various tissues. We investigated the impact of SHED-conditioned medium (SHED-CM) on myocardial injury in a mouse model of ischemia-reperfusion (I/R). Wild-type (WT) mice were subjected to myocardial ischemia followed by reperfusion. SHED-CM was intravenously injected at 5 min after reperfusion. Administration of SHED-CM reduced myocardial infarct size as well as decreased apoptosis and inflammatory cytokine levels, such as TNF-α, IL-6, and IL-β, in the myocardium following I/R. In cultured cardiac myocytes, SHED-CM significantly suppressed apoptosis under hypoxia/serum-deprivation and reduced LPS-induced expression of pro-inflammatory genes. Furthermore, anti-apoptotic action of SHED-CM was stronger than bone marrow-derived stem cell (BMSC)-CM or adipose-derived stem cell (ADSC)-CM in cardiac myocytes. SHED-CM contains a higher concentration of hepatocyte growth factor (HGF) than BMSC-CM and ADSC-CM, and neutralization of HGF attenuated the inhibitory actions of SHED-CM on apoptosis in cardiac myocytes. Finally, WT mice were intravenously treated with an HGF-depleted SHED-CM, followed by myocardial I/R. HGF depletion significantly attenuated the inhibitory actions of SHED-CM on myocardial infarct size and apoptosis after I/R. SHED-CM protects the heart from acute ischemic injury because it suppresses inflammation and apoptosis. SHED-CM could be a useful treatment option for acute myocardial infarction. PMID:26542315

  10. Conditional Derepression of Ferritin Synthesis in Cells Expressing a Constitutive IRP1 Mutant

    PubMed Central

    Wang, Jian; Pantopoulos, Kostas

    2002-01-01

    Iron regulatory protein 1 (IRP1), a major posttranscriptional regulator of cellular iron and energy metabolism, is controlled by an iron-sulfur cluster switch. Cysteine-437 is critical for coordinating the cluster, and its replacement yields mutants that do not respond to iron perturbations and constitutively bind to cognate mRNA iron-responsive elements (IREs). The expression of IRP1C437S in cells has been associated with aberrations in iron homeostasis and toxicity. We have established clones of human lung (H1299) and breast (MCF7) cancer cells that express high levels of IRP1C437S in a tetracycline-inducible manner. As expected, IRP1C437S stabilizes transferrin receptor mRNA and inhibits translation of ferritin mRNA in both cell types by binding to their respective IREs. However, H1299 transfectants grown at high densities are able to overcome the IRP1C437S-mediated inhibition in ferritin synthesis. The mechanism involves neither alteration in ferritin mRNA levels nor utilization of alternative transcription start sites to eliminate the IRE or relocate it in less inhibitory downstream positions. The derepression of ferritin mRNA translation occurs under conditions where global protein synthesis appears to be impaired, as judged by a significant enrichment in the expression of the underphosphorylated form of the translational regulator 4E-BP1. Collectively, these data document an example where ferritin mRNA translation evades control of the IRE-IRP system. The physiological implications of this response are reflected in protection against iron-mediated toxicity, oxidative stress, and apoptosis. PMID:12052872

  11. In vivo immunomodulatory effects of adipose-derived mesenchymal stem cells conditioned medium in experimental autoimmune encephalomyelitis.

    PubMed

    Yousefi, Forouzan; Ebtekar, Massoumeh; Soudi, Sara; Soleimani, Masoud; Hashemi, Seyed Mahmoud

    2016-04-01

    Mesenchymal stem cells (MSCs) are well known to possess neuroprotective and immunomodulatory effects, due to cell-to-cell interaction and their soluble factors. We conducted a comparative analysis of the immunomodulatory properties of adipose tissue mesenchymal stem cells (AT-MSCs) and their conditioned media (CM), derived from C57/BL6 mice, for mitigating the adverse clinical course of experimental autoimmune encephalomyelitis (EAE). We measure IL4, IL17 and IFNɣ production of supernatant from spleen cells. We analyzed brain cell infiltration, splenocyte proliferation and evaluated the percentage of CD4+CD25+FOXP3+splenic cell population in all EAE C57/BL6 mice. AT-MSCs and its conditioned medium induced CD4+CD25+FOXP3+regulatory T cells after in vitro co-culture with naïve T cells. There is no significant difference in the clinical scores and body weight of EAE mice treated with AT-MSCs and CM. The reduction in proliferative responses and brain cell infiltration was more pronounced in mice injected with CM than other groups. It is found that the percentage of splenic CD4+CD25+FOXP3+ population as well as the level of IL4 production in mice administrated with AT-MSCs is increased compared to other animals. Our results suggest that AT-MSCs-derived CM is promising in stem cell therapy, due to their neuroprotective and immunomudulatory properties. PMID:26930038

  12. Modeling of Thermal Performance of Multiphase Nuclear Fuel Cell Under Variable Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Ding, Z.; Anghaie, S.

    1996-01-01

    A unique numerical method has been developed to model the dynamic processes of bulk evaporation and condensation processes, associated with internal heat generation and natural convection under different gravity levels. The internal energy formulation, for the bulk liquid-vapor phase change problems in an encapsulated container, was employed. The equations, governing the conservation of mass, momentum and energy for both phases involved in phase change, were solved. The thermal performance of a multiphase uranium tetra-fluoride fuel element under zero gravity, micro-gravity and normal gravity conditions has been investigated. The modeling yielded results including the evolution of the bulk liquid-vapor phase change process, the evolution of the liquid-vapor interface, the formation and development of the liquid film covering the side wall surface, the temperature distribution and the convection flow field in the fuel element. The strong dependence of the thermal performance of such multiphase nuclear fuel cell on the gravity condition has been revealed. Under all three gravity conditions, 0-g, 10(exp -3)-g, and 1-g, the liquid film is formed and covers the entire side wall. The liquid film covering the side wall is more isothermalized at the wall surface, which can prevent the side wall from being over-heated. As the gravity increases, the liquid film is thinner, the temperature gradient is larger across the liquid film and smaller across the vapor phase. This investigation provides valuable information about the thermal performance of multi-phase nuclear fuel element for the potential space and ground applications.

  13. Intravenous Busulfan-Based Myeloablative Conditioning Regimens Prior to Hematopoietic Cell Transplantation for Hematologic Malignancies.

    PubMed

    Pasquini, Marcelo C; Le-Rademacher, Jennifer; Zhu, Xiaochun; Artz, Andrew; DiPersio, John; Fernandez, Hugo F; Mineishi, Shin; Kamishohara, Masaru; Mehta, Jayesh; Nakamura, Yuki; Ratanatharathorn, Voravit; Sobecks, Ronald; Burkart, Jeanne; Bredeson, Christopher

    2016-08-01

    Busulfan (Bu)-containing regimens are commonly used in myeloablative conditioning regimens before allogeneic hematopoietic cell transplantation (HCT). Yet, there is considerable variability on how Bu is administered related to frequency (4 times a day [Q6] or daily [Q24]) and combinations with other chemotherapeutic agents (cyclophosphamide [Cy] or fludarabine [Flu]). We performed a prospective cohort study of recipients of Bu-based conditioning according to contemporary practices to compare different approaches (BuCy Q6, n = 495; BuFlu Q24, n = 331; BuCy Q24, n = 96; BuFlu Q6, n = 91) in patients with myeloid malignancies between 2009 and 2011. BuFlu Q24 recipients were more likely to be older and tended to have worse performance status and a higher comorbid burden. The cumulative incidences of hepatic veno-occlusive disease (P = .40), idiopathic pneumonia (P = .50), and seizures (P = .50) did not differ across groups. One-year HCT-related mortality ranged from 12% to 16% (P = .80), 3-year relapse incidence ranged from 32% to 36% (P = .80), and 3-year overall survival ranged from 51% to 58% (P = .20) across groups. This study demonstrates that HCT conditioning regimens using i.v. Bu Q6 or Q24 alone or in combination with Cy or Flu have similar outcomes in the myeloablative setting for treatment of myeloid malignancies. PMID:27154848

  14. Hepatic injury after nonmyeloablative conditioning followed by allogeneic hematopoietic cell transplantation: a study of 193 patients.

    PubMed

    Hogan, William J; Maris, Michael; Storer, Barry; Sandmaier, Brenda M; Maloney, David G; Schoch, H Gary; Woolfrey, Ann E; Shulman, Howard M; Storb, Rainer; McDonald, George B

    2004-01-01

    Liver injury is a frequent, serious complication of allogeneic hematopoietic cell transplantation (HCT) following myeloablative preparative regimens. We sought to determine the frequency and severity of hepatic injury after nonmyeloablative conditioning and its relationship to outcomes. One hundred ninety-three consecutive patients who received 2 Gy total body irradiation with or without fludarabine were evaluated for end points related to liver injury. Patients with diseases treatable by HCT who were ineligible for conventional myeloablative allogeneic HCT because of advanced age and/or comorbid conditions were included. Fifty-one patients (26%) developed hyperbilirubinemia of 68.4 microM (4 mg/dL) or greater, most commonly resulting from cholestasis due to graft-versus-host disease (GVHD) or sepsis. Pretransplantation factors associated with liver dysfunction were a diagnosis of aggressive malignancy (hazard ratio [HR] 1.9; P =.04) and the inclusion of fludarabine in the conditioning regimen (HR 1.8; P =.07). Overall survival at 1 year was superior for patients who had maximal serum bilirubin levels in the normal (78%) or minimally elevated (22.23-66.69 microM [1.3-3.9 mg/dL]) ranges (69%) compared with those in the 68.4 to 117.99 microM (4-6.9 mg/dL; 20%), 119.7 to 169.29 microM (7.0-9.9 mg/dL; 17%), and 171.0 microM (10 mg/dL; 19%) or greater groups. In summary, significant jaundice occurred in 26% of patients and was predominantly due to cholestasis resulting from GVHD and/or sepsis. Aggressive malignancies (mainly advanced disease) and later development of jaundice after transplantation predicted inferior survival. PMID:12969980

  15. Maximizing productivity of CHO cell-based fed-batch culture using chemically defined media conditions and typical manufacturing equipment.

    PubMed

    Huang, Yao-Ming; Hu, WeiWei; Rustandi, Eddie; Chang, Kevin; Yusuf-Makagiansar, Helena; Ryll, Thomas

    2010-01-01

    A highly productive chemically defined fed-batch process was developed to maximize titer and volumetric productivity for Chinese hamster ovary cell-based recombinant protein manufacturing. Two cell lines producing a recombinant antibody (cell line A) and an Fc-fusion protein (cell line B) were used for development. Both processes achieved product titers of 10 g/L on day 18 under chemically defined conditions. For cell line B, the use of plant derived hydrolysates combined with the optimized chemically defined medium increased the titer to 13 g/L. Volumetric productivities were increased from a base line of about 200 mg/L/d to about 500 mg/L/d under chemically defined conditions and as high as 700 mg/L/d with cell line B using plant derived hydrolysates. Peak cell densities reached greater than 20E6 vc/mL, and cell viabilities were maintained above 80% on day 18 without the use of antiapoptotic genes or temperature shift. A rapid compound screening method was developed to effectively test positive factors within 72 h. Peak volumetric oxygen uptake rates (OUR) more than tripled from the baseline condition. Oxygen demand continued to increase after maximum cell density was reached with a maximal OUR of 3.7 mmol/L/h. The new process format was scaled up and verified at 100 L pilot scale using reactor equipment of similar configuration as used at manufacturing scale. PMID:20945494

  16. Relation between cell death progression, reactive oxygen species production and mitochondrial membrane potential in fermenting Saccharomyces cerevisiae cells under heat-shock conditions.

    PubMed

    Pyatrikas, Darya V; Fedoseeva, Irina V; Varakina, Nina N; Rusaleva, Tatyana M; Stepanov, Alexei V; Fedyaeva, Anna V; Borovskii, Gennadii B; Rikhvanov, Eugene G

    2015-06-01

    Moderate heat shock increased reactive oxygen species (ROS) production that led to cell death in glucose-grown Saccharomyces cerevisiae cells. Conditions that disturb mitochondrial functions such as treatment by uncouplers and petite mutation were shown to inhibit ROS production and protects cell from thermal death. Hence, mitochondria are responsible for ROS production and play an active role in cell death. An increase in ROS production was accompanied by hyperpolarization of inner mitochondrial membrane. All agents suppressing hyperpolarization also suppressed heat-induced ROS production. It was supposed that generation of ROS under moderate heat shock in glucose-grown S. cerevisiae cells is driven by the mitochondrial membrane potential. PMID:25991811

  17. AN INVESTIGATION TO RESOLVE THE INTERACTION BETWEEN FUEL CELL, POWER CONDITIONING SYSTEM AND APPLICATION LOADS

    SciTech Connect

    Sudip K. Mazumder; Chuck McKintyre; Dan Herbison; Doug Nelson; Comas Haynes; Michael von Spakovsky; Joseph Hartvigsen; S. Elangovan

    2003-11-03

    Solid-Oxide Fuel Cell (SOFC) stacks respond quickly to changes in load and exhibit high part- and full-load efficiencies due to its rapid electrochemistry. However, this is not true for the thermal, mechanical, and chemical balance-of-plant subsystem (BOPS), where load-following time constants are, typically, several orders of magnitude higher. This dichotomy diminishes the reliability and performance of the electrode with increasing demand of load. Because these unwanted phenomena are not well understood, the manufacturers of SOFC use conservative schemes (such as, delayed load-following to compensate for slow BOPS response or expensive inductor filtering) to control stack responses to load variations. This limits the applicability of SOFC systems for load-varying stationary and transportation applications from a cost standpoint. Thus, a need exists for the synthesis of component- and system-level models of SOFC power-conditioning systems and the development of methodologies for investigating the system-interaction issues (which reduce the lifetime and efficiency of a SOFC) and optimizing the responses of each subsystem, leading to optimal designs of power-conditioning electronics and optimal control strategies, which mitigate the electrical-feedback effects. Equally important are ''multiresolution'' finite-element modeling and simulation studies, which can predict the impact of changes in system-level variables (e.g., current ripple and load-transients) on the local current densities, voltages, and temperature (these parameters are very difficult or cumbersome, if not impossible to obtain) within a SOFC cell. Towards that end, for phase I of this project, sponsored by the U.S. DOE (NETL), we investigate the interactions among fuel cell, power-conditioning system, and application loads and their effects on SOFC reliability (durability) and performance. A number of methodologies have been used in Phase I to develop the steady-state and transient nonlinear models of

  18. G-CSF and hypoxic conditioning improve the proliferation, neural differentiation and migration of canine bone marrow mesenchymal stem cells

    PubMed Central

    Yu, Jing; Liu, Xing-Long; Cheng, Qi-Guang; Lu, Shan-Shan; Xu, Xiao-Quan; Zu, Qing-Quan; Liu, Sheng

    2016-01-01

    Transplantation using bone marrow mesenchymal stem cells (BMSCs) is emerging as a potential regenerative therapy after ischemic attacks in the brain. However, it has been questioned because very few transplanted BMSCs are detected homing to and survived in the ischemic region. Improving the cell viability and migration ability under the complex ischemic condition seems very important. The aim of our study is to identify whether hypoxic condition and granulocyte colony-stimulating factor (G-CSF) could improve the cell survival and migration ability of transplanted cells or hypoxic condition could promote BMSC's neural differentiation. BMSCs were treated under either normoxic (21% O2) or hypoxic (1% O2) (HP-BMSCs) conditions, no significant apoptosis was observed in hypoxic precondition (HP) group, our study confirmed that HP improves BMSCs proliferation and migration. Meanwhile, neural induction of BMSCs under hypoxic condition exhibited significant superior results than normoxic condition. Additionally, the addition of G-CSF in HP-BMSCs culture media promoted HP efficiency on BMSCs. These findings shed light on novel efficient strategy on the prosperity of BMSCs. Hypoxic preconditioning and cultured with G-CSF may become a promising therapeutics for cell-based therapy in the treatments of ischemia stroke. PMID:27588100

  19. Immune reconstitution after haploidentical hematopoietic cell transplantation: impact of reduced intensity conditioning and CD3/CD19 depleted grafts.

    PubMed

    Federmann, B; Hägele, M; Pfeiffer, M; Wirths, S; Schumm, M; Faul, C; Vogel, W; Handgretinger, R; Kanz, L; Bethge, W A

    2011-01-01

    Haploidentical hematopoietic cell transplantation (HHCT) using CD34 selected grafts is complicated by slow engraftment and immune reconstitution. Engraftment and immune reconstitution might be improved using CD3/CD19-depleted grafts and reduced intensity conditioning (RIC). We report on 28 patients after HHCT with CD3/CD19-depleted grafts using RIC, which were prospectively evaluated for engraftment and immune reconstitution. Engraftment was rapid with full chimerism reached on day +15 after HHCT. T-cell reconstitution was delayed with a median of 205 CD3+ cells/μl, 70 CD3+CD4+ cells/μl and 66 CD3+ CD8+ cells/μl on day +100, respectively. A skewed T-cell receptor-Vβ repertoire with oligoclonal T-cell expansions to day +100 and normalization after day +200 was observed. B-cell reconstitution was slow with a median of 100 CD19+ CD20+ cells/μl on day +150. Natural killer (NK) cell engraftment was fast reaching normal values on day +20. An increased natural cytotoxicity receptor and NKG2A, but decreased NKG2D and KIR expressions were observed on NK cells until day +100. We observed a positive impact of donor lymphocyte infusions on immune reconstitution. In conclusion, after HHCT, using CD3/CD19-depleted grafts and RIC, T- and B-cell reconstitution is delayed, whereas NK-cell reconstitution occurs early and fast. PMID:20944677

  20. Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space.

