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  1. Early specification of dopaminergic phenotype during ES cell differentiation

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    Li Meng

    2007-07-01

    Full Text Available Abstract Background Understanding how lineage choices are made during embryonic stem (ES cell differentiation is critical for harnessing strategies for controlled production of therapeutic somatic cell types for cell transplantation and pharmaceutical drug screens. The in vitro generation of dopaminergic neurons, the type of cells lost in Parkinson's disease patients' brains, requires the inductive molecules sonic hedgehog and FGF8, or an unknown stromal cell derived inducing activity (SDIA. However, the exact identity of the responding cells and the timing of inductive activity that specify a dopaminergic fate in neural stem/progenitors still remain elusive. Results Using ES cells carrying a neuroepithelial cell specific vital reporter (Sox1-GFP and FACS purification of Sox1-GFP neural progenitors, we have investigated the temporal aspect of SDIA mediated dopaminergic neuron specification during ES cell differentiation. Our results establish that SDIA induces a dopaminergic neuron fate in nascent neural stem or progenitor cells at, or prior to, Sox1 expression and does not appear to have further instructive role or neurotrophic activity during late neuronal differentiation of neural precursors. Furthermore, we show that dopaminergic neurons could be produced efficiently in a monolayer differentiation paradigm independent of SDIA activity or exogenous signalling molecules. In this case, the competence for dopaminergic neuron differentiation is also established at the level of Sox1 expression. Conclusion Dopaminergic neurons are specified early during mouse ES cell differentiation. The subtype specification seems to be tightly linked with the acquisition of a pan neuroectoderm fate.

  2. Wnt/ß-catenin signalling and the dynamics of fate decisions in early mouse embryos and embryonic stem (ES) cells.

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    Muñoz-Descalzo, Silvia; Hadjantonakis, Anna-Katerina; Arias, Alfonso Martinez

    2015-12-01

    Wnt/ß-catenin signalling is a widespread cell signalling pathway with multiple roles during vertebrate development. In mouse embryonic stem (mES) cells, there is a dual role for ß-catenin: it promotes differentiation when activated as part of the Wnt/ß-catenin signalling pathway, and promotes stable pluripotency independently of signalling. Although mES cells resemble the preimplantation epiblast progenitors, the first requirement for Wnt/ß-catenin signalling during mouse development has been reported at implantation [1,2]. The relationship between ß-catenin and pluripotency and that of mES cells with epiblast progenitors suggests that ß-catenin might have a functional role during preimplantation development. Here we summarize the expression and function of Wnt/ß-catenin signalling elements during the early stages of mouse development and consider the reasons why the requirement in ES cells do not reflect the embryo. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  3. Live cell imaging of the nascent inactive X chromosome during the early differentiation process of naive ES cells towards epiblast stem cells.

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    Guyochin, Aurélia; Maenner, Sylvain; Chu, Erin Tsi-Jia; Hentati, Asma; Attia, Mikael; Avner, Philip; Clerc, Philippe

    2014-01-01

    Random X-chromosome inactivation ensures dosage compensation in mammals through the transcriptional silencing of one of the two X chromosomes present in each female cell. Silencing is initiated in the differentiating epiblast of the mouse female embryos through coating of the nascent inactive X chromosome by the non-coding RNA Xist, which subsequently recruits the Polycomb Complex PRC2 leading to histone H3-K27 methylation. Here we examined in mouse ES cells the early steps of the transition from naive ES cells towards epiblast stem cells as a model for inducing X chromosome inactivation in vitro. We show that these conditions efficiently induce random XCI. Importantly, in a transient phase of this differentiation pathway, both X chromosomes are coated with Xist RNA in up to 15% of the XX cells. In an attempt to determine the dynamics of this process, we designed a strategy aimed at visualizing the nascent inactive X-chromosome in live cells. We generated transgenic female XX ES cells expressing the PRC2 component Ezh2 fused to the fluorescent protein Venus. The fluorescent fusion protein was expressed at sub-physiological levels and located in nuclei of ES cells. Upon differentiation of ES cell towards epiblast stem cell fate, Venus-fluorescent territories appearing in interphase nuclei were identified as nascent inactive X chromosomes by their association with Xist RNA. Imaging of Ezh2-Venus for up to 24 hours during the differentiation process showed survival of some cells with two fluorescent domains and a surprising dynamics of the fluorescent territories across cell division and in the course of the differentiation process. Our data reveal a strategy for visualizing the nascent inactive X chromosome and suggests the possibility for a large plasticity of the nascent inactive X chromosome.

  4. Live cell imaging of the nascent inactive X chromosome during the early differentiation process of naive ES cells towards epiblast stem cells.