    PubMed

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki; Kotake, Toshihisa; Yamazaki, Takashi; Higashibata, Akira; Ishioka, Noriaki; Shimazu, Toru; Fukui, Keiji; Osada, Ikuko; Kasahara, Haruo; Kamada, Motoshi

    2015-01-01

    Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions. PMID:26378793

  1. Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space

    PubMed Central

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki; Kotake, Toshihisa; Yamazaki, Takashi; Higashibata, Akira; Ishioka, Noriaki; Shimazu, Toru; Fukui, Keiji; Osada, Ikuko; Kasahara, Haruo; Kamada, Motoshi

    2015-01-01

    Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions. PMID:26378793

  2. Transcript profiles in cortical cells of maize primary root during ethylene-induced lysigenous aerenchyma formation under aerobic conditions

    PubMed Central

    Takahashi, Hirokazu; Yamauchi, Takaki; Rajhi, Imene; Nishizawa, Naoko K.; Nakazono, Mikio

    2015-01-01

    Background and Aims Internal aeration is important for plants to survive during periods of waterlogging, and the ability to form aerenchyma contributes by creating a continuous gas space between the shoots and the roots. Roots of maize (Zea mays) react to prolonged waterlogging by forming aerenchyma in root cortical cells by programmed cell death (PCD) in response to ethylene. The aim of this study was to understand the molecular mechanisms of ethylene-induced aerenchyma formation by identifying genes that are either up- or downregulated by ethylene treatment in maize root cortical cells. Methods Three-day-old maize seedlings were treated with ethylene for several hours under aerobic conditions. Cortical cells were isolated from the primary roots using laser microdissection (LM), and transcript profiles with and without ethylene treatment were compared by microarray. In addition, the effect on ethylene-induced aerenchyma formation of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, was examined in order to assess the involvement of reactive oxygen species (ROS). Key Results A total of 223 genes were identified whose transcript levels were significantly increased or decreased by ethylene treatment in root cortical cells under aerobic conditions. Subsequent tissue-specific quantitative reverse-transcription PCR analyses revealed that ethylene increased the transcript levels of genes related to ethylene signalling in all of the root tissues examined (stelar cells, cortical cells and outer cell layers), whereas it increased the transcript levels of genes related to cell wall modification and proteolysis specifically in the cortical cells. DPI treatment inhibited the ethylene-induced aerenchyma formation and suppressed expression of some cell wall modification-related genes. Conclusions Several genes related to cell wall modification and proteolysis are specifically up- or downregulated in cortical cells during lysigenous aerenchyma formation under aerobic

  3. Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

    PubMed Central

    Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

    2016-01-01

    Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384

  4. Monomeric adiponectin increases cell viability in porcine aortic endothelial cells cultured in normal and high glucose conditions: Data on kinases activation.

    PubMed

    Grossini, Elena; Farruggio, Serena; Qoqaiche, Fatima; Raina, Giulia; Camillo, Lara; Sigaudo, Lorenzo; Mary, David; Surico, Nicola; Surico, Daniela

    2016-09-01

    We found that monomeric adiponectin was able to increase cell viability in porcine aortic endothelial cells (PAE) cultured both in normal and high glucose condition. Moreover, in normal glucose condition monomeric adiponectin increased p38MAPK, Akt, ERK1/2 and eNOS phosphorylation in a dose- and time-dependent way. Also in high glucose condition monomeric adiponectin increased eNOS and above kinases phosphorylation with similar patterns but at lower extent. For interpretation of the data presented in this article, please see the research article "Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions" (Grossini et al., in press) [1]. PMID:27583345

  5. Lentiviral vectors for induction of self-differentiation and conditional ablation of dendritic cells.

    PubMed

    Pincha, M; Salguero, G; Wedekind, D; Sundarasetty, B S; Lin, A; Kasahara, N; Brugman, M H; Jirmo, A C; Modlich, U; Gutzmer, R; Büsche, G; Ganser, A; Stripecke, R

    2011-08-01

    Development of lentiviral vectors (LVs) in the field of immunotherapy and immune regeneration will strongly rely on biosafety of the gene transfer. We demonstrated previously the feasibility of ex vivo genetic programming of mouse bone marrow precursors with LVs encoding granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), which induced autonomous differentiation of long-lived dendritic cells (DCs), referred to as self-differentiated myeloid-derived antigen-presenting-cells reactive against tumors (SMART-DCs). Here, LV biosafety was enhanced by using a DC-restricted and physiological promoter, the major histocompatibility complex (MHC) II promoter, and including co-expression of the herpes simplex virus-thymidine kinase (sr39HSV-TK) conditional suicide gene. Tricistronic vectors co-expressing sr39HSV-TK, GM-CSF and IL-4 transcriptionally regulated by the MHCII promoter or the ubiquitous cytomegalovirus (CMV) promoter were compared. Despite the different gene transfer effects, such as the kinetics, levels of transgene expression and persistency of integrated vector copies, both vectors induced highly viable SMART-DCs, which persisted for at least 70 days in vivo and could be ablated with the pro-drug Ganciclovir (GCV). SMART-DCs co-expressing the tyrosine-related protein 2 melanoma antigen administered subcutaneously generated antigen-specific, anti-melanoma protective and therapeutic responses in the mouse B16 melanoma model. GCV administration after immunotherapy did not abrogate DC vaccination efficacy. This demonstrates proof-of-principle of genetically programmed DCs that can be ablated pharmacologically. PMID:21412283

  6. Optical-cell evidence for superheated ice under gas-hydrate-forming conditions

    USGS Publications Warehouse

    Stern, L.A.; Hogenboom, D.L.; Durham, W.B.; Kirby, S.H.; Chou, I.-Ming

    1998-01-01

    We previously reported indirect but compelling evidence that fine-grained H2O ice under elevated CH4 gas pressure can persist to temperatures well above its ordinary melting point while slowly reacting to form methane clathrate hydrate. This phenomenon has now been visually verified by duplicating these experiments in an optical cell while observing the very slow hydrate-forming process as the reactants were warmed from 250 to 290 K at methane pressures of 23 to 30 MPa. Limited hydrate growth occurred rapidly after initial exposure of the methane gas to the ice grains at temperatures well within the ice subsolidus region. No evidence for continued growth of the hydrate phase was observed until samples were warmed above the equilibrium H2O melting curve. With continued heating, no bulk melting of the ice grains or free liquid water was detected anywhere within the optical cell until hydrate dissociation conditions were reached (292 K at 30 MPa), even though full conversion of the ice grains to hydrate requires 6-8 h at temperatures approaching 290 K. In a separate experimental sequence, unreacted portions of H2O ice grains that had persisted to temperatures above their ordinary melting point were successfully induced to melt, without dissociating the coexisting hydrate in the sample tube, by reducing the pressure overstep of the equilibrium phase boundary and thereby reducing the rate of hydrate growth at the ice-hydrate interface. Results from similar tests using CO2 as the hydrate-forming species demonstrated that this superheating effect is not unique to the CH4-H2O system.

  7. Conditions for diffusion-limited and reaction-limited recombination in nanostructured solar cells

    SciTech Connect

    Ansari-Rad, Mehdi; Anta, Juan A.; Arzi, Ezatollah

    2014-04-07

    The performance of Dye-sensitized solar cells (DSC) and related devices made of nanostructured semiconductors relies on a good charge separation, which in turn is achieved by favoring charge transport against recombination. Although both processes occur at very different time scales, hence ensuring good charge separation, in certain cases the kinetics of transport and recombination can be connected, either in a direct or an indirect way. In this work, the connection between electron transport and recombination in nanostructured solar cells is studied both theoretically and by Monte Carlo simulation. Calculations using the Multiple-Trapping model and a realistic trap distribution for nanostructured TiO{sub 2} show that for attempt-to-jump frequencies higher than 10{sup 11}–10{sup 13} Hz, the system adopts a reaction limited (RL) regime, with a lifetime which is effectively independent from the speed of the electrons in the transport level. For frequencies lower than those, and depending on the concentration of recombination centers in the material, the system enters a diffusion-limited regime (DL), where the lifetime increases if the speed of free electrons decreases. In general, the conditions for RL or DL recombination depend critically on the time scale difference between recombination kinetics and free-electron transport. Hence, if the former is too rapid with respect to the latter, the system is in the DL regime and total thermalization of carriers is not possible. In the opposite situation, a RL regime arises. Numerical data available in the literature, and the behavior of the lifetime with respect to (1) density of recombination centers and (2) probability of recombination at a given center, suggest that a typical DSC in operation stays in the RL regime with complete thermalization, although a transition to the DL regime may occur for electrolytes or hole conductors where recombination is especially rapid or where there is a larger dispersion of energies of

  8. Second solid cancers after allogeneic hematopoietic cell transplantation using reduced intensity conditioning

    PubMed Central

    Ringdén, Olle; Brazauskas, Ruta; Wang, Zhiwei; Ahmed, Ibrahim; Atsuta, Yoshiko; Buchbinder, David; Burns, Linda J.; Cahn, Jean-Yves; Duncan, Christine; Hale, Gregory A.; Halter, Joerg; Hayashi, Robert J.; Hsu, Jack W.; Jacobsohn, David A.; Kamble, Rammurti T.; Kamani, Naynesh R.; Kasow, Kimberly A.; Khera, Nandita; Lazarus, Hillard M.; Loren, Alison W.; Marks, David I.; Myers, Kasiani C.; Ramanathan, Muthalagu; Saber, Wael; Savani, Bipin N.; Schouten, Harry C.; Socie, Gérard; Sorror, Mohamed L.; Steinberg, Amir; Popat, Uday; Wingard, John R.; Mattsson, Jonas; Majhail, Navneet S.

    2014-01-01

    We examined risk of second solid cancers after allogeneic hematopoietic cell transplantation (AHCT) using reduced intensity/non-myeloablative conditioning (RIC/NMC). RIC/NMC recipients with leukemia/myelodysplastic syndrome (MDS) (n=2833) and lymphoma (n=1436) between 1995–2006 were included. In addition, RIC/NMC recipients 40–60 years of age (n=2138) were compared with patients of the same age receiving myeloablative conditioning (MAC, n=6428). The cumulative incidence of solid cancers was 3.35% at 10-years. There was no increase in overall cancer risk compared to the general population (standardized incidence ratio [SIR] 0.99, P=1.00 for leukemia/MDS and 0.92, P=0.75 for lymphoma). However, risks were significantly increased in leukemia/MDS patients for cancers of lip (SIR 14.28), tonsil (SIR 8.66), oropharynx (SIR 46.70), bone (SIR 23.53), soft tissue (SIR 12.92), and vulva (SIR 18.55) and skin melanoma (SIR 3.04). Lymphoma patients had significantly higher risks of oropharyngeal cancer (SIR 67.35) and skin melanoma (SIR 3.52). Among RIC/NMC recipients, age >50 years was the only independent risk factor for solid cancers (hazard ratio [HR] 3.02, P<0.001). Among patients age 40–60 years, when adjusted for other factors, there was no difference in cancer risks between RIC/NMC and MAC in leukemia/MDS patients (HR 0.98, P=0.905). In lymphoma patients, risks were lower after RIC/NMC (HR 0.51, P=0.047). In conclusion, the overall risks of second solid cancers in RIC/NMC recipients are similar to the general population, although there is an increased risk of cancer at some sites. Studies with longer follow-up are needed to realize the complete risks of solid cancers after RIC/NMC AHCT. PMID:25042734

  9. In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75{sup +} stem cells with dental follicle cell conditioned medium

    SciTech Connect

    Wen, Xiujie; Liu, Luchuan; Deng, Manjing; Liu, Rui; Zhang, Li; Nie, Xin

    2015-09-10

    Cranial neural crest-derived cells (CNCCs) play important role in epithelial–mesenchymal interactions during tooth morphogenesis. However, the heterogeneity of CNCCs and their tendency to spontaneously differentiate along smooth muscle or osteoblast lineages in vitro limit further understanding of their biological properties. We studied the differentiation properties of isolated rat embryonic postmigratory CNCCs, expressing p75 neurotrophin receptor (p75NTR). These p75NTR positive (p75{sup +}) CNCCs, isolated using fluorescence activated cell sorter, exhibited fibroblast-like morphology and characteristics of mesenchymal stem cells. Incubation of p75{sup +} CNCCs in dental follicle cell conditioned medium (DFCCM) combined with dentin non-collagenous proteins (dNCPs), altered their morphological features to cementoblast-like appearance. These cells also showed low proliferative activity, high ALP activity and significantly increased calcified nodule formation. Markers related to mineralization or specific to cementoblast lineage were highly expressed in dNCPs/DFCCM-treated p75{sup +} cells, suggesting their differentiation along cementoblast-like lineage. p75{sup +} stem cells selected from postmigratory CNCCs represent a pure stem cell population and could be used as a stem cell model for in vitro studies due to their intrinsic ability to differentiate to neuronal cells and transform from neuroectoderm to ectomesenchyme. They can provide a potential stem cell resource for tooth engineering studies and help to further investigate mechanisms of epithelial–mesenchymal interactions in tooth morphogenesis. - Highlights: • Cranial neural crest-derived cells (CNCCs) take part in tooth morphogenesis. • positive (p75{sup +}) CNCCs are fibroblast-like and resemble mesenchymal stem cells. • p75{sup +} CNCCs in dental follicle cell medium (DFCCM/dNCP) appear like cementoblasts. • DFCCM/dNCP-treated p75{sup +} cells express cementoblast specific mineralization

  10. B Cells Are Critical to T-cell-Mediated Antitumor Immunity Induced by a Combined Immune-Stimulatory/Conditionally Cytotoxic Therapy for Glioblastoma12

    PubMed Central

    Candolfi, Marianela; Curtin, James F; Yagiz, Kader; Assi, Hikmat; Wibowo, Mia K; Alzadeh, Gabrielle E; Foulad, David; Muhammad, AKM G; Salehi, Sofia; Keech, Naomi; Puntel, Mariana; Liu, Chunyan; Sanderson, Nicholas R; Kroeger, Kurt M; Dunn, Robert; Martins, Gislaine; Lowenstein, Pedro R; Castro, Maria G

    2011-01-01

    We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6-/- mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6-/- mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression. PMID:22028620

  11. Dynamics of Metal Partitioning at the Cell-Solution Interface: Implications for Toxicity Assessment under Growth-Inhibiting Conditions.

    PubMed

    Duval, Jérôme F L; Paquet, Nathalie; Lavoie, Michel; Fortin, Claude

    2015-06-01

    Metal toxicity toward microorganisms is usually evaluated by determining growth inhibition. To achieve a mechanistic interpretation of such toxic effects, the intricate coupling between cell growth kinetics and metal partitioning dynamics at the cell-solution interface over time must be considered on a quantitative level. A formalism is elaborated to evaluate cell-surface-bound, internalized, and extracellular metal fractions in the limit where metal uptake kinetics is controlled by internalization under noncomplexing medium conditions. Cell growth kinetics is tackled using the continuous logistic equation modified to include growth inhibition by metal accumulation to intracellular or cell surface sites. The theory further includes metal-proton competition for adsorption at cell-surface binding sites, as well as possible variation of cell size during exposure to metal ions. The formalism elucidates the dramatic impacts of initial cell concentration on metal bioavailability and toxicity over time, in agreement with reported algae bioassays. It further highlights that appropriate definition of toxicity endpoints requires careful inspection of the ratio between exposure time scale and time scale of metal depletion from bulk solution. The latter depends on metal internalization-excretion rate constants, microorganism growth, and the extent of metal adsorption on nonspecific, transporter, and growth inhibitory sites. As an application of the theory, Cd toxicity in the algae Pseudokirchneriella subcapitata is interpreted from constrained modeling of cell growth kinetics and of interfacial Cd-partitioning dynamics measured under various exposure conditions. PMID:25945520

  12. Stability of housekeeping genes in human intervertebral disc, endplate and articular cartilage cells in multiple conditions for reliable transcriptional analysis.

    PubMed

    Lopa, S; Ceriani, C; Cecchinato, R; Zagra, L; Moretti, M; Colombini, A

    2016-01-01

    Quantitative gene expression analysis is widely used to evaluate the expression of specific tissue markers. To obtain reliable data it is essential to select stable housekeeping genes whose expression is not influenced by the anatomical origin of cells or by the culture conditions. No studies have evaluated housekeeping gene stability in intervertebral disc (IVD) cells and only few studies using cartilaginous endplate (CEP) and articular cartilage (AC) cells are present in the literature. We analysed the stability of four candidate housekeeping genes (GAPDH, TBP, YWHAZ and RPL13A) in human cells isolated from nucleus pulposus (NP) and annulus fibrosus (AF), CEP and AC. Cell isolation, expansion, cryoconservation, and differentiation in 3D pellets were tested. GeNorm, NormFinder, BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability. In each cell population, TBP alone or combined with YWHAZ was identified as the best normaliser in both monolayer and 3D pellets. GAPDH was the best performer only for AC cells in monolayer. In most culture conditions considering groups of two or more cell types, TBP was the most stable and YWHAZ was the second choice. GAPDH was the best performer only in 3D pellets with factors for AC and AF combined with CEP cells. RPL13A was the most stable only for AF with CEP cells at isolation. Our findings will be useful to properly design the experimental set-up of studies involving IVD, CEP or AC cells in different culture conditions, in order to obtain accurate and high quality data from quantitative gene expression analysis. PMID:27232666

  13. The Autophagoproteasome a Novel Cell Clearing Organelle in Baseline and Stimulated Conditions.

    PubMed

    Lenzi, Paola; Lazzeri, Gloria; Biagioni, Francesca; Busceti, Carla L; Gambardella, Stefano; Salvetti, Alessandra; Fornai, Francesco

    2016-01-01

    Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be

  14. The Autophagoproteasome a Novel Cell Clearing Organelle in Baseline and Stimulated Conditions

    PubMed Central

    Lenzi, Paola; Lazzeri, Gloria; Biagioni, Francesca; Busceti, Carla L.; Gambardella, Stefano; Salvetti, Alessandra; Fornai, Francesco

    2016-01-01

    Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be

  15. Conditions for the generation of cytotoxic CD4(+) Th cells that enhance CD8(+) CTL-mediated tumor regression.

    PubMed

    Li, Kunyu; Baird, Margaret; Yang, Jianping; Jackson, Chris; Ronchese, Franca; Young, Sarah

    2016-08-01

    Adoptive cell therapies (ACTs) using tumor-reactive T cells have shown clinical benefit and potential for cancer treatment. While the majority of the current ACT are focused on using CD8(+) cytotoxic T lymphocytes (CTL), others have shown that the presence of tumor-reactive CD4(+) T helper (Th) cells can greatly enhance the anti-tumor activity of CD8(+) CTL. However, difficulties in obtaining adequate numbers of CD4(+) Th cells through in vitro expansion can limit the application of CD4 Th cells in ACT. This study aims to optimize the culture conditions for mouse CD4 T cells to provide basic information for animal studies of ACT using CD4 T cells. Taking advantage of the antigen-specificity of CD4(+) Th cells from OT-II transgenic mice, we examined different methodologies for generating antigen-specific CD4(+) Th1 cells in vitro. We found that cells grown in complete advanced-DMEM/F12 medium supplemented with low-dose IL-2 and IL-7 induced substantial cell expansion. These Th cells were Th1-like, as they expressed multiple Th1-cytokines and exhibited antigen-specific cytotoxicity. In addition co-transfer of these CD4(+) Th1-like cells with CD8(+) CTL significantly enhanced tumor regression, leading to complete cure in 80% of mice bearing established B16-OVA. These observations indicate that the CD4(+) Th1-like cells generated using the method we optimized are functionally active to eliminate their target cells, and can also assist CD8(+) CTL to enhance tumor regression. The findings of this study provide valuable data for further research into in vitro expansion of CD4(+) Th1-like cells, with potential applications to cancer treatment involving ACT. PMID:27588200

  16. Conditions for the generation of cytotoxic CD4+ Th cells that enhance CD8+ CTL-mediated tumor regression

    PubMed Central

    Li, Kunyu; Baird, Margaret; Yang, Jianping; Jackson, Chris; Ronchese, Franca; Young, Sarah

    2016-01-01

    Adoptive cell therapies (ACTs) using tumor-reactive T cells have shown clinical benefit and potential for cancer treatment. While the majority of the current ACT are focused on using CD8+ cytotoxic T lymphocytes (CTL), others have shown that the presence of tumor-reactive CD4+ T helper (Th) cells can greatly enhance the anti-tumor activity of CD8+ CTL. However, difficulties in obtaining adequate numbers of CD4+ Th cells through in vitro expansion can limit the application of CD4 Th cells in ACT. This study aims to optimize the culture conditions for mouse CD4 T cells to provide basic information for animal studies of ACT using CD4 T cells. Taking advantage of the antigen-specificity of CD4+ Th cells from OT-II transgenic mice, we examined different methodologies for generating antigen-specific CD4+ Th1 cells in vitro. We found that cells grown in complete advanced-DMEM/F12 medium supplemented with low-dose IL-2 and IL-7 induced substantial cell expansion. These Th cells were Th1-like, as they expressed multiple Th1-cytokines and exhibited antigen-specific cytotoxicity. In addition co-transfer of these CD4+ Th1-like cells with CD8+ CTL significantly enhanced tumor regression, leading to complete cure in 80% of mice bearing established B16-OVA. These observations indicate that the CD4+ Th1-like cells generated using the method we optimized are functionally active to eliminate their target cells, and can also assist CD8+ CTL to enhance tumor regression. The findings of this study provide valuable data for further research into in vitro expansion of CD4+ Th1-like cells, with potential applications to cancer treatment involving ACT. PMID:27588200

  17. Effect of Heat-Inactivated Clostridium sporogenes and Its Conditioned Media on 3-Dimensional Colorectal Cancer Cell Models.

    PubMed

    Bhave, Madhura Satish; Hassanbhai, Ammar Mansoor; Anand, Padmaja; Luo, Kathy Qian; Teoh, Swee Hin

    2015-01-01

    Traditional cancer treatments, such as chemotherapy and radiation therapy continue to have limited efficacy due to tumor hypoxia. While bacterial cancer therapy has the potential to overcome this problem, it comes with the risk of toxicity and infection. To circumvent these issues, this paper investigates the anti-tumor effects of non-viable bacterial derivatives of Clostridium sporogenes. These non-viable derivatives are heat-inactivated C. sporogenes bacteria (IB) and the secreted bacterial proteins in culture media, known as conditioned media (CM). In this project, the effects of IB and CM on CT26 and HCT116 colorectal cancer cells were examined on a 2-Dimensional (2D) and 3-Dimensional (3D) platform. IB significantly inhibited cell proliferation of CT26 to 6.3% of the control in 72 hours for the 2D monolayer culture. In the 3D spheroid culture, cell proliferation of HCT116 spheroids notably dropped to 26.2%. Similarly the CM also remarkably reduced the cell-proliferation of the CT26 cells to 2.4% and 20% in the 2D and 3D models, respectively. Interestingly the effect of boiled conditioned media (BCM) on the cells in the 3D model was less inhibitory than that of CM. Thus, the inhibitive effect of inactivated C. sporogenes and its conditioned media on colorectal cancer cells is established. PMID:26507312

  18. Patient-specific modeling and analysis of dynamic behavior of individual sickle red blood cells under hypoxic conditions

    NASA Astrophysics Data System (ADS)

    Li, Xuejin; Du, E.; Li, Zhen; Tang, Yu-Hang; Lu, Lu; Dao, Ming; Karniadakis, George

    2015-11-01

    Sickle cell anemia is an inherited blood disorder exhibiting heterogeneous morphology and abnormal dynamics under hypoxic conditions. We developed a time-dependent cell model that is able to simulate the dynamic processes of repeated sickling and unsickling of red blood cells (RBCs) under physiological conditions. By using the kinetic cell model with parameters derived from patient-specific data, we present a mesoscopic computational study of the dynamic behavior of individual sickle RBCs flowing in a microfluidic channel with multiple microgates. We investigate how individual sickle RBCs behave differently from healthy ones in channel flow, and analyze the alteration of cellular behavior and response to single-cell capillary obstruction induced by cell rheologic rigidification and morphological change due to cell sickling under hypoxic conditions. We also simulate the flow dynamics of sickle RBCs treated with hydroxyurea (HU) and quantify the relative enhancement of hemodynamic performance of HU. This work was supported by the National Institutes of Health (NIH) Grant U01HL114476.

  19. CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions.

    PubMed

    McVey, Duncan; Aronov, Michael; Rizzi, Giovanni; Cowan, Alexis; Scott, Charo; Megill, John; Russell, Reb; Tirosh, Boaz

    2016-09-01

    The kinase mTOR operates in two cellular complexes, mTORC1 and mTORC2. mTORC1 adjusts metabolic activity according to external growth conditions and nutrients availability. When conditions are prosperous, mTOR facilitates protein and lipid biosyntheses and inhibits autophagy, while under metabolic constraints, however, its attenuation induces a catabolic program, energy preservation and autophagy. CHO is a key cell line for manufacturing of biologics owing to its remarkable ability to grow to high densities and maintain protein production and secretion for extended times. While high mTOR activity has been associated with high productivity in CHO cells, its inhibition by rapamycin has also been documented to augment productivity via promotion of viability. Here using CRISPR/Cas9 editing we engineered CHO cells to enforce high mTORC1 activity by knocking-out TSC2, a major mTOR inhibitory protein, or PTEN, a phosphatase that attenuates the PI3K/AKT/mTOR pathway. Only TSC2-deleted cells exhibited a constitutive activation of mTORC1 under fed batch conditions. Cells grew larger in size, synthesized more proteins and displayed an over twofold elevation in their specific productivity. While peak viable cell density was compromised, overall titers increased to an extent dependent upon the parental clone. Our data underscore manipulation of TSC as a strategy to improve performance of CHO cell in bioreactors. Biotechnol. Bioeng. 2016;113: 1942-1952. © 2016 Wiley Periodicals, Inc. PMID:26888596

  20. 211Astatine-Conjugated Monoclonal CD45 Antibody-Based Nonmyeloablative Conditioning for Stem Cell Gene Therapy

    PubMed Central

    Burtner, Christopher R.; Chandrasekaran, Devikha; Santos, Erlinda B.; Beard, Brian C.; Adair, Jennifer E.; Hamlin, Donald K.; Wilbur, D. Scott; Sandmaier, Brenda M.

    2015-01-01

    Abstract Most hematopoietic stem cell gene therapy studies require host conditioning to allow for efficient engraftment of gene-modified cells. Conditioning regimens with lower treatment-related toxicities are especially relevant for the treatment of nonmalignant blood disorders, such as hemoglobinopathies and immunodeficiencies, and for patients who are otherwise ineligible for conventional high-dose conditioning. Radioimmunotherapy, which employs an α- or a β-emitting radionuclide conjugated to a targeting antibody, is effective for delivering cytotoxic doses of radiation to a cell type of interest while minimizing off-target toxicity. Here, we demonstrate the feasibility of using a nonmyeloablative dose of a monoclonal anti-CD45 antibody conjugated to the α-emitter Astatine-211 (211At) to promote engraftment of an autologous gene-modified stem cell graft in the canine model. The doses used provided myelosuppression with rapid autologous recovery and minimal off-target toxicity. Engraftment levels were low in all dogs and reflected the low numbers of gene-modified cells infused. Our data suggest that a cell dose exceeding 1×106 cells/kg be used with nonmyeloablative doses of 211At-anti-CD45 monoclonal antibodies for sustained engraftment in the dog model. PMID:25919226

  1. (211)Astatine-Conjugated Monoclonal CD45 Antibody-Based Nonmyeloablative Conditioning for Stem Cell Gene Therapy.

    PubMed

    Burtner, Christopher R; Chandrasekaran, Devikha; Santos, Erlinda B; Beard, Brian C; Adair, Jennifer E; Hamlin, Donald K; Wilbur, D Scott; Sandmaier, Brenda M; Kiem, Hans-Peter

    2015-06-01

    Most hematopoietic stem cell gene therapy studies require host conditioning to allow for efficient engraftment of gene-modified cells. Conditioning regimens with lower treatment-related toxicities are especially relevant for the treatment of nonmalignant blood disorders, such as hemoglobinopathies and immunodeficiencies, and for patients who are otherwise ineligible for conventional high-dose conditioning. Radioimmunotherapy, which employs an α- or a β-emitting radionuclide conjugated to a targeting antibody, is effective for delivering cytotoxic doses of radiation to a cell type of interest while minimizing off-target toxicity. Here, we demonstrate the feasibility of using a nonmyeloablative dose of a monoclonal anti-CD45 antibody conjugated to the α-emitter Astatine-211 ((211)At) to promote engraftment of an autologous gene-modified stem cell graft in the canine model. The doses used provided myelosuppression with rapid autologous recovery and minimal off-target toxicity. Engraftment levels were low in all dogs and reflected the low numbers of gene-modified cells infused. Our data suggest that a cell dose exceeding 1×10(6) cells/kg be used with nonmyeloablative doses of (211)At-anti-CD45 monoclonal antibodies for sustained engraftment in the dog model. PMID:25919226

  2. Arg tyrosine kinase modulates TGF-β1 production in human renal tubular cells under high-glucose conditions.

    PubMed

    Torsello, Barbara; Bianchi, Cristina; Meregalli, Chiara; Di Stefano, Vitalba; Invernizzi, Lara; De Marco, Sofia; Bovo, Giorgio; Brivio, Rinaldo; Strada, Guido; Bombelli, Silvia; Perego, Roberto A

    2016-08-01

    Renal tubular cells are involved in the tubular interstitial fibrosis observed in diabetic nephropathy. It is debated whether epithelial-mesenchymal transition (EMT) affects tubular cells, which under high-glucose conditions overproduce transforming growth factor-β (TGF-β), a fibrogenic cytokine involved in interstitial fibrosis development. Our study investigated the involvement of non-receptor tyrosine kinase Arg (also called Abl2) in TGF-β production. Human primary tubular cell cultures exposed to high-glucose conditions were used. These cells showed an elongated morphology, stress fibers and vimentin increment but maintained most of the epithelial marker expression and distribution. In these cells exposed to high glucose, which overexpressed and secreted active TGF-β1, Arg protein and activity was downregulated. A further TGF-β1 increase was induced by Arg silencing with siRNA, as with the Arg tyrosine kinase inhibitor Imatinib. In the cells exposed to high glucose, reactive oxygen species (ROS)-dependent Arg kinase downregulation induced both RhoA activation, through p190RhoGAPA (also known as ARHGAP35) modulation, and proteasome activity inhibition. These data evidence a new specific involvement of Arg kinase into the regulation of TGF-β1 expression in tubular cells under high-glucose conditions and provide cues for new translational approaches in diabetic nephropathy. PMID:27298228

  3. Effect of Heat-Inactivated Clostridium sporogenes and Its Conditioned Media on 3-Dimensional Colorectal Cancer Cell Models

    PubMed Central

    Bhave, Madhura Satish; Hassanbhai, Ammar Mansoor; Anand, Padmaja; Luo, Kathy Qian; Teoh, Swee Hin

    2015-01-01

    Traditional cancer treatments, such as chemotherapy and radiation therapy continue to have limited efficacy due to tumor hypoxia. While bacterial cancer therapy has the potential to overcome this problem, it comes with the risk of toxicity and infection. To circumvent these issues, this paper investigates the anti-tumor effects of non-viable bacterial derivatives of Clostridium sporogenes. These non-viable derivatives are heat-inactivated C. sporogenes bacteria (IB) and the secreted bacterial proteins in culture media, known as conditioned media (CM). In this project, the effects of IB and CM on CT26 and HCT116 colorectal cancer cells were examined on a 2-Dimensional (2D) and 3-Dimensional (3D) platform. IB significantly inhibited cell proliferation of CT26 to 6.3% of the control in 72 hours for the 2D monolayer culture. In the 3D spheroid culture, cell proliferation of HCT116 spheroids notably dropped to 26.2%. Similarly the CM also remarkably reduced the cell-proliferation of the CT26 cells to 2.4% and 20% in the 2D and 3D models, respectively. Interestingly the effect of boiled conditioned media (BCM) on the cells in the 3D model was less inhibitory than that of CM. Thus, the inhibitive effect of inactivated C. sporogenes and its conditioned media on colorectal cancer cells is established. PMID:26507312

  4. A Reduced-Intensity Conditioning Regimen for Patients with Dyskeratosis Congenita Undergoing Hematopoietic Stem Cell Transplantation.

    PubMed

    Nelson, Adam S; Marsh, Rebecca A; Myers, Kasiani C; Davies, Stella M; Jodele, Sonata; O'Brien, Tracey A; Mehta, Parinda A

    2016-05-01

    Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative option for progressive marrow failure, myelodysplastic syndrome, or leukemia associated with dyskeratosis congenita (DC). HSCT for DC is limited by a high incidence of treatment-related mortality, thought to be related to underlying chromosomal instability and sensitivity to chemotherapy and radiation. We report our experience in 7 patients with DC who underwent allogeneic transplantation using a reduced-intensity conditioning (RIC) preparative regimen that contained chemotherapy only (no radiation). This RIC regimen, designed specifically for patients with DC, contained alemtuzumab, fludarabine, and melphalan (with melphalan at 50% reduced dosing), with the goal of decreasing toxicity and improving outcome. All 7 patients engrafted, with none developing mixed chimerism or rejection. Two patients experienced acute graft-versus-host disease (GVHD) and 1 went on to develop limited chronic GVHD of the skin. Five patients remain alive and well at a median follow-up of 44 months (range, 14 to 57 months). We conclude that a radiation-free RIC regimen results in durable engraftment, acceptable toxicity, and improved overall survival in patients with DC undergoing allogeneic HSCT. PMID:26845033

  5. Conditioned Medium From Human Amniotic Mesenchymal Stromal Cells Limits Infarct Size and Enhances Angiogenesis

    PubMed Central

    Danieli, Patrizia; Malpasso, Giuseppe; Ciuffreda, Maria Chiara; Cervio, Elisabetta; Calvillo, Laura; Copes, Francesco; Pisano, Federica; Mura, Manuela; Kleijn, Lennaert; de Boer, Rudolf A.; Viarengo, Gianluca; Rosti, Vittorio; Spinillo, Arsenio; Roccio, Marianna

    2015-01-01

    The paracrine properties of human amniotic membrane-derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiogenesis. Moreover, we aimed to identify the putative active paracrine mediators. hAMCs were isolated, expanded, and characterized. In vitro, conditioned medium from hAMC (hAMC-CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC-CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, and strongly promoted capillary formation at the infarct border zone. Gene array analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factors were detected in hAMC-CM by cytokine array. Our results strongly support the concept that the administration of hAMC-CM favors the repair process after acute myocardial infarction. PMID:25824141

  6. Conditioned medium from human amniotic mesenchymal stromal cells limits infarct size and enhances angiogenesis.

    PubMed

    Danieli, Patrizia; Malpasso, Giuseppe; Ciuffreda, Maria Chiara; Cervio, Elisabetta; Calvillo, Laura; Copes, Francesco; Pisano, Federica; Mura, Manuela; Kleijn, Lennaert; de Boer, Rudolf A; Viarengo, Gianluca; Rosti, Vittorio; Spinillo, Arsenio; Roccio, Marianna; Gnecchi, Massimiliano

    2015-05-01

    The paracrine properties of human amniotic membrane-derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiogenesis. Moreover, we aimed to identify the putative active paracrine mediators. hAMCs were isolated, expanded, and characterized. In vitro, conditioned medium from hAMC (hAMC-CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC-CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, and strongly promoted capillary formation at the infarct border zone. Gene array analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factors were detected in hAMC-CM by cytokine array. Our results strongly support the concept that the administration of hAMC-CM favors the repair process after acute myocardial infarction. PMID:25824141

  7. Increased power generation from primary sludge by a submersible microbial fuel cell and optimum operational conditions.