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    Aurélia Guyochin

    Full Text Available Random X-chromosome inactivation ensures dosage compensation in mammals through the transcriptional silencing of one of the two X chromosomes present in each female cell. Silencing is initiated in the differentiating epiblast of the mouse female embryos through coating of the nascent inactive X chromosome by the non-coding RNA Xist, which subsequently recruits the Polycomb Complex PRC2 leading to histone H3-K27 methylation. Here we examined in mouse ES cells the early steps of the transition from naive ES cells towards epiblast stem cells as a model for inducing X chromosome inactivation in vitro. We show that these conditions efficiently induce random XCI. Importantly, in a transient phase of this differentiation pathway, both X chromosomes are coated with Xist RNA in up to 15% of the XX cells. In an attempt to determine the dynamics of this process, we designed a strategy aimed at visualizing the nascent inactive X-chromosome in live cells. We generated transgenic female XX ES cells expressing the PRC2 component Ezh2 fused to the fluorescent protein Venus. The fluorescent fusion protein was expressed at sub-physiological levels and located in nuclei of ES cells. Upon differentiation of ES cell towards epiblast stem cell fate, Venus-fluorescent territories appearing in interphase nuclei were identified as nascent inactive X chromosomes by their association with Xist RNA. Imaging of Ezh2-Venus for up to 24 hours during the differentiation process showed survival of some cells with two fluorescent domains and a surprising dynamics of the fluorescent territories across cell division and in the course of the differentiation process. Our data reveal a strategy for visualizing the nascent inactive X chromosome and suggests the possibility for a large plasticity of the nascent inactive X chromosome.

  5. Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives

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    Hummel Oliver

    2009-02-01

    Full Text Available Abstract Background Mouse embryonic stem (ES cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF. Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency. Results By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed. Conclusion We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment, which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG is still expressed and which might be down

  6. TGF-β/Smad2/3 signaling directly regulates several miRNAs in mouse ES cells and early embryos.

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    Nicholas Redshaw

    Full Text Available The Transforming Growth Factor-β (TGF-β signaling pathway is one of the major pathways essential for normal embryonic development and tissue homeostasis, with anti-tumor but also pro-metastatic properties in cancer. This pathway directly regulates several target genes that mediate its downstream functions, however very few microRNAs (miRNAs have been identified as targets. miRNAs are modulators of gene expression with essential roles in development and a clear association with diseases including cancer. Little is known about the transcriptional regulation of the primary transcripts (pri-miRNA, pri-miR from which several mature miRNAs are often derived. Here we present the identification of miRNAs regulated by TGF-β signaling in mouse embryonic stem (ES cells and early embryos. We used an inducible ES cell system to maintain high levels of the TGF-β activated/phosphorylated Smad2/3 effectors, which are the transcription factors of the pathway, and a specific inhibitor that blocks their activation. By performing short RNA deep-sequencing after 12 hours Smad2/3 activation and after 16 hours inhibition, we generated a database of responsive miRNAs. Promoter/enhancer analysis of a subset of these miRNAs revealed that the transcription of pri-miR-181c/d and the pri-miR-341∼3072 cluster were found to depend on activated Smad2/3. Several of these miRNAs are expressed in early mouse embryos, when the pathway is known to play an essential role. Treatment of embryos with TGF-β inhibitor caused a reduction of their levels confirming that they are targets of this pathway in vivo. Furthermore, we showed that pri-miR-341∼3072 transcription also depends on FoxH1, a known Smad2/3 transcription partner during early development. Together, our data show that miRNAs are regulated directly by the TGF-β/Smad2/3 pathway in ES cells and early embryos. As somatic abnormalities in functions known to be regulated by the TGF-β/Smad2/3 pathway underlie tumor

  7. Transcriptome analysis of chicken ES, blastodermal and germ cells reveals that chick ES cells are equivalent to mouse ES cells rather than EpiSC

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    Christian Jean

    2015-01-01

    Full Text Available Pluripotent Embryonic Stem cell (ESC lines can be derived from a variety of sources. Mouse lines derived from the early blastocyst and from primordial germ cells (PGCs can contribute to all somatic lineages and to the germ line, whereas cells from slightly later embryos (EpiSC no longer contribute to the germ line. In chick, pluripotent ESCs can be obtained from PGCs and from early blastoderms. Established PGC lines and freshly isolated blastodermal cells (cBC can contribute to both germinal and somatic lineages but established lines from the former (cESC can only produce somatic cell types. For this reason, cESCs are often considered to be equivalent to mouse EpiSC. To define these cell types more rigorously, we have performed comparative microarray analysis to describe a transcriptomic profile specific for each cell type. This is validated by real time RT-PCR and in situ hybridisation. We find that both cES and cBC cells express classic pluripotency-related genes (including cPOUV/OCT4, NANOG, SOX2/3, KLF2 and SALL4, whereas expression of DAZL, DND1, DDX4 and PIWIL1 defines a molecular signature for germ cells. Surprisingly, contrary to the prevailing view, our results also suggest that cES cells resemble mouse ES cells more closely than mouse EpiSC.