    PubMed

    Vologni, Valentina; Kakarla, Ramesh; Angelidaki, Irini; Min, Booki

    2013-05-01

    Microbial fuel cells (MFCs) have received attention as a promising renewable energy technology for waste treatment and energy recovery. We tested a submersible MFC with an innovative design capable of generating a stable voltage of 0.250 ± 0.008 V (with a fixed 470 Ω resistor) directly from primary sludge. In a polarization test, the maximum power density was 0.18 W/m(2) at a current density of 0.8 A/m(2) with an external resistor of 300 Ω. The anodic solution of the primary sludge needs to be adjusted to a pH 7 for high power generation. The modified primary sludge with an added phosphate buffer prolonged the current generation and increased the power density by 7 and 1.5 times, respectively, in comparison with raw primary sludge. These findings suggest that energy recovery from primary sludge can be maximized using an advanced MFC system with optimum conditions. PMID:23420478

  8. Antisenescence effect of mouse embryonic stem cell conditioned medium through a PDGF/FGF pathway.

    PubMed

    Bae, Yun-Ui; Choi, Joon-Hyuk; Nagy, Andras; Sung, Hoon-Ki; Kim, Jae-Ryong

    2016-03-01

    Cellular senescence, an irreversible state of growth arrest, underlies organismal aging and age-related diseases. Recent evidence suggests that aging intervention based on inhibition of cellular senescence might be a promising strategy for treatment of aging and age-related diseases. Embryonic stem cells (ESCs) and ESC conditioned medium (CM) have been suggested as a desirable source for regenerative medicine. However, effects of ESC-CM on cellular senescence remain to be determined. We found that treatment of senescent human dermal fibroblasts with CM from mouse ESCs (mESCs) decreases senescence phenotypes. We found that platelet-derived growth factor BB in mESC-CM plays a critical role in antisenescence effect of mESC-CM through up-regulation of fibroblast growth factor 2. We confirmed that mESC-CM treatment accelerates the wound-healing process by down-regulating senescence-associated p53 expression in in vivo models. Taken together, our results suggest that mESC-CM has the ability to suppress cellular senescence and maintain proliferative capacity. Therefore, this strategy might emerge as a novel therapeutic strategy for aging and age-related diseases. PMID:26675707

  9. Three-dimensional spherical spatial boundary conditions differentially regulate osteogenic differentiation of mesenchymal stromal cells

    PubMed Central

    Lo, Yin-Ping; Liu, Yi-Shiuan; Rimando, Marilyn G.; Ho, Jennifer Hui-Chun; Lin, Keng-hui; Lee, Oscar K.

    2016-01-01

    The spatial boundary condition (SBC) arising from the surrounding microenvironment imposes specific geometry and spatial constraints that affect organogenesis and tissue homeostasis. Mesenchymal stromal cells (MSCs) sensitively respond to alterations of mechanical cues generated from the SBC. However, mechanical cues provided by a three-dimensional (3D) environment are deprived in a reductionist 2D culture system. This study investigates how SBC affects osteogenic differentiation of MSCs using 3D scaffolds with monodispersed pores and homogenous spherical geometries. MSCs cultured under SBCs with diameters of 100 and 150 μm possessed the greatest capability of osteogenic differentiation. This phenomenon was strongly correlated with MSC morphology, organization of actin cytoskeleton, and distribution of focal adhesion involving α2 and α5 integrins. Further silencing either α2 or α5 integrin significantly reduced the above mentioned mechanosensitivity, indicating that the α2 and α5 integrins as mechano-sensitive molecules mediate MSCs’ ability to provide enhanced osteogenic differentiation in response to different spherical SBCs. Taken together, the findings provide new insights regarding how MSCs respond to mechanical cues from the surrounding microenvironment in a spherical SBC, and such biophysical stimuli should be taken into consideration in tissue engineering and regenerative medicine in conjunction with biochemical cues. PMID:26884253

  10. SP1 and USF differentially regulate ADAMTS1 gene expression under normoxic and hypoxic conditions in hepatoma cells.

    PubMed

    Turkoglu, Sumeyye Aydogan; Kockar, Feray

    2016-01-01

    ADAM metallopeptidase with thrombospondin type I motif, 1 (ADAMTS1) that has both antiangiogenic and aggrecanase activity was dysregulated in many pathophysiologic circumstances. However, there is limited information available on the transcriptional regulation of ADAMTS1 gene. Therefore, this study mainly aimed to identify regulatory regions important for the regulation of ADAMTS1 gene under normoxic and hypoxic conditions in human hepatoma cells (HEP3B). Cultured HEP3B cells were exposed to normal oxygen condition, and Cobalt chloride (CoCl2) induced the hypoxic condition, which is an HIF-1 inducer. The cocl2-induced hypoxic condition led to the induced ADAMTS1 mRNA and protein expression in Hepatoma cells. Differential regulation of SP1 and USF transcription factors on ADAMTS1 gene expression was determined by transcriptional activity, mRNA and protein level of ADAMTS1 gene. Ectopic expression of SP1 and USF transcription factors resulted in the decrease in ADAMTS1 transcriptional activity of all promoter constructs consistent with mRNA and protein level in normoxic condition. However, overexpression of SP1 and USF led to the increase of ADAMTS1 gene expressions at mRNA and protein level in hypoxic condition. On the other hand, C/EBPα transcription factor didn't show any statistically significant effect on ADAMTS1 gene expression at mRNA, protein and transcriptional level under normoxic and hypoxic condition. PMID:26299656

  11. Purified human pancreatic duct cell culture conditions defined by serum-free high-content growth factor screening.

    PubMed

    Hoesli, Corinne A; Johnson, James D; Piret, James M

    2012-01-01

    The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells. PMID:22442738

  12. Impact of Scaffold Micro and Macro Architecture on Schwann Cell Proliferation under Dynamic Conditions in a Rotating Wall Vessel Bioreactor

    PubMed Central

    Valmikinathan, Chandra M.; Hoffman, John; Yu, Xiaojun

    2011-01-01

    Over the last decade tissue engineering has emerged as a powerful alternative to regenerate lost tissues owing to trauma or tumor. Evidence shows that Schwann cell containing scaffolds have improved performance in vivo as compared to scaffolds that depend on cellularization post implantation. However, owing to limited supply of cells from the patients themselves, several approaches have been taken to enhance cell proliferation rates to produce complete and uniform cellularization of scaffolds. The most common approach is the application of a bioreactor to enhance cell proliferation rate and therefore reduce the time needed to obtain sufficiently significant number of glial cells, prior to implantation. In this study, we show the application of a rotating wall bioreactor system for studying Schwann cell proliferation on nanofibrous spiral shaped scaffolds, prepared by solvent casting and salt leaching techniques. The scaffolds were fabricated from polycaprolactone (PCL), which has ideal mechanical properties and upon degradation does not produce acidic byproducts. The spiral scaffolds were coated with aligned or random nanofibers, produced by electrospinning, to provide a substrate that mimics the native extracellular matrix and the essential contact guidance cues. At the 4 day time point, an enhanced rate of cell proliferation was observed on the open structured nanofibrous spiral scaffolds in a rotating wall bioreactor, as compared to static culture conditions. However, the cell proliferation rate on the other contemporary scaffolds architectures such as the tubular and cylindrical scaffolds show reduced cell proliferation in the bioreactor as compared to static conditions, at the same time point. Moreover, the rotating wall bioreactor does not alter the orientation or the phenotype of the Schwann cells on the aligned nanofiber containing scaffolds, wherein, the cells remain aligned along the length of the scaffolds. Therefore, these open structured spiral

  13. Conditioning the cochlea to facilitate survival and integration of exogenous cells into the auditory epithelium.

    PubMed

    Park, Yong-Ho; Wilson, Kevin F; Ueda, Yoshihisa; Tung Wong, Hiu; Beyer, Lisa A; Swiderski, Donald L; Dolan, David F; Raphael, Yehoash

    2014-04-01

    The mammalian auditory epithelium (AE) cannot replace supporting cells and hair cells once they are lost. Therefore, sensorineural hearing loss associated with missing cells is permanent. This inability to regenerate critical cell types makes the AE a potential target for cell replacement therapies such as stem cell transplantation. Inserting stem cells into the AE of deaf ears is a complicated task due to the hostile, high potassium environment of the scala media in the cochlea, and the robust junctional complexes between cells in the AE that resist stem cell integration. Here, we evaluate whether temporarily reducing potassium levels in the scala media and disrupting the junctions in the AE make the cochlear environment more receptive and facilitate survival and integration of transplanted cells. We used sodium caprate to transiently disrupt the AE junctions, replaced endolymph with perilymph, and blocked stria vascularis pumps with furosemide. We determined that these three steps facilitated survival of HeLa cells in the scala media for at least 7 days and that some of the implanted cells formed a junctional contact with native AE cells. The data suggest that manipulation of the cochlear environment facilitates survival and integration of exogenously transplanted HeLa cells in the scala media. PMID:24394296

  14. CD226 reduces endothelial cell glucose uptake under hyperglycemic conditions with inflammation in type 2 diabetes mellitus

    PubMed Central

    Dong, Zilong; Zhang, Jinxue; Sun, Yizheng; Jin, Boquan; Gao, Feng; Guo, Shuzhong; Zhuang, Ran

    2016-01-01

    CD226 is a co-stimulatory adhesion molecule found on immune and endothelial cells. Here, we evaluated a possible role for CD226 in inhibiting glucose uptake in isolated human umbilical vein endothelial cells (HUVECs) and in wild-type (WT) and CD226 knockout (KO) mice with high-fat diet (HFD)-induced type 2 diabetes (T2DM). CD226 expression increased under hyperglycemic conditions in the presence of TNF-α. Furthermore, CD226 knockdown improved glucose uptake in endothelial cells, and CD226 KO mice exhibited increased glucose tolerance. Levels of soluble CD226 in plasma were higher in T2DM patients following an oral glucose tolerance test (OGTT) than under fasting conditions. Our results indicate that low-grade inflammation coupled with elevated blood glucose increases CD226 expression, resulting in decreased endothelial cell glucose uptake in T2DM. PMID:26910838

  15. On the Influence of the Furnace and Cell Conditions on the Phase Transition of the Eutectic Co-C

    NASA Astrophysics Data System (ADS)

    Bloembergen, P.; Battuello, M.; Girard, F.; Machin, G.; Wright, L.

    2015-08-01

    This paper considers the influence, in terms of temperature and time, of the following three factors on the phase transition of high-temperature fixed points: the temperature gradient of the furnace along the fixed-point cell, the thermal inertia of the furnace, and the thermal condition of the cell itself. Melting and freezing experiments have been performed for the eutectic Co-C in a three-zone furnace, of which the temperature gradient along the cell could be varied from positive to negative. To help the interpretation of these experiments, a realistic 2D finite-element model has been implemented, simulating the three influence factors. The model allowed us to understand the features observed in the melts and associated freezes, for the different furnace conditions, not only qualitatively but also quantitatively. This research identifies the essential requirements to facilitate the optimum configuration of the furnace and cell design.

  16. A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach.

    PubMed

    Chou, Bin-Kuan; Gu, Haihui; Gao, Yongxing; Dowey, Sarah N; Wang, Ying; Shi, Jun; Li, Yanxin; Ye, Zhaohui; Cheng, Tao; Cheng, Linzhao

    2015-04-01

    Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10-50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications. PMID:25742692

  17. TRAP1 controls cell migration of cancer cells in metabolic stress conditions: Correlations with AKT/p70S6K pathways.

    PubMed

    Agliarulo, Ilenia; Matassa, Danilo Swann; Amoroso, Maria Rosaria; Maddalena, Francesca; Sisinni, Lorenza; Sepe, Leandra; Ferrari, Maria Carla; Arzeni, Diana; Avolio, Rosario; Paolella, Giovanni; Landriscina, Matteo; Esposito, Franca

    2015-10-01

    Cell motility is a highly dynamic phenomenon that is essential to physiological processes such as morphogenesis, wound healing and immune response, but also involved in pathological conditions such as metastatic dissemination of cancers. The involvement of the molecular chaperone TRAP1 in the regulation of cell motility, although still controversial, has been recently investigated along with some well-characterized roles in cancer cell survival and drug resistance in several tumour types. Among different functions, TRAP1-dependent regulation of protein synthesis seems to be involved in the migratory behaviour of cancer cells and, interestingly, the expression of p70S6K, a kinase responsible for translation initiation, playing a role in cell motility, is regulated by TRAP1. In this study, we demonstrate that TRAP1 silencing enhances cell motility in vitro but compromises the ability of cells to overcome stress conditions, and that this effect is mediated by the AKT/p70S6K pathway. In fact: i) inhibition of p70S6K activity specifically reduces migration in TRAP1 knock-down cells; ii) nutrient deprivation affects p70S6K activity thereby impairing cell migration only in TRAP1-deficient cells; iii) TRAP1 regulates the expression of both AKT and p70S6K at post-transcriptional level; and iii) TRAP1 silencing modulates the expression of genes involved in cell motility and epithelial-mesenchymal transition. Notably, a correlation between TRAP1 and AKT expression is found in vivo in human colorectal tumours. These results provide new insights into TRAP1 role in the regulation of cell migration in cancer cells, tumour progression and metastatic mechanisms. PMID:26071104

  18. Regulatory role of hexosamine biosynthetic pathway on hepatic cancer stem cell marker CD133 under low glucose conditions

    NASA Astrophysics Data System (ADS)

    Lin, Shu-Hai; Liu, Tengfei; Ming, Xiaoyan; Tang, Zhi; Fu, Li; Schmitt-Kopplin, Philippe; Kanawati, Basem; Guan, Xin-Yuan; Cai, Zongwei

    2016-02-01

    Cancer was hypothesized to be driven by cancer stem cells (CSCs), but the metabolic determinants of CSC-like phenotype still remain elusive. Here, we present that hexosamine biosynthetic pathway (HBP) at least in part rescues cancer cell fate with inactivation of glycolysis. Firstly, metabolomic analysis profiled cellular metabolome in CSCs of hepatocellular carcinoma using CD133 cell-surface marker. The metabolic signatures of CD133-positive subpopulation compared to CD133-negative cells highlighted HBP as one of the distinct metabolic pathways, prompting us to uncover the role of HBP in maintenance of CSC-like phenotype. To address this, CSC-like phenotypes and cell survival were investigated in cancer cells under low glucose conditions. As a result, HBP inhibitor azaserine reduced CD133-positive subpopulation and CD133 expression under high glucose condition. Furthermore, treatment of N-Acetylglucosamine in part restores CD133-positive subpopulation when either 2.5 mM glucose in culture media or glycolytic inhibitor 2-deoxy-D-glucose in HCC cell lines was applied, enhancing CD133 expression as well as promoting cancer cell survival. Together, HBP might be a key metabolic determinant in the functions of hepatic CSC marker CD133.

  19. Nonmyeloablative Conditioning Regimen to Increase Engraftment of Gene-modified Hematopoietic Stem Cells in Young Rhesus Monkeys

    PubMed Central

    Tarantal, Alice F; Giannoni, Francesca; I Lee, C Chang; Wherley, Jennifer; Sumiyoshi, Teiko; Martinez, Michele; Kahl, Christoph A; Elashoff, David; Louie, Stan G; Kohn, Donald B

    2012-01-01

    Immune responses to transgene products may lead to rejection of transduced cells, limiting successful gene therapy for genetic diseases. While moderate dosages of chemotherapeutic agents such as busulfan may increase hematopoietic stem cells (HSC) engraftment, they are not immune suppressive and do not abrogate immune responses to transgene products. Studies focused on nonmyeloablative conditioning with busulfan ± fludarabine in a clinically relevant monkey model to induce immune suppression to allow cells expressing a foreign transgene product to persist. Bone marrow CD34+ HSC were transduced in two equal fractions using simian immunodeficiency virus (SIV)-based lentiviral vectors carrying a nonexpressed DNA sequence tag (NoN) and the green fluorescent protein (GFP) reporter gene. Post-transplant there was no evidence of elimination of cells containing the potentially immunogenic GFP gene; several recipients had stable persistence of cells, and no differences were detected with fludarabine, which was rapidly cleared. Antibodies and cellular immune responses to GFP developed in recipients with the highest levels of GFP-marked cells, although these cells were not eliminated. These studies establish a clinically relevant pediatric primate model to assess the effects of conditioning regimens on the engraftment of transduced HSC and the immune responses to cells expressing a foreign gene product. PMID:22294147

  20. In vitro production of estradiol by bovine granulosa cells: evaluation of culture condition, stage of follicular development, and location of cells within follicles.

    PubMed

    Roberts, A J; Echternkamp, S E

    1994-08-01

    In vitro estradiol (E2) production by bovine granulosa cells was evaluated under several culture conditions, which included the presence or absence of fetal bovine serum (FBS; 2.5 and 10%), serum substitutes (1% Nutridoma [Boehringer-Mannheim, Indianapolis, IN], 2% UltroSer G [IBF Biotechnics, Villenue-la Garenne, France]), selenium (Se; 10 ng/ml), lipoprotein (0.25% Excyte/ml), O2 concentration (5 and 20%), and two attachment factors (Pronectin F and PepTite-2000). Dulbecco's Modified Eagle's medium:Ham's F-12 medium (1:1 mixture) containing 1 microM androstenedione, 1 microgram/ml insulin, and 0.1% BSA was the basal medium evaluated. The optimum conditions determined were the basal medium in 5% O2. These conditions were then used to ascertain whether or not E2 production by granulosa cells varied with respect to location of cells within a follicle. Follicular fluid was aspirated and centrifuged to obtain granulosa cells expected to be primarily luminal and cumulus cells. Follicles were then bisected, and remaining mural granulosa cells were removed by scraping the follicle wall with a fine plastic loop. Aspirated granulosa cells secreted more (p < 0.01) E2 than scraped cells. Production of E2 during Days 0 to 2 of culture by aspirated (0.15 +/- 0.05 ng/microgram DNA) and scraped (0.02 +/- 0.01 ng/microgram DNA) granulosa cells from small follicles (< 8 mm) was less than that by aspirated (6.30 +/- 2.20 ng/micrograms DNA) and scraped cells (1.90 +/- 1.00 ng/microgram DNA) from large follicles (> or = 8 mm). During Days 2 to 4 of culture when compared to Days 0 to 2, E2 production increased for aspirated (but not scraped) granulosa cells from small follicles (0.66 +/- 0.23 ng/microgram DNA)). In contrast, E2 production decreased (p < 0.05) over time in culture for aspirated (2.10 +/- 0.50 ng/microgram DNA) and scraped (0.16 +/- 0.07 ng/microgram DNA) granulosa cells from large follicles. Thus, granulosa cells proximal to the basement membrane may be less

  1. Studies on the mechanisms of mammalian cell killing by a freeze-thaw cycle: conditions that prevent cell killing using nucleated freezing

    SciTech Connect

    Shier, W.T.

    1988-04-01

    Normally a freeze-thaw cycle is a very efficient method of killing mammalian cells. However, this report describes conditions that prevent killing of cultured mammalian cells by nucleated freezing at -24 degrees C. Optimal protection from cell killing at -24 degrees C was obtained in isotonic solutions containing an organic cryoprotectant such as dimethyl sulfoxide (DMSO; 10%, v/v), a saccharide such as sucrose over a broad concentration range from 50 to 150 mM, and glucose. Glycerol was also an effective cryoprotectant but other organic solvents were ineffective, although in some cases they appeared to protect cell membranes, while not protecting other vital components. A wide variety of saccharide structures were effective at protecting cells from freeze-thaw killing, with trehalose being particularly effective. The degree of resistance to killing by a freeze-thaw cycle under these conditions varied widely among different cell lines. If toxicity of DMSO was responsible for this variability of cryoprotection, it must have been due to short-term, not longer term, toxicity of DMSO. Studies on the mechanism by which cells are protected from killing under these conditions indicated that neither vitrification of the medium nor the concentrating of components during freezing were involved. One model not eliminated by the mechanistic studies proposes that the organic solvent cryoprotectant component acts by fluidizing membranes under the thawing conditions, so that any holes produced by ice crystals propagating through membranes can reseal during the thawing process. In this model one of the mechanisms by which the saccharide component could act is by entering the cells and stabilizing vital intracellular components. Consistent with this, a freeze-thaw cycle promoted the uptake of labeled sucrose into cultured cells.

  2. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

    PubMed

    Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

    2011-11-15

    The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications. PMID:22065768

  3. Conditional deletion of Abca3 in alveolar type II cells alters surfactant homeostasis in newborn and adult mice

    PubMed Central

    Besnard, Valérie; Matsuzaki, Yohei; Clark, Jean; Xu, Yan; Wert, Susan E.; Ikegami, Machiko; Stahlman, Mildred T.; Weaver, Timothy E.; Hunt, Alan N.; Postle, Anthony D.

    2010-01-01

    ATP-binding cassette A3 (ABCA3) is a lipid transport protein required for synthesis and storage of pulmonary surfactant in type II cells in the alveoli. Abca3 was conditionally deleted in respiratory epithelial cells (Abca3Δ/Δ) in vivo. The majority of mice in which Abca3 was deleted in alveolar type II cells died shortly after birth from respiratory distress related to surfactant deficiency. Approximately 30% of the Abca3Δ/Δ mice survived after birth. Surviving Abca3Δ/Δ mice developed emphysema in the absence of significant pulmonary inflammation. Staining of lung tissue and mRNA isolated from alveolar type II cells demonstrated that ∼50% of alveolar type II cells lacked ABCA3. Phospholipid content and composition were altered in lung tissue, lamellar bodies, and bronchoalveolar lavage fluid from adult Abca3Δ/Δ mice. In adult Abca3Δ/Δ mice, cells lacking ABCA3 had decreased expression of mRNAs associated with lipid synthesis and transport. FOXA2 and CCAAT enhancer-binding protein-α, transcription factors known to regulate genes regulating lung lipid metabolism, were markedly decreased in cells lacking ABCA3. Deletion of Abca3 disrupted surfactant lipid synthesis in a cell-autonomous manner. Compensatory surfactant synthesis was initiated in ABCA3-sufficient type II cells, indicating that surfactant homeostasis is a highly regulated process that includes sensing and coregulation among alveolar type II cells. PMID:20190032

  4. Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells.

    PubMed

    Liu, Li; Liu, Ying-Feng; Zhang, Jing; Duan, Yin-Zhong; Jin, Yan

    2016-06-01

    Induced pluripotent stem (iPS) cells possess the ability of self-renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum-free conditioned medium (ASF-CM), we successfully generated ameloblast-like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF-CM supplemented with BMP4 (ASF-BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast-specific as well as odontoblast-specific genes. On the other hand, culturing mouse iPS cells in ASF-CM supplemented with noggin (ASF-noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF-BMP4 to differentiate into ameloblast-like and odontoblast-like cells. The results of our study raise the possibility of using patient-specific iPS cells for tooth regeneration in the future. Copyright © 2016 John Wiley & Sons, Ltd. PMID:23606575

  5. Direct production of organic acids from starch by cell surface-engineered Corynebacterium glutamicum in anaerobic conditions

    PubMed Central

    2013-01-01

    We produced organic acids, including lactate and succinate, directly from soluble starch under anaerobic conditions using high cell-density cultures of Corynebacterium glutamicum displaying α-amylase (AmyA) from Streptococcus bovis 148 on the cell surface. Notably, reactions performed under anaerobic conditions at 35 and 40°C, which are higher than the optimal growth temperature of 30°C, showed 32% and 19%, respectively, higher productivity of the organic acids lactate, succinate, and acetate compared to that at 30°C. However, α-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30°C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30°C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated. PMID:24342107

  6. Direct production of organic acids from starch by cell surface-engineered Corynebacterium glutamicum in anaerobic conditions.

    PubMed

    Tsuge, Yota; Tateno, Toshihiro; Sasaki, Kengo; Hasunuma, Tomohisa; Tanaka, Tsutomu; Kondo, Akihiko

    2013-01-01

    We produced organic acids, including lactate and succinate, directly from soluble starch under anaerobic conditions using high cell-density cultures of Corynebacterium glutamicum displaying α-amylase (AmyA) from Streptococcus bovis 148 on the cell surface. Notably, reactions performed under anaerobic conditions at 35 and 40°C, which are higher than the optimal growth temperature of 30°C, showed 32% and 19%, respectively, higher productivity of the organic acids lactate, succinate, and acetate compared to that at 30°C. However, α-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30°C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30°C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated. PMID:24342107

  7. Quantitative & qualitative analysis of endothelial cells of donor cornea before & after penetrating keratoplasty in different pathological conditions

    PubMed Central

    Gupta, Aruna K.R.; Gupta, Roopam K.R.

    2016-01-01

    Background & objectives: Endothelial cells of the donor cornea are known to be affected quantitatively and qualitatively in different pathological conditions after penetrating keratoplasty (PK) and this has direct effect on the clarity of vision obtained after PK. This study was undertaken to analyze the qualitative and quantitative changes in donor endothelial cells before and after PK in different pathological conditions. Methods: A prospective investigational analysis of 100 consecutive donor corneas used for penetrating keratoplasty between June 2006 and June 2008, was conducted. The patients were evaluated on the first day, at the end of first week, first month, third and six months and one year. Results: A decrease was observed in endothelial cell count in all pathological conditions. After one year of follow up the loss was 33.1 per cent in corneal opacity, 45.9 per cent in acute infective keratitis (AIK), 58.5 per cent in regrafts, 28.5 per cent in pseudophakic bullous keratopathy (PBK), 37 per cent in descemetocele, 27 per cent in keratoconus and 35.5 per cent in aphakic bullous keratopathy (ABK) cases. Interpretation & conclusions: The endothelial cell loss was highest in regraft cases which was significant (P<0.05), while the least endothelial cell loss was seen in keratoconus cases. The cell loss was associated with increase in coefficient of variation (CV), i.e. polymegathism and pleomorphism. Inspite of this polymegathism and pleomorphism, the clarity of the graft was maintained. PMID:27121519

  8. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

    SciTech Connect

    Walter, M.N.M.; Wright, K.T.; Fuller, H.R.; MacNeil, S.; Johnson, W.E.B.

    2010-04-15

    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-{beta}1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

  9. Conditional Disruption of Raptor Reveals an Essential Role for mTORC1 in B Cell Development, Survival, and Metabolism

    PubMed Central

    Iwata, Terri N.; Ramírez, Julita A.; Tsang, Mark; Park, Heon; Margineantu, Daciana H.; Hockenbery, David M.

    2016-01-01

    Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism. PMID:27521345

  10. Directed Endothelial Progenitor Differentiation from Human Pluripotent Stem Cells Via Wnt Activation Under Defined Conditions.

    PubMed

    Bao, Xiaoping; Lian, Xiaojun; Palecek, Sean P

    2016-01-01

    Efficient derivation of endothelial cells and their progenitors from human pluripotent stem cells (hPSCs) can facilitate studies of human vascular development, disease modeling, drug discovery, and cell-based therapy. Here we provide a detailed protocol for directing hPSCs to functional endothelial cells and their progenitors in a completely defined, growth factor- and serum-free system by temporal modulation of Wnt/β-catenin signaling via small molecules. We demonstrate a 10-day, two-stage process that recapitulates endothelial cell development, in which hPSCs first differentiate to endothelial progenitors that then generate functional endothelial cells and smooth muscle cells. Methods to characterize endothelial cell identity and function are also described. PMID:27590162

  11. Changes of vessel-cells complex in zones of adaptive remodeling of the bone tissue under microgravity conditions

    NASA Astrophysics Data System (ADS)

    Rodionova, N.; Oganov, V.; Nosova, L.

    The development and differentiation of osteogenic cells in organism happen in closely topographical and functional connection with blood capillaries. We formerly proofed, that small-differentiated cells, which are in the population of perivascular cells are osteogenic cells -precursors . At the present time it is actually to clear up, how these biostructures react on conditions of less of biomechanical load on skeleton bones. We researched peculiarities of blood-bed structure and perivascular cells in metaphises of thighbones and tibial bones in rats, which were onboard the American space station SLS-2 and in experiments of modeling hypokinesia. There were used methods of cytochemistry, histology and electron microscopy. We established, that under the support and functional load decreasing in zones of bones adaptive remodeling, comparatively to control, on histosections the own volume of sinusoid capillaries reduces. The small vessels prevail here. The spaces of sinusoid capillaries are limited by 1 2 cells of the endothelia. Endotheliocytes in- general have the typical ultrastructure. Basal membranes are expressed not-distinctly. Perivascular cells don't create the unbroken layer. The population of these cells is not-homogeneous. It includes enclosed to endothelia small-differentiated forms and separating cells with sings of fibroblastic differentiation (the own volume of rough endoplasmic reticulum in cytoplasm induces). The part of these cells reacts on the alkaline phosphatase (the marker of the osteogenic differentiation). Under the conditions of support load decreasing (especially under the microgravity) there is a tendency to reducing of separating osteogenic cells number. We noted the priority of differentiating fibroblasts. It leads to further development in zones of bone remodeling of hearths of fibrous tissue, that doesn't mineralize. The obtained data are seen as one of mechanisms of osteoporosis and osteopenia development under the deficite of support

  12. Investigating the neuroglial differentiation effect of neuroblastoma conditioned medium in human endometrial stem cells cultured on 3D nanofibrous scaffold.

    PubMed

    Ebrahimi-Barough, Somayeh; Hoveizi, Elham; Norouzi Javidan, Abbas; Ai, Jafar

    2015-08-01

    Neural tissue engineering is an important area of research in the field of tissue-engineering especially for neurodegenerative disease such as spinal cord injury. The differentiation capacity of human endometrial stem cells (hEnSCs) into neuronal cells has yet to be elucidated. Here, the major aim of the present study was to investigate the differentiation ability of hEnSCs cultured on polylactic acid/chitosan (PLA/CS) nanofibrous scaffold into neuroglial cells in response to conditioned medium of BE(2)-C human neuroblastoma cells and growth factors. Here we investigated the use PLA/CS scaffold as a three dimensional (3D) system that increased neuro-glial cells differentiation. Human EnSCs after three passages were differentiated in neuro-glial like cells under neuroblastoma conditioned medium with FGF2/PDGF-AA on PLA/CS scaffold. By day 18, differentiated cells were analyzed for expression of neuroglial markers by qRT-PCR and immunofluorescence. The results revealed that hEnSCs attach, grow and differentiation on the nanofibrous PLA/CS scaffold. Additionally, our study showed the expression of neural and glial lineage markers such as Nestin, NF-L, MAP2, PDGFRa, CNP, Olig2, MBP, and GFAP in the level of mRNA and MAP2, Tuj-1, and NF-L in the protein level after 18 days. Our results demonstrate that hEnSCs cultured on PLA/CS nanofibrous scaffold have the potential to differentiate in neuronal and glial cells in presence of neuroblastoma conditioned medium on PLA/CS scaffold. The result of this study may have impact in tissue engineering and cells-base therapy of neurodegenerative diseases and have a great potential for wide application. PMID:25611196

  13. An Investigation to Resolve the Interaction Between Fuel Cell, Power Conditioning System and Application Loads

    SciTech Connect

    Sudip K. Mazumder

    2005-12-31

    Development of high-performance and durable solidoxide fuel cells (SOFCs) and a SOFC power-generating system requires knowledge of the feedback effects from the power-conditioning electronics and from application-electrical-power circuits that may pass through or excite the power-electronics subsystem (PES). Therefore, it is important to develop analytical models and methodologies, which can be used to investigate and mitigate the effects of the electrical feedbacks from the PES and the application loads (ALs) on the reliability and performance of SOFC systems for stationary and non-stationary applications. However, any such attempt to resolve the electrical impacts of the PES on the SOFC would be incomplete unless one utilizes a comprehensive analysis, which takes into account the interactions of SOFC, PES, balance-of-plant system (BOPS), and ALs as a whole. SOFCs respond quickly to changes in load and exhibit high part- and full-load efficiencies due to its rapid electrochemistry, which is not true for the thermal and mechanical time constants of the BOPS, where load-following time constants are, typically, several orders of magnitude higher. This dichotomy can affect the lifetime and durability of the SOFCSs and limit the applicability of SOFC systems for load-varying stationary and transportation applications. Furthermore, without validated analytical models and investigative design and optimization methodologies, realizations of cost-effective, reliable, and optimal PESs (and power-management controls), in particular, and SOFC systems, in general, are difficult. On the whole, the research effort can lead to (a) cost-constrained optimal PES design for high-performance SOFCS and high energy efficiency and power density, (b) effective SOFC power-system design, analyses, and optimization, and (c) controllers and modulation schemes for mitigation of electrical impacts and wider-stability margin and enhanced system efficiency.

  14. Effects of Bisphosphonates on Glucose Transport in a Conditionally Immortalized Rat Retinal Capillary Endothelial Cell Line (TR-iBRB Cells)

    PubMed Central

    Lee, Na-Young; Park, Hyun-Joo; Kang, Young-Sook

    2016-01-01

    The objective of the present study was to elucidate the effect of bisphosphonates, anti-osteoporosis agents, on glucose uptake in retinal capillary endothelial cells under normal and high glucose conditions. The change of glucose uptake by pre-treatment of bisphosphonates at the inner blood-retinal barrier (iBRB) was determined by measuring cellular uptake of [3H]3-O-methyl glucose (3-OMG) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) under normal and high glucose conditions. [3H]3-OMG uptake was inhibited by simultaneous treatment of unlabeled D-glucose and 3-OMG as well as glucose transport inhibitor, cytochalasin B. On the other hand, simultaneous treatment of alendronate or pamidronate had no significant inhibitory effect on [3H]3-OMG uptake by TR-iBRB cells. Under high glucose condition of TR-iBRB cells, [3H]3-OMG uptake was increased at 48 h. However, [3H]3-OMG uptake was decreased significantly by pre-treatment of alendronate or pamidronate compared with the values for normal and high glucose conditions. Moreover, geranylgeraniol (GGOH), a mevalonate pathway intermediate, increased the uptake of [3H]3-OMG reduced by bisphosphonates pre-treatment. But, pre-treatment of histamine did not show significant inhibition of [3H]3-OMG uptake. The glucose uptake may be down regulated by inhibiting the mevalonate pathway with pre-treatment of bisphosphonates in TR-iBRB cells at high glucose condition. PMID:26759707

  15. A flow cell for in situ synchrotron x-ray diffraction studies of scale formation under Bayer processing conditions.

    PubMed

    Webster, Nathan A S; Madsen, Ian C; Loan, Melissa J; Scarlett, Nicola V Y; Wallwork, Kia S

    2009-08-01

    The design, construction, and commissioning of a stainless steel flow cell for in situ synchrotron x-ray diffraction studies of scale formation under Bayer processing conditions is described. The use of the cell is demonstrated by a study of Al(OH)(3) scale formation on a mild steel substrate from synthetic Bayer liquor at 70 degrees C. The cell design allows for interchangeable parts and substrates and would be suitable for the study of scale formation in other industrial processes. PMID:19725670

  16. Activity of interferon-dependent 2',5'-oligoadenylate synthetase in rat lymphoid cells under transformed environment conditions

    NASA Astrophysics Data System (ADS)

    Ostapchenko, L. I.; Mikhailik, I. V.; Prokopova, K. V.