  8. Mouse ES cell culture system as a model of development.

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    Niwa, Hitoshi

    2010-04-01

    Mouse embryonic stem (mES) cells are pluripotent stem cells derived from pre-implantation embryos. They are regarded as an essential tool for studying mouse development, as they provide a means for generating knock-out mouse lines. This, however, is not the sole utility of the mES cell system. They undergo differentiation in culture, mimicking the morphological differentiation of peri-implantation embryos from epiblast to egg-cylinder stage. Moreover, they retain the capacity to respond to triggers of differentiation toward trophectoderm and primitive endoderm by forced activation. For these reasons, mES cells can be regarded as a useful tool for analyzing molecular mechanisms underlying early mouse development.

  9. Human ES cells: starting culture from frozen cells.

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    Trish, Erin; Dimos, John; Eggan, Kevin

    2006-11-09

    Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80 C freezer is sourced and quickly submerged in a 37 C water bath for quick thawing. Cells in freezing media are then removed from the vial and placed in a large volume of HuES media. The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate. Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate. It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate. The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

  10. The Epigenetic Paradox of Pluripotent ES Cells.

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    Festuccia, Nicola; Gonzalez, Inma; Navarro, Pablo

    2017-05-19

    The propagation and maintenance of gene expression programs are at the foundation of the preservation of cell identity. A large and complex set of epigenetic mechanisms enables the long-term stability and inheritance of transcription states. A key property of authentic epigenetic regulation is being independent from the instructive signals used for its establishment. This makes epigenetic regulation, particularly epigenetic silencing, extremely robust and powerful to lock regulatory states and stabilise cell identity. In line with this, the establishment of epigenetic silencing during development restricts cell potency and maintains the cell fate choices made by transcription factors (TFs). However, how more immature cells that have not yet established their definitive fate maintain their transitory identity without compromising their responsiveness to signalling cues remains unclear. A paradigmatic example is provided by pluripotent embryonic stem (ES) cells derived from a transient population of cells of the blastocyst. Here, we argue that ES cells represent an interesting "epigenetic paradox": even though they are captured in a self-renewing state characterised by extremely efficient maintenance of their identity, which is a typical manifestation of robust epigenetic regulation, they seem not to heavily rely on classical epigenetic mechanisms. Indeed, self-renewal strictly depends on the TFs that previously instructed their undifferentiated identity and relies on a particular signalling-dependent chromatin state where repressive chromatin marks play minor roles. Although this "epigenetic paradox" may underlie their exquisite responsiveness to developmental cues, it suggests that alternative mechanisms to faithfully propagate gene regulatory states might be prevalent in ES cells. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  11. Human ES cells: Starting Culture from Frozen Cells

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    Trish, Erin; Dimos, John; Eggan, Kevin

    2006-01-01

    Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submer...

  12. Zfp322a Regulates mouse ES cell pluripotency and enhances reprogramming efficiency.

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    Hui Ma

    2014-02-01

    Full Text Available Embryonic stem (ES cells derived from the inner cell mass (ICM of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotency maintenance of mouse ES cells. Here, we report that Zfp322a is required for mES cell identity since depletion of Zfp322a directs mES cells towards differentiation. Chromatin immunoprecipitation (ChIP and dual-luciferase reporter assays revealed that Zfp322a binds to Pou5f1 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of mES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated mES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits mES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka's factors (OSKM. It can be even used in combination with Yamanaka's factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of mouse ES cells.

  13. Improved Establishment of Embryonic Stem (ES) Cell Lines from the Chinese Kunming Mice by Hybridization with 129 Mice

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    Yu, Shumin; Yan, Xingrong; Liu, Huanhuan; Cai, Xin; Cao, Suizhong; Shen, Liuhong; Zuo, Zhicai; Deng, Junliang; Ma, Xiaoping; Wang, Ya; Ren, Zhihua