    It is detected that interferon-dependent 2',5'-oligoadenylate synthetase is a sensitive index of immunocompetent cells functional state under transformed environment conditions. Microgravitation and ionising radiation induce increase of investigated enzyme activity in rat lymphocytes, which can be a result of lymphoid cells compensatory mechanisms starting in response to stress factors action. Administration of interferon inductors permits to stimulate the 2',5'-oligoadenylate synthetase, which enables one to correct pathological changes in the cells and to intensify adaptive reactions of immune systems.

  17. PD-1 expression conditions T cell avidity within an antigen-specific repertoire

    PubMed Central

    Simon, Sylvain; Vignard, Virginie; Florenceau, Laetitia; Dreno, B.; Khammari, A.; Lang, F.; Labarriere, N.

    2016-01-01

    ABSTRACT Despite its negative regulatory role on tumor-specific T cells, Programmed cell death 1 (PD-1) is also a marker of activated tumor-infiltrating T cells. In cancer, PD-1 blockade partially reverses T cell dysfunction allowing the amplification of tumor reactive T cells. Here, we investigated the role of PD-1 signaling on effector/memory human T cells specific for shared melanoma antigens, derived from blood. We documented for the first time the existence of melanoma-specific T cell clones unable to express PD-1. This stable feature was due to the persistent methylation of the PDCD1 promoter. These PD-1neg clones were of lower avidity than their PD-1pos counterparts, suggesting that high-affinity-specific T cell clones unable to express PD-1 are not or rarely present in peripheral blood, as they are probably eliminated by negative selection, due to their high reactivity. We also documented the existence of such PD-1neg T cell clones in melanoma tumor-infiltrating lymphocytes (TIL), which also exhibited a lower functional avidity than PD-1pos TIL clones. This clearly shows that PD-1 expression identifies antigen-specific T cell clonotypes of high functional avidity. Finally, we demonstrated that PD-1 blockade during the in vitro selection process of Melan-A-specific T cells favored the amplification of higher avidity T cell clonotypes. This preferential amplification of high-avidity memory T cells upon PD-1 blockade resonates with the expansion of reactive T cells, including neo-antigen-specific T cells observed in anti-PD-1-treated patients. This feature should also be a useful biomarker of clinical efficiency, while providing new insights for adoptive transfer treatments. PMID:26942093

  18. Optimum flash lamp annealing conditions for fabrication of low dose ion-implanted Si solar cells

    NASA Astrophysics Data System (ADS)

    Usami, A.; Nishioka, H.; Inoue, Y.

    1984-06-01

    A method to fabricate silicon solar cells was developed around low dose (nonamorphized) ion implantation and Xe flash lamp annealing under assist heating (350-550 C). Solar cells fabricated by low dose B-11/+/ (p+/n-type cell) and P-31/+/ (n+/p-type cell) implantation and flash lamp annealing showing a high efficiency of about 10.4 percent (AM2) without AR coating and BSF structures. The results were compared with those of high dose implanted cells reported previously (in this transaction). Effects of assist heating temperature were also examined.

  19. Expansion and activation kinetics of immune cells during early phase of GVHD in mouse model based on chemotherapy conditioning.

    PubMed

    Sadeghi, Behnam; Al-Hashmi, Suleiman; Hassan, Zuzana; Rozell, Bjorn; Concha, Hernan; Lundmark, Carin; Grönvik, Kjell-Olov; Abedi-Valugerdi, Manuchehr; Hassan, Moustapha

    2010-01-01

    In the present paper, we have investigated early pathophysiological events in graft-versus-host disease (GVHD), a major complication to hematopoietic stem cell transplantation (HSCT). BLLB/c female mice conditioned with busulfan/cyclophosphamide (Bu-Cy) were transplanted with allogeneic male C57BL/6. Control group consisted of syngeneic transplanted Balb/c mice. In allogeneic settings, significant expansion and maturation of donor dendritic cells (DCs) were observed at day +3, while donor T-cells CD8+ were increased at day +5 (230%) compared to syngeneic HSCT. Highest levels of inflammatory cytokines IL-2, IFN-gamma, and TNF-alfa at day +5 matched T-cell activation. Concomitantly naïve T-cells gain effecr-memory phenotype and migrated from spleen to peripheral lymphoid organs. Thus, in the very early phase of GHVD following Bu-Cy conditioning donor, DCs play an important role in the activation of donor T cells. Subsequently, donor naïve T-cells gain effector-memory phenotype and initiate GVHD. PMID:21197273

  20. Expansion and Activation Kinetics of Immune Cells during Early Phase of GVHD in Mouse Model Based on Chemotherapy Conditioning

    PubMed Central

    Sadeghi, Behnam; Al-Hashmi, Suleiman; Hassan, Zuzana; Rozell, Bjorn; Concha, Hernan; Lundmark, Carin; Grönvik, Kjell-Olov; Abedi-Valugerdi, Manuchehr; Hassan, Moustapha

    2010-01-01

    In the present paper, we have investigated early pathophysiological events in graft-versus-host disease (GVHD), a major complication to hematopoietic stem cell transplantation (HSCT). BLLB/c female mice conditioned with busulfan/cyclophosphamide (Bu-Cy) were transplanted with allogeneic male C57BL/6. Control group consisted of syngeneic transplanted Balb/c mice. In allogeneic settings, significant expansion and maturation of donor dendritic cells (DCs) were observed at day +3, while donor T-cells CD8+ were increased at day +5 (230%) compared to syngeneic HSCT. Highest levels of inflammatory cytokines IL-2, IFN-gamma, and TNF-alfa at day +5 matched T-cell activation. Concomitantly naïve T-cells gain effecr-memory phenotype and migrated from spleen to peripheral lymphoid organs. Thus, in the very early phase of GHVD following Bu-Cy conditioning donor, DCs play an important role in the activation of donor T cells. Subsequently, donor naïve T-cells gain effector-memory phenotype and initiate GVHD. PMID:21197273

  1. Endomucin prevents leukocyte–endothelial cell adhesion and has a critical role under resting and inflammatory conditions

    PubMed Central

    Zahr, Alisar; Alcaide, Pilar; Yang, Jinling; Jones, Alexander; Gregory, Meredith; dela Paz, Nathaniel G.; Patel-Hett, Sunita; Nevers, Tania; Koirala, Adarsha; Luscinskas, Francis W.; Saint-Geniez, Magali; Ksander, Bruce; D'Amore, Patricia A.; Argüeso, Pablo

    2016-01-01

    Endomucin is a membrane-bound glycoprotein expressed luminally by endothelial cells that line postcapillary venules, a primary site of leukocyte recruitment during inflammation. Here we show that endomucin abrogation on quiescent endothelial cells enables neutrophils to adhere firmly, via LFA-1-mediated binding to ICAM-1 constitutively expressed by endothelial cells. Moreover, TNF-α stimulation downregulates cell surface expression of endomucin concurrent with increased expression of adhesion molecules. Adenovirus-mediated expression of endomucin under inflammatory conditions prevents neutrophil adhesion in vitro and reduces the infiltration of CD45+ and NIMP-R14+ cells in vivo. These results indicate that endomucin prevents leukocyte contact with adhesion molecules in non-inflamed tissues and that downregulation of endomucin is critical to facilitate adhesion of leukocytes into inflamed tissues. PMID:26831939

  2. Endomucin prevents leukocyte-endothelial cell adhesion and has a critical role under resting and inflammatory conditions.

    PubMed

    Zahr, Alisar; Alcaide, Pilar; Yang, Jinling; Jones, Alexander; Gregory, Meredith; dela Paz, Nathaniel G; Patel-Hett, Sunita; Nevers, Tania; Koirala, Adarsha; Luscinskas, Francis W; Saint-Geniez, Magali; Ksander, Bruce; D'Amore, Patricia A; Argüeso, Pablo

    2016-01-01

    Endomucin is a membrane-bound glycoprotein expressed luminally by endothelial cells that line postcapillary venules, a primary site of leukocyte recruitment during inflammation. Here we show that endomucin abrogation on quiescent endothelial cells enables neutrophils to adhere firmly, via LFA-1-mediated binding to ICAM-1 constitutively expressed by endothelial cells. Moreover, TNF-α stimulation downregulates cell surface expression of endomucin concurrent with increased expression of adhesion molecules. Adenovirus-mediated expression of endomucin under inflammatory conditions prevents neutrophil adhesion in vitro and reduces the infiltration of CD45(+) and NIMP-R14(+) cells in vivo. These results indicate that endomucin prevents leukocyte contact with adhesion molecules in non-inflamed tissues and that downregulation of endomucin is critical to facilitate adhesion of leukocytes into inflamed tissues. PMID:26831939

  3. Effect of spiperone on mesenchymal multipotent stromal and hemopoietic stem cells under conditions of pulmonary fibrosis.

    PubMed

    Skurikhin, E G; Khmelevskaya, E S; Ermakova, N N; Pershina, O V; Reztsova, A M; Krupin, V A; Stepanova, I E; Reztsova, V M; Reikhart, D V; Dygai, A M

    2014-05-01

    The antifibrotic properties of spiperone and its effect on stem and progenitor cells were studied on the model of reversible bleomycin-induced pulmonary fibrosis in C57Bl/6 mice. Spiperone reduced infiltration of the alveolar interstitium and alveolar ducts with inflammatory cells and prevented the growth of the connective tissue in the parenchyma of bleomycin lungs. Apart from anti-inflammatory effect, spiperone suppressed bone marrow hemopoietic cells (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)-, Sca-1+, c-Kit+, CD34- and progenitor hemopoietic cells (granulocyte-erythroid-macrophage-megakaryocytic and granulocyte CFU). Spiperone-induced disturbances of fi brogenesis were paralleled by restoration of endothelial cells in the lung parenchyma, reduction of the number of circulating bone marrow cells and lung mesenchymopoietic cells (mesenchymal multipotent stromal cells (CD31-, CD34-, CD45-, CD44+, CD73+, CD90+, CD106+) and progenitor fi broblast cells), and suppression of multilineage differentiation of multipotent mesenchymal stromal cells (including fi broblast-lineage cells). PMID:24913578

  4. An analysis of T cell intrinsic roles of E2A by conditional gene disruption in the thymus.

    PubMed

    Pan, Lihua; Hanrahan, Jenifer; Li, Jie; Hale, Laura P; Zhuang, Yuan

    2002-04-15

    The importance of E2A transcription factors in T cell development has been demonstrated in studies of E2A-deficient mice, which display abnormal T cell development and a high frequency of T cell lymphomas. Because E2A expression is not restricted to the T cell lineage, the primary cause of the T cell phenotype in E2A-deficient mice was not fully determined. To further investigate the role of E2A in T cell lineage, we generated mice with the E2A gene disrupted exclusively during thymocyte development using the Cre-lox system. We show that this system allows E2A gene disruption to occur throughout the double-negative stage of thymocyte development. E2A deletion appears to be completed before development reaches the double-positive stage. Consistent with the gene disruption, these mice reveal a T cell intrinsic role for E2A during the transition from the double-negative stage to the double-positive stage of thymocyte development. In contrast to germline E2A knockout mice, conditional E2A knockout mice do not develop T cell lymphoma. This work establishes a new model for further investigating E2A function in T cell development and leukemiogenesis. PMID:11937548

  5. Listeria monocytogenes Scott A: Cell Surface Charge, Hydrophobicity, and Electron Donor and Acceptor Characteristics under Different Environmental Growth Conditions

    PubMed Central

    Briandet, Romain; Meylheuc, Thierry; Maher, Catherine; Bellon-Fontaine, Marie Noëlle

    1999-01-01

    We determined the variations in the surface physicochemical properties of Listeria monocytogenes Scott A cells that occurred under various environmental conditions. The surface charges, the hydrophobicities, and the electron donor and acceptor characteristics of L. monocytogenes Scott A cells were compared after the organism was grown in different growth media and at different temperatures; to do this, we used microelectrophoresis and the microbial adhesion to solvents method. Supplementing the growth media with glucose or lactic acid affected the electrical, hydrophobic, and electron donor and acceptor properties of the cells, whereas the growth temperature (37, 20, 15, or 8°C) primarily affected the electrical and electron donor and acceptor properties. The nonlinear effects of the growth temperature on the physicochemical properties of the cells were similar for cells cultivated in two different growth media, but bacteria cultivated in Trypticase soy broth supplemented with 6 g of yeast extract per liter (TSYE) were slightly more hydrophobic than cells cultivated in brain heart infusion medium (P < 0.05). Adhesion experiments conducted with L. monocytogenes Scott A cells cultivated in TSYE at 37, 20, 15, and 8°C and then suspended in a sodium chloride solution (1.5 × 10−1 or 1.5 × 10−3 M NaCl) confirmed that the cell surface charge and the electron donor and acceptor properties of the cells had an influence on their attachment to stainless steel. PMID:10583984

  6. Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53

    SciTech Connect

    Mercer, W.E.; Shields, M.T.; Amin, M.; Sauve, G.J. ); Appella, E.; Romano, J.W.; Ullrich, S.J. )

    1990-08-01

    To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation the authors constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastomas multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. The authors show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits G{sub 0}/G{sub 1} progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the G{sub 0}/G{sub 1} phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.

  7. The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

    SciTech Connect

    Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas; Simos, George

    2010-07-16

    Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.

  8. Culture condition improvement for whole-cell lipase production in submerged fermentation by Rhizopus chinensis using statistical method.

    PubMed

    Teng, Yun; Xu, Yan

    2008-06-01

    Rhizopus chinensis CCTCC M201021 was a versatile strain capable of producing whole-cell lipase with synthetic activity in submerged fermentation. In order to improve the production of whole-cell lipase and study the culture conditions systematically, the combination of taguchi method and response surface methodology was performed. Taguchi method was used for the initial optimization, and eight factors viz., maltose, olive oil, peptone, K2HPO4, agitation, inoculum size, fermentation volume and pH were selected for this study. The whole-cell lipase activity yield was two times higher than the control experiment under initial optimal conditions, and four significant factors (inoculum, olive oil, fermentation volume and peptone) were selected to test the effect on the lipase production using response surface methodology. The optimal fermentation parameters for enhanced whole-cell lipase yield were found to be: inoculum 4.25 x 10(8) spores/L, olive oil 2.367% (w/v), fermentation volume 18 mL/250 mL flask, peptone 4.06% (w/v). Subsequent experimental trails confirmed the validity of the model. These optimal culture conditions in the shake flask led to a lipase yield of 13875 U/L, which 120% increased compare with the non-optimized conditions. PMID:17888652

  9. Severe fludarabine neurotoxicity after reduced intensity conditioning regimen to allogeneic hematopoietic stem cell transplantation: a case report.

    PubMed

    Annaloro, Claudio; Costa, Antonella; Fracchiolla, Nicola S; Mometto, Gabriella; Artuso, Silvia; Saporiti, Giorgia; Tagliaferri, Elena; Grifoni, Federica; Onida, Francesco; Cortelezzi, Agostino

    2015-07-01

    We present a case of severe, irreversible neurotoxicity in a 55-year-old-patient with myelofibrosis undergoing hematopoietic stem cell transplantation following a reduced intensity conditioning including fludarabine. The patient developed progressive sensory-motor, visual and consciousness disturbances, eventually leading to death. MRI imaging pattern was unique and attributable to fludarabine neurotoxicity. PMID:26273463

  10. Severe fludarabine neurotoxicity after reduced intensity conditioning regimen to allogeneic hematopoietic stem cell transplantation: a case report

    PubMed Central

    Annaloro, Claudio; Costa, Antonella; Fracchiolla, Nicola S; Mometto, Gabriella; Artuso, Silvia; Saporiti, Giorgia; Tagliaferri, Elena; Grifoni, Federica; Onida, Francesco; Cortelezzi, Agostino

    2015-01-01

    Key Clinical Message We present a case of severe, irreversible neurotoxicity in a 55-year-old-patient with myelofibrosis undergoing hematopoietic stem cell transplantation following a reduced intensity conditioning including fludarabine. The patient developed progressive sensory-motor, visual and consciousness disturbances, eventually leading to death. MRI imaging pattern was unique and attributable to fludarabine neurotoxicity. PMID:26273463

  11. Survival of mammalian cells under high vacuum condition for ion bombardment.

    PubMed

    Feng, Huiyun; Wu, Lijun; Xu, An; Hu, Burong; Hei, Tom K; Yu, Zengliang

    2004-12-01

    An ion beam has been used to irradiate various organisms and its effects have been studied. Because of the poor tolerance that mammalian cells have for vacuum, such studies have not been carried out on living mammalian cells until now. However, this work is important both for elucidating the mechanism of mutation in response to low-energy ions and in exploring possible new applications of ion beam technology. The current paper describes an investigation of the survival of mammalian cells (the A(L) cell line) in a high-vacuum chamber in preparation for ion bombardment studies. The ion beam facility is described and the actual vacuum profile that the cells endured in the target chamber is reported. Cells were damaged immediately following vacuum exposure; the injury was characterized by alteration of the membrane permeability, loss of firm adhesion to the dish, and increased fragility. Three cryoprotective agents were tested (glycerol, propylene glycol, and trehalose) and of these, glycerol showed the highest potency for protecting cells against vacuum stress. This was revealed by an increase in the cell survival level from <1 to >10% with a glycerol concentration of 15 and 20%. Two glycerol-based protocols were investigated (freezing-vacuum vs. non-freezing-vacuum), but there was no significant difference (P > 0.1) in their ability to improve cell survival, the values being 10.31 +/- 4.5 and 12.7 +/- 3.37%, respectively with 20% glycerol concentration. These cells had a normal growth capability, and also retained integrity of the cell surface antigen CD59. These initial experiments indicate that mammalian cells can withstand vacuum to the degree that is needed to study the effect of the ion beam. In addition to the improvements made in this study, other factors are discussed that may increase the survival of mammalian cells exposed to a vacuum in future studies. PMID:15615610

  12. Induction of Salmonella pathogenicity island 1 under different growth conditions can affect Salmonella–host cell interactions in vitro

    PubMed Central

    Ibarra, J. Antonio; Knodler, Leigh A.; Sturdevant, Daniel E.; Virtaneva, Kimmo; Carmody, Aaron B.; Fischer, Elizabeth R.; Porcella, Stephen F.; Steele-Mortimer, Olivia

    2010-01-01

    Salmonella invade non-phagocytic cells by inducing massive actin rearrangements, resulting in membrane ruffle formation and phagocytosis of the bacteria. This process is mediated by a cohort of effector proteins translocated into the host cell by type III secretion system 1, which is encoded by genes in the Salmonella pathogenicity island (SPI) 1 regulon. This network is precisely regulated and must be induced outside of host cells. In vitro invasive Salmonella are prepared by growth in synthetic media although the details vary. Here, we show that culture conditions affect the frequency, and therefore invasion efficiency, of SPI1-induced bacteria and also can affect the ability of Salmonella to adapt to its intracellular niche following invasion. Aerobically grown late-exponential-phase bacteria were more invasive and this was associated with a greater frequency of SPI1-induced, motile bacteria, as revealed by single-cell analysis of gene expression. Culture conditions also affected the ability of Salmonella to adapt to the intracellular environment, since they caused marked differences in intracellular replication. These findings show that induction of SPI1 under different pre-invasion growth conditions can affect the ability of Salmonella to interact with eukaryotic host cells. PMID:20035008

  13. Derivation of embryonic stem cells from Kunming mice IVF blastocyst in feeder- and serum-free condition.

    PubMed

    Liu, Xiaokun; Wei, Qiang; Zhang, Junhong; Yang, Wanli; Zhao, Xiaoe; Ma, Baohua

    2015-06-01

    Kunming mice are widely used in China; however, it is difficult to isolate embryonic stem cells (ESCs) in conventional derivation condition containing feeder cells and serum. 6-Bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, could facilitate the maintenance of pluripotency of ESCs. Therefore, BIO could be considered as a candidate to replace feeder cells and serum. On the other hand, in vitro fertilization (IVF) is an important technology in assisted reproduction. It is reported that there was some difference in gene expression between IVF and in vivo developed blastocyst. ESCs derived from IVF blastocyst could provide a valuable tool to research the effect of IVF on differentiation and development. In the present study, we established two novel ESC lines from IVF blastocyst of Kunming mice in a feeder- and serum-free condition containing 2.5 μM BIO. In this condition, expanded IVF blastocyst could spontaneously hatch from zonae pellucidae and attached to the gelatin-coated bottom of dishes. ESC-like outgrowth could be observed without overfull trophoblast cells. After further propagation, two Kunming mice ESC lines, designated as KMES1 and KMES2, were obtained. These two novel ESCs shared common morphological characteristics with other rodent ESCs, showed strong alkaline phosphatase activity, and expressed pluripotent markers, including Oct-4, Nanog, and SSEA-1. Embryoid body (EB) and teratoma test indicated that these ESCs could spontaneously differentiate into cells representative of all three embryonic germ layers. PMID:25592083

  14. Method for Producing Non-Neoplastic, Three Dimensional, Mammalian Tissue and Cell Aggregates Under Microgravity Culture Conditions and the Products Produced Therefrom

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Prewett, Tracey L. (Inventor)

    1996-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural, and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  15. Conditional Radioresistance of tet-Inducible Manganese Superoxide Dismutase Bone Marrow Stromal Cell Lines

    PubMed Central

    Epperly, Michael W.; Chaillet, J. Richard; Kalash, Ronny; Shaffer, Ben; Goff, Julie; Franicola, Darcy; Zhang, Xichen; Dixon, Tracy; Houghton, Frank; Wang, Hong; Berhane, Hebist; Romero, Cynthia; Kim, Jee-Hong; Greenberger, Joel S.

    2013-01-01

    Mitochondrial targeted manganese superoxide dismutase is a major antioxidant enzyme, the levels of which modulate the response of cells, tissues and organs to ionizing irradiation. We developed a Tet-regulated MnSOD mouse (MnSODtet) to examine the detailed relationship between cellular MnSOD concentration and radioresistance and carried out in vitro studies using bone marrow culture derived stromal cell lines (mesenchymal stem cells). Homozygous MnSODtet/tet cells had low levels of MnSOD, reduced viability and proliferation, increased radiosensitivity, elevated overall antioxidant stores, and defects in cell proliferation and DNA strand-break repair. Doxycycline (doxy) treatment of MnSODtet/tet cells increased MnSOD levels and radioresistance from ñ of 2.79 ± 1.04 to 8.69 ± 1.09 (P = 0.0060) and normalized other biologic parameters. In contrast, MnSODtet/tet cells showed minimal difference in baseline and radiation induced mRNA and protein levels of TGF-β, Nrf2 and NF-κB and radiation induced cell cycle arrest was not dependent upon MnSOD level. These novel MnSODtet/tet mouse derived cells should be valuable for elucidating several parameters of the oxidative stress response to ionizing radiation. PMID:23862693

  16. Conditional radioresistance of Tet-inducible manganese superoxide dismutase bone marrow stromal cell lines.

    PubMed

    Epperly, Michael W; Chaillet, J Richard; Kalash, Ronny; Shaffer, Ben; Goff, Julie; Franicola, Darcy; Zhang, Xichen; Dixon, Tracy; Houghton, Frank; Wang, Hong; Berhane, Hebist; Romero, Cynthia; Kim, Jee-Hong; Greenberger, Joel S

    2013-08-01

    Mitochondrial targeted manganese superoxide dismutase is a major antioxidant enzyme, the levels of which modulate the response of cells, tissues and organs to ionizing irradiation. We developed a Tet-regulated MnSOD mouse (MnSOD(tet)) to examine the detailed relationship between cellular MnSOD concentration and radioresistance and carried out in vitro studies using bone marrow culture derived stromal cell lines (mesenchymal stem cells). Homozygous MnSOD(tet/tet) cells had low levels of MnSOD, reduced viability and proliferation, increased radiosensitivity, elevated overall antioxidant stores, and defects in cell proliferation and DNA strand-break repair. Doxycycline (doxy) treatment of MnSOD(tet/tet) cells increased MnSOD levels and radioresistance from ñ of 2.79 ± 1.04 to 8.69 ± 1.09 (P = 0.0060) and normalized other biologic parameters. In contrast, MnSOD(tet/tet) cells showed minimal difference in baseline and radiation induced mRNA and protein levels of TGF-β, Nrf2 and NF-κB and radiation induced cell cycle arrest was not dependent upon MnSOD level. These novel MnSOD(tet/tet) mouse derived cells should be valuable for elucidating several parameters of the oxidative stress response to ionizing radiation. PMID:23862693

  17. Fuel sensor-less control of a liquid feed fuel cell under dynamic loading conditions for portable power sources (II)

    NASA Astrophysics Data System (ADS)

    Chang, C. L.; Chen, C. Y.; Sung, C. C.; Liou, D. H.; Chang, C. Y.; Cha, H. C.

    This work presents a new fuel sensor-less control scheme for liquid feed fuel cells that is able to control the supply to a fuel cell system for operation under dynamic loading conditions. The control scheme uses cell-operating characteristics, such as potential, current, and power, to regulate the fuel concentration of a liquid feed fuel cell without the need for a fuel concentration sensor. A current integral technique has been developed to calculate the quantity of fuel required at each monitoring cycle, which can be combined with the concentration regulating process to control the fuel supply for stable operation. As verified by systematic experiments, this scheme can effectively control the fuel supply of a liquid feed fuel cell with reduced response time, even under conditions where the membrane electrolyte assembly (MEA) deteriorates gradually. This advance will aid the commercialization of liquid feed fuel cells and make them more adaptable for use in portable and automotive power units such as laptops, e-bikes, and handicap cars.

  18. Cyclooxygenase-2 level and culture conditions influence NS398-induced apoptosis and caspase activation in lung cancer cells.

    PubMed

    Chang, H C; Weng, C F

    2001-01-01

    Cyclooxygenases (COXs) catalyze the synthesis of prostaglandins (PGs) from arachidonic acid. Overexpression of COX-2 is frequently found in human cancers and is suggested to play an important role in tumorigenesis. Recent studies indicated that COX-2 inhibitors exert potent anti-cancer effects on a number of cancers. Interestingly, some COX-2 inhibitors potently induce apoptosis, while other COX-2 inhibitors primarily induce growth inhibition. Therefore, there is a variability in the effects that different COX-2 inhibitors have on cancer cells. In this study, we demonstrated that induction of apoptosis of high COX-2-expressing A549 lung cancer cells by a specific COX-2 inhibitor NS398 was observed in cells cultured under serum-free condition. However, this drug induced G1 growth arrest rather than apoptosis in A549 cells maintained in 10% serum medium. Conversely, low COX-2-expressing H226 lung cancer cells were resistant to NS398-induced apoptosis under both serum-free and serum-containing conditions. Moreover, our results showed that NS398-induced apoptosis is associated with activation of caspase-3, a cysteine protease that plays a crucial role in the execution phase of apoptosis. These results suggest that the cytotoxic effect of COX-2 inhibitors on cancer cells may be influenced by extracellular environments and the anti-cancer action of these inhibitors in vivo needs careful evaluation. Additionally, a correlation between the level of COX-2 expression and the extent of apoptosis induced by COX-2 inhibitors was found. PMID:11605058

  19. Performance of Busulfan Dosing Guidelines for Pediatric Hematopoietic Stem Cell Transplant Conditioning.

    PubMed

    Zao, Jamie H; Schechter, Tal; Liu, Wenchao Jessica; Gerges, Sandra; Gassas, Adam; Egeler, R Maarten; Grunebaum, Eyal; Dupuis, L Lee

    2015-08-01

    Achievement of a busulfan area-under-the-concentration versus time curve (AUC) of 900 to 1500 μM·min is associated with improved hematopoietic stem cell transplant (HSCT) outcomes. Multiple pediatric busulfan dosing guidelines aim to achieve this target. The authors' objective was to describe the AUCs achieved after simulated dosing using available pediatric i.v. busulfan dosing guidelines. The health records of children who received i.v. busulfan for HSCT conditioning at The Hospital for Sick Children were reviewed. Busulfan AUCs were calculated for each patient based on plasma busulfan concentrations using either a 1-compartment model or a validated limited-sampling strategy. Published pediatric busulfan dosing guidelines were identified. Initial busulfan doses were determined for all patients using each dosing guideline and total body weight (TBW). For overweight patients (TBW-to-ideal body weight [IBW] ≥ 1.25), initial busulfan doses were also determined using IBW and adjusted IBW (IBWadj). The resulting AUCs were simulated. The proportion of subjects (TBW/IBW < 1.25, TBW/IBW ≥ 1.25, and infants) with an AUC within target (900 to 1500 μM·min) after dosing simulation with each guideline was compared. One hundred eleven children (mean age, 6.2 years [SD, ±5.2]) who received i.v. busulfan were included. When dosing with each of the 12 i.v. busulfan dosing guidelines identified was simulated using TBW in 97 non-overweight patients, the proportion of patients with an AUC within the target range varied from 51% to 74% and from 45% to 64% in infants. Use of IBW or IBWadj to calculate initial busulfan doses in overweight children improved the performance of most guidelines. Current busulfan dosing guidelines vary in their ability to achieve AUCs within the target range. For children who are not overweight, we recommend 1 of 3 high-performing guidelines that allow individualization of the target busulfan AUC. Use of either IBW or IBWadj in overweight children

  20. Conditioned medium from concanavalin A-stimulated spleen cells inhibits the IgE-dependent sensitization of murine peritoneal mast cells in vitro.

    PubMed Central

    Coleman, J W

    1990-01-01

    Conditioned medium (CM) from concanavalin A (Con A)-stimulated murine spleen cells inhibited release of histamine and 5-HT from murine peritoneal mast cells sensitized with monoclonal IgE anti-DNP antibody and challenged with DNP-human serum albumin (HSA) antigen. Inhibition was seen when the CM was added to the mast cells either 24 hr before or simultaneous with, but not 24 hr subsequent to, the IgE, thus showing that inhibition was at the IgE-dependent stage of mast cell sensitization. Unconditioned medium, prepared in the same way as CM but not exposed to spleen cells was without activity, demonstrating that inhibition was due to a spleen cell-derived factor. CM from unstimulated spleen cells was likewise without activity. The sensitization inhibitory factor appears to be a protein, since it was retained upon dialysis, and destroyed by heating at 70 degrees and above. The factor does not appear to be IgE, since it was stable at 56 degrees, and is not IL-1 or IL-2, since recombinant human IL-1 alpha and IL-1 beta, and recombinant mouse IL-1 alpha and IL-2 were without inhibitory activity. The active CM and all recombinant IL-1 and IL-2 preparations did not release histamine or 5-HT directly from mast cells during 48 hr of culture, and did not modulate the histamine content of these cells, nor their capacity to incorporate [3H]5-HT. PMID:2312153

  1. Generation of Induced Pluripotent Stem Cells from Human Amniotic Fluid Cells by Reprogramming with Two Factors in Feeder-free Conditions

    PubMed Central

    LI, Qing; FAN, Yong; SUN, Xiaofang; YU, Yanhong

    2012-01-01

    Abstract The ectopic expression of transcription factors for reprogramming human somatic cells to a pluripotent state represents a valuable resource for the development of in vitro-based models for human disease and has great potential in regenerative therapies. However, the majority of studies have used skin fibroblasts to generate induced pluripotent stem cells (iPSCs) that typically require the enforced expression of several transcription factors, thereby posing a mutagenesis risk by the insertion of viral transgenes. To reduce this risk, iPSCs have been generated with OCT4 and KLF4 from human neural stem cells that endogenously express the remaining reprogramming factors. However, human neural stem cells are rare and difficult to obtain. Here, we show that iPSCs can be generated from human amniotic fluid cells (hAFCs) with two transcription factors: OCT4 and KLF4. Furthermore, iPSCs can be readily derived from hAFCs in a feeder-free conditions, thereby eliminating the potential variability caused by using feeder cells. Our results indicate that hAFCs represent an accessible source of cells that can be reprogrammed into pluripotent stem cells with two Yamanaka factors. Therefore, hAFCs may become a preferred cell type in the future for safe reprogramming without any exogenous genetic material. PMID:23138118

  2. Hypoxia inducible factor-1α-dependent epithelial to mesenchymal transition under hypoxic conditions in prostate cancer cells.

    PubMed

    Li, Mingchuan; Wang, Yong Xing; Luo, Yong; Zhao, Jiahui; Li, Qing; Zhang, Jiao; Jiang, Yongguang

    2016-07-01

    Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer death. Hypoxia is an environmental stimulus that plays an important role in the development and cancer progression especially for solid tumors. The key regulator under hypoxic conditions is stabilized hypoxia-inducible factor (HIF)-1α. In the present study, immune-fluorescent staining, siRNAs, qRT-PC, immunoblotting, cell migration and invasion assays were carried out to test typical epithelial to mesenchymal transition under hypoxia and the key regulators of this process in PC3, a human prostate cancer cell line. Our data demonstrated that hypoxia induces diverse molecular, phenotypic and functional changes in prostate cancer cells that are consistent with EMT. We also showed that a cell signal factor such as HIF-1α, which might be stabilized under hypoxic environment, is involved in EMT and cancer cell invasive potency. The induced hypoxia could be blocked by HIF-1α gene silencing and reoxygenation of EMT in prostate cancer cells, hypoxia partially reversed accompanied by a process of mesenchymal-epithelial reverting transition (MErT). EMT might be induced by activation of HIF-1α-dependent cell signaling in hypoxic prostate cancer cells. PMID:27108616

  3. Pemetrexed alters folate phenotype and inflammatory profile in EA.hy 926 cells grown under low-folate conditions

    PubMed Central

    Hammons, Andrea L.; Summers, Carolyn M.; Jochems, Jeanine; Arora, Jasbir S.; Zhang, Suhong; Blair, Ian A.; Whitehead, Alexander S.

    2014-01-01

    Elevated homocysteine is a risk marker for several major human pathologies. Emerging evidence suggests that perturbations of folate/homocysteine metabolism can directly modify production of inflammatory mediators. Pemetrexed acts by inhibiting thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). EA.hy 926 cells grown under low (“Lo”) and high (“Hi”) folate conditions were treated with pemetrexed. The concentrations of several intracellular folate derivatives were measured using LC-MRM/MS. Lo cells had lower total folate concentrations and a different distribution of the intracellular folate derivatives than Hi cells. Treatment with pemetrexed caused a decrease in individual folate analytes. Microarray analysis showed that several genes were significantly up or down-regulated in pemetrexed treated Lo cells. Several of the significantly up-regulated transcripts were inflammatory. Changes in transcript levels of selected targets, including C3, IL-8, and DHFR, were confirmed by quantitative RT-PCR. C3 and IL-8 transcript levels were increased in pemetrexed-treated Lo cells relative to Lo controls; DHFR transcript levels were decreased. In Lo cells, IL-8 and C3 protein concentrations were increased following pemetrexed treatment. Pemetrexed drug treatment was shown in this study to have effects that lead to an increase in pro-inflammatory mediators in Lo cells. No such changes were observed in Hi cells, suggesting that pemetrexed could not modify the inflammatory profile in the context of cellular folate sufficiency. PMID:22975265

  4. Prediction of Low-Voltage Tetrafluoromethane Emissions Based on the Operating Conditions of an Aluminium Electrolysis Cell

    NASA Astrophysics Data System (ADS)

    Dion, Lukas; Kiss, László I.; Poncsák, Sándor; Lagacé, Charles-Luc

    2016-08-01

    Greenhouse gas (GHG) generation is inherent in the production of aluminium by a technology that uses carbon anodes. Most of those GHG are composed of CO2 produced by redox reaction that occurs in the cell. However, a significant fraction of the annual GHG production is composed of perfluorocarbons (PFC) resulting from anode effects (AE). Multiple investigations have shown that tetrafluoromethane (CF4) can be generated under low-voltage conditions in the electrolysis cells, without global anode effect. The aim of this paper is to find a quantitative relationship between monitored cell parameters and the emissions of CF4. To achieve this goal, a predictive algorithm has been developed using seven cell indicators. These indicators are based on the cell voltage, the noise level and other parameters calculated from individual anode current monitoring. The predictive algorithm is structured into three different steps. The first two steps give qualitative information while the third one quantitatively describes the expected CF4 concentration at the duct end of the electrolysis cells. Validations after each step are presented and discussed. Finally, a sensitivity analysis was performed to understand the effect of each indicator on the onset of low-voltage PFC emissions. The standard deviation of individual anode currents was found to be the dominant variable. Cell voltage, noise level, and maximum individual anode current also showed a significant correlation with the presence of CF4 in the output gas of an electrolysis cell.

  5. Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

    PubMed

    Lidgerwood, Grace E; Lim, Shiang Y; Crombie, Duncan E; Ali, Ray; Gill, Katherine P; Hernández, Damián; Kie, Josh; Conquest, Alison; Waugh, Hayley S; Wong, Raymond C B; Liang, Helena H; Hewitt, Alex W; Davidson, Kathryn C; Pébay, Alice

    2016-04-01

    We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening. PMID:26589197

  6. [Phenotypic switching of Escherichia coli cells containing cyclic digenic systems with negative feedback upon changes in cultivation conditions].

    PubMed

    Stupak, E E; Stupak, I V

    2010-05-01

    One of the mechanisms for the epigenetic control of cell phenotypes is based on switching the functioning regimes of bistable gene networks, which can maintain the two alternative levels of gene expression under the same conditions. Cyclic digenic systems with negative feedback represent an example of a simple bistable gene network. Cells carrying artificial cyclic digenic systems on plasmids inherit each alternative phenotype upon exponential growth on rich medium during several cell generations. The action of specific inducers is necessary for switching. In this work, the impact of changes in cell cultivation conditions on the phenotypic composition of the clonal Escherichia coli cell population containing artificial cyclic digenic systems with negative feedback was studied. Phenotypes differ with respect to the expression level of marker proteins: beta-galactosidase and GFP. Slow growth on a medium containing little-available carbon sources was shown to cause the transition from the phenotype Lac- to Lac+ in the absence of inducers. Phenotypic switching cannot be explained by transcriptional activation of the lactose operon, because 80 +/- 15% of cells inherit the acquired phenotype after replating bacteria on rich medium. Inheritance of the phenotype Lac- in batch culture depends on the medium and duration of cultivation. Dynamics of changes in the activity of beta-galactosidase and culture fluorescence suggests that a decrease in the level of metabolism resulted in the switch of these cyclic systems from bistable to monostable functioning regime, which corresponds to the Lac+ phenotype with respect to the ratio of regulatory proteins. Thus, the instability of growth conditions may cause phenotypic heterogeneity in the clonal population of cells containing bistable gene networks. PMID:20583595

  7. Detection of activity in the conditioned medium of ethanol-treated HepG2 cells which stimulates collagen synthesis in IMR-90 cells.

    PubMed

    Inui, N; Kato, J; Kohgo, Y; Katsuki, S; Niitsu, Y

    1996-02-01

    Hepatic fibrosis often occurs in alcoholic liver diseases without accompanying tissue necrosis or inflammation. However, the precise mechanism of this fibrosis has not been fully clarified. In the present study, using the hepatoblastoma cell line HepG2 as a model for hepatocytes, we identified a factor that stimulates collagen synthesis of fibroblasts in a conditioned medium of HepG2 cells after treatment with ethanol. Type 1 procollagen peptide (PIC) in a culture of human fibroblast IMR-90 markedly increased after incubation with the conditioned medium of ethanol-treated HepG2 cells. The stimulating activity on the production of PIC by IMR-90 remained after the dialysis and evaporation of the conditioned medium of HepG2 cells, indicating this factor was not as volatile from low molecular substances such as acetaldehyde, acetate, or lactate. The activity of this factor diminished with heat or trypsin treatment. A gel chromatographic analysis disclosed that the molecular weight of this factor was approximately 8000 Da. These results suggest that a polypeptide factor secreted from HepG2 cells by treatment with ethanol stimulates collagen synthesis of fibroblasts. PMID:8659693

  8. Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation

    PubMed Central

    Köffel, René; Meshcheryakova, Anastasia; Warszawska, Joanna; Hennig, Annika; Wagner, Karin; Jörgl, Almut; Gubi, Daniela; Moser, Doris; Hladik, Anastasiya; Hoffmann, Ulrike; Fischer, Michael B.; van den Berg, Wim; Koenders, Marije; Scheinecker, Clemens; Gesslbauer, Bernhard; Knapp, Sylvia

    2014-01-01

    During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly “transdifferentiate” into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal–induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor–dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF–pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo–isolated G-CSF–mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils. PMID:25214442

  9. Unique characteristics of human mesenchymal stem/progenitor cells (MSC) pre-activated in 3D cultures under different conditions

    PubMed Central

    Ylostalo, Joni H.; Bartosh, Thomas J.; Tiblow, April; Prockop, Darwin J.

    2014-01-01

    Background Human mesenchymal stem cells (MSCs) are being employed in clinical trials, but the best protocol to prepare the cells for administration to patients remains unclear. We previously demonstrated that MSCs could be pre-activated to express therapeutic factors by culturing the cells in 3D. Here we compared the activation of MSCs in 3D in fetal bovine serum (FBS) containing medium and in multiple xeno-free media formulations. Methods MSC aggregation and sphere formation was studied using hanging drop cultures with medium containing FBS or with various commercially available stem cell media with or without human serum albumin (HSA). Activation of MSCs was studied with gene expression and protein secretion measurements and with functional studies using macrophages and cancer cells. Results MSCs did not condense into tight spheroids and express a full complement of therapeutic genes in MEMα or several commercial stem-cell media. However, we identified a chemically-defined xeno-free media that when supplemented with HSA from blood or recombinant HSA, resulted in compact spheres with high cell viability, together with high expression of anti-inflammatory (PGE2, TSG-6) and anti-cancer molecules (TRAIL, IL-24). Furthermore, spheres cultured in this medium showed potent anti-inflammatory effects in an LPS-stimulated macrophage system, and suppressed the growth of prostate cancer cells by promoting cell-cycle arrest and cell death. Discussion We demonstrated that cell activation in 3D depends critically on the culture medium. The conditions developed here for 3D culture of MSCs should be useful in further research on MSCs and their potential therapeutic applications. PMID:25231893

  10. Stem cells for spinal cord regeneration: Current status

    PubMed Central

    Sobani, Zain A.; Quadri, Syed A.; Enam, S. Ather

    2010-01-01

    Background: Nearly 11,000 cases of spinal cord injury (SCI) are reported in the United States annually. Current management options give a median survival time of 38 years; however, no rehabilitative measures are available. Stem cells have been under constant research given their ability to differentiate into neural cell lines replacing non functional tissue. Efforts have been made to establish new synapses and provide a conducive environment, by grafting cells from autologous and fetal sources; including embryonic or adult stem cells, Schwann cells, genetically modified fibroblasts, bone stromal cells, and olfactory ensheathing cells and combinations/ variants thereof. Methods: In order to discuss the underlying mechanism of SCI along with the previously mentioned sources of stem cells in context to SCI, a simple review of literature was conducted. An extensive literature search was conducted using the PubMed data base and online search engines and articles published in the last 15 years were considered along with some historical articles where a background was required. Results: Stem cell transplantation for SCI is at the forefront with animal and in vitro studies providing a solid platform to enable well-designed human studies. Olfactory ensheathing cells seem to be the most promising; whilst bone marrow stromal cells appear as strong candidates for an adjunctive role. Conclusion: The key strategy in developing the therapeutic basis of stem cell transplantation for spinal cord regeneration is to weed out the pseudo-science and opportunism. All the trials should be based on stringent scientific criteria and effort to bypass that should be strongly discouraged at the international level. PMID:21246060

  11. Optimum Thickness Conditions of TiO2 Nanotubes Layer for Efficient Electrochemical Luminescence Cells Application.

    PubMed

    Choi, Min-Ki; Sung, Youl-Moon; Park, Min-Woo

    2015-02-01

    We report a TiO2 nanotubes (NTs)-based Electrochemical luminescence (ECL) cell. The ECL cell was fabricated using the electrode of TiO2 NTs and Ru(II) complex (Ru(bpy)2+(3)) as a luminescence materials. The fabricated ECL cell is composed of F-doped SnO2 (FTO) glass/Ru(II)/TiO2 NTs/Ti plate. At a bias voltage of 3 V, the measured ECL efficiencies were 0 Im/W for cell without NTs, 0.03 Im/W for NTs-6.5 µm, 0.07 Im/W for NTs-8 µm and 0.1 Im/W for NTs-10 µm, respectively. The use of Ti02 NTs increases ECL intensities by about 2 times compared to the typical ECL cell without the use of TiO2 NTs. PMID:26353679

  12. Chemically defined conditions for human iPS cell derivation and culture

    PubMed Central

    Chen, Guokai; Gulbranson, Daniel R.; Hou, Zhonggang; Bolin, Jennifer M.; Ruotti, Victor; Probasco, Mitchell D.; Smuga-Otto, Kimberly; Howden, Sara E.; Diol, Nicole R.; Propson, Nicholas E.; Wagner, Ryan; Lee, Garrett O.; Antosiewicz-Bourget, Jessica; Teng, Joyce M. C.; Thomson, James A.

    2011-01-01

    We reexamine the individual components for human ES and iPS cell culture, and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces, and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal or human sourced albumin batches have previously plagued human ES and iPS cell culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPS cells with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ES and iPS cells and their derivatives, and should be applicable to other reprogramming methods. PMID:21478862

  13. Growth Conditions and Cell Cycle Phase Modulate Phase Transition Temperatures in RBL-2H3 Derived Plasma Membrane Vesicles

    PubMed Central

    Gray, Erin M.; Díaz-Vázquez, Gladys; Veatch, Sarah L.

    2015-01-01

    Giant plasma membrane vesicle (GPMV) isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune their plasma membrane

  14. Hypoxic culture conditions induce increased metabolic rate and collagen gene expression in ACL-derived cells.

    PubMed

    Kowalski, Tomasz J; Leong, Natalie L; Dar, Ayelet; Wu, Ling; Kabir, Nima; Khan, Adam Z; Eliasberg, Claire D; Pedron, Andrew; Karayan, Ashant; Lee, Siyoung; Di Pauli von Treuheim, Theodor; Jiacheng, Jin; Wu, Ben M; Evseenko, Denis; McAllister, David R; Petrigliano, Frank A

    2016-06-01

    There has been substantial effort directed toward the application of bone marrow and adipose-derived mesenchymal stromal cells (MSCs) in the regeneration of musculoskeletal tissue. Recently, resident tissue-specific stem cells have been described in a variety of mesenchymal structures including ligament, tendon, muscle, cartilage, and bone. In the current study, we systematically characterize three novel anterior cruciate ligament (ACL)-derived cell populations with the potential for ligament regeneration: ligament-forming fibroblasts (LFF: CD146(neg) , CD34(neg) CD44(pos) , CD31(neg) , CD45(neg) ), ligament perivascular cells (LPC: CD146(pos) CD34(neg) CD44(pos) , CD31(neg) , CD45(neg) ) and ligament interstitial cells (LIC: CD34(pos) CD146(neg) , CD44(pos) , CD31(neg) , CD45(neg) )-and describe their proliferative and differentiation potential, collagen gene expression and metabolism in both normoxic and hypoxic environments, and their trophic potential in vitro. All three groups of cells (LIC, LPC, and LFF) isolated from adult human ACL exhibited progenitor cell characteristics with regard to proliferation and differentiation potential in vitro. Culture in low oxygen tension enhanced the collagen I and III gene expression in LICs (by 2.8- and 3.3-fold, respectively) and LFFs (by 3- and 3.5-fold, respectively) and increased oxygen consumption rate and extracellular acidification rate in LICs (by 4- and 3.5-fold, respectively), LFFs (by 5.5- and 3-fold, respectively), LPCs (by 10- and 4.5-fold, respectively) as compared to normal oxygen concentration. In summary, this study demonstrates for the first time the presence of three novel progenitor cell populations in the adult ACL that demonstrate robust proliferative and matrix synthetic capacity; these cells may play a role in local ligament regeneration, and consequently represent a potential cell source for ligament engineering applications. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc

  15. In Situ Irradiation and Measurement of Triple Junction Solar Cells at Low Intensity, Low Temperature (LILT) Conditions

    NASA Technical Reports Server (NTRS)

    Harris, R.D.; Imaizumi, M.; Walters, R.J.; Lorentzen, J.R.; Messenger, S.R.; Tischler, J.G.; Ohshima, T.; Sato, S.; Sharps, P.R.; Fatemi, N.S.

    2008-01-01

    The performance of triple junction InGaP/(In)GaAs/Ge space solar cells was studied following high energy electron irradiation at low temperature. Cell characterization was carried out in situ at the irradiation temperature while using low intensity illumination, and, as such, these conditions reflect those found for deep space, solar powered missions that are far from the sun. Cell characterization consisted of I-V measurements and quantum efficiency measurements. The low temperature irradiations caused substantial degradation that differs in some ways from that seen after room temperature irradiations. The short circuit current degrades more at low temperature while the open circuit voltage degrades more at room temperature. A room temperature anneal after the low temperature irradiation produced a substantial recovery in the degradation. Following irradiation at both temperatures and an extended room temperature anneal, quantum efficiency measurement suggests that the bulk of the remaining damage is in the (In)GaAs sub-cell

  16. Giant Cell Tumor of Bone: A Neoplasm or a Reactive Condition?

    PubMed Central

    Haque, Anwar Ul; Moatasim, Ambreen

    2008-01-01

    Giant cell tumor of bone (GCTB) is a benign but locally aggressive bone tumor of young adults. It typically presents as a large lytic mass at the end of the epiphysis of long bones. Grossly it is comprised of cystic and hemorrhagic areas with little or no periosteal reaction. Microscopically areas of frank hemorrhage, numerous multinucleated giant cells and spindly stromal cells are present. Telomeric fusions, increased telomerase activity and karyotypic aberrations have been advanced as a proof of its neoplastic nature. However such findings are not universal and can be seen in rapidly proliferating normal cells as well as in several osseous lesions of developmental and/or reactive nature, and the true neoplastic nature of GCTB remains controversial. The ancillary studies have generally not reached to the point where these alone can be taken as sole diagnostic and discriminatory criteria. While giant cells and stromal cells have been extensively studied, little attention has been paid to the overwhelming hemorrhagic component. If examined carefully intact and partially degenerated red blood cells are almost invariably seen in many giant cells as well as in the stroma. While hemorrhage in many patients may be resolved without leaving any trace over time, in some it gives rise to giant cell formation, and in others it may lead to proliferation of fibroblasts and histiocytes. At times one sees xanthomatous cells due to intracytoplasmic cholesterol deposits and sharp cholesterol clefts. Individual genetic makeup, local tissue factors as well as the amount of hemorrhage may play a key role in the final effects and outcome. Malignancy usually does not occur in GCTB and when discover, it usually represents primary bone sarcomas missed at original diagnosis. Embolization therapy to curtail hemorrhage and insertion of cement substance to support matrix are helpful in reducing recurrences. Aneurysmal bone cyst (ABC) shares many features with GCTB. There had been unique

  17. Cytokine profile of conditioned medium from human tumor cell lines after acute and fractionated doses of gamma radiation and its effect on survival of bystander tumor cells.

    PubMed

    Desai, Sejal; Kumar, Amit; Laskar, S; Pandey, B N

    2013-01-01

    Cytokines are known to play pivotal roles in cancer initiation, progression and pathogenesis. Accumulating evidences suggest differences in basal and stress-induced cytokine profiles of cancers with diverse origin. However, a comprehensive investigation characterising the cytokine profile of various tumor types after acute and fractionated doses of gamma-irradiation, and its effect on survival of bystander cells is not well known in literature. In the present study, we have evaluated the cytokine secretion profile of human tumor cell lines (HT1080, U373MG, HT29, A549 and MCF-7) either before (basal) or after acute (2, 6 Gy) and fractionated doses (3×2 Gy) of gamma-irradiation in culture medium obtained from these cells by multiplex bead array/ELISA. Moreover, clonogenic assays were performed to evaluate the effect of conditioned medium (CM) on the survival and growth of respective cells. Based on the screening of 28 analytes, our results showed that the basal profiles of these cell lines varied considerably in terms of the number and magnitude of secreted factors, which was minimum in MCF-7. Interestingly, TNF-α, IL-1β, PDGF-AA, TGF-β1, fractalkine, IL-8, VEGF and GCSF were found in CM of all the cell lines. However, secretion of certain cytokines was cell line-specific. Moreover, CM caused increase in clonogenic survival of respective tumor cells (in the order HT1080>U373MG>HT29>A549>MCF-7), which was correlated with th