    2014-01-01

    Chinese Kunming mice (Mus musculus Km), widely used as laboratory animals throughout China, remain very refractory for embryonic stem (ES) cell isolation. The present study was aimed to evaluate the effects of hybridization with 129/Sv mice, and culture media containing fetal bovine serum (FBS) or Knockout serum replacement (KSR) on ES cell isolation from Kunming mice. The results demonstrated that ES cells had been effectively isolated from the hybrid embryos of Kunming and 129/Sv mice using all three media containing 15% FBS, 15% KSR and their mixture of 14% KSR and 1% FBS, individually. These isolated ES cells had maintained in vitro undifferentiated for a long time, exhibiting all features specific for mouse ES cells. In addition, the rates of ES cell isolation in the medium containing 14% KSR and 1% FBS, was 46.67% and significantly higher than those in another two media containing only FBS or KSR (p < 0.05). Contrarily, no ES cell line had been established from Kunming mouse inbred embryos using the same protocols. These results suggested that ES cells with long-term self-renewal ability could be efficiently generated from hybrid embryos of Kunming and 129/Sv mice, and a small volume of FBS was necessary to isolate ES cells in the KSR medium when embryos and early ES cells cultured. PMID:24573251

  14. Improved Establishment of Embryonic Stem (ES Cell Lines from the Chinese Kunming Mice by Hybridization with 129 Mice

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    Shumin Yu

    2014-02-01

    Full Text Available Chinese Kunming mice (Mus musculus Km, widely used as laboratory animals throughout China, remain very refractory for embryonic stem (ES cell isolation. The present study was aimed to evaluate the effects of hybridization with 129/Sv mice, and culture media containing fetal bovine serum (FBS or Knockout serum replacement (KSR on ES cell isolation from Kunming mice. The results demonstrated that ES cells had been effectively isolated from the hybrid embryos of Kunming and 129/Sv mice using all three media containing 15% FBS, 15% KSR and their mixture of 14% KSR and 1% FBS, individually. These isolated ES cells had maintained in vitro undifferentiated for a long time, exhibiting all features specific for mouse ES cells. In addition, the rates of ES cell isolation in the medium containing 14% KSR and 1% FBS, was 46.67% and significantly higher than those in another two media containing only FBS or KSR (p < 0.05. Contrarily, no ES cell line had been established from Kunming mouse inbred embryos using the same protocols. These results suggested that ES cells with long-term self-renewal ability could be efficiently generated from hybrid embryos of Kunming and 129/Sv mice, and a small volume of FBS was necessary to isolate ES cells in the KSR medium when embryos and early ES cells cultured.

  15. Cloning of ES cells and mice by nuclear transfer.

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    Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

    2009-01-01

    We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

  16. Risk Factors of Early Childhood Caries among Dar es Salaam ...

    African Journals Online (AJOL)

    Background: Early childhood caries (ECC) describes caries experience on at least one primary tooth in children under six years of age. It is among the most common chronic diseases in young children and may develop as soon as the teeth erupt. Thus it presents a serious problem in pediatric dentistry not only because of ...

  17. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

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    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  18. Connexin expression and gap-junctional intercellular communication in ES cells and iPS cells.

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    Oyamada, Masahito; Takebe, Kumiko; Endo, Aya; Hara, Sachiko; Oyamada, Yumiko

    2013-01-01

    Pluripotent stem cells, i.e., embryonic stem (ES) and induced pluripotent stem (iPS) cells, can indefinitely proliferate without commitment and differentiate into all cell lineages. ES cells are derived from the inner cell mass of the preimplantation blastocyst, whereas iPS cells are generated from somatic cells by overexpression of a few transcription factors. Many studies have demonstrated that mouse and human iPS cells are highly similar but not identical to their respective ES cell counterparts. The potential to generate basically any differentiated cell types from these cells offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. ES cells and iPS cells also provide useful models to study connexin expression and gap-junctional intercellular communication (GJIC) during cell differentiation and reprogramming. In 1996, we reported connexin expression and GJIC in mouse ES cells. Because a substantial number of papers on these subjects have been published since our report, this Mini Review summarizes currently available data on connexin expression and GJIC in ES cells and iPS cells during undifferentiated state, differentiation, and reprogramming.

  19. Cell to Cell Signalling via Exosomes Through esRNA

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    Lotvall, Jan; Valadi, Hadi

    2007-01-01

    Exosomes are small vesicles of endosomal origin that can be released by many different cells to the microenvironment. Exosomes have been shown to participate in the immune system, by mediating antigen presentation. We have recently shown the presence of both mRNA and microRNA in exosomes, specifically in exosomes derived from mast cells. This RNA can be transferred between one mast cell to another, most likely through fusion of the exosome to the recipient cell membrane. The delivered RNA is ...

  20. Mesenchymal stem cells originating from ES cells show high telomerase activity and therapeutic benefits.

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    Ninagawa, Nana; Murakami, Rumi; Isobe, Eri; Tanaka, Yusuke; Nakagawa, Hiroki; Torihashi, Shigeko

    2011-10-01

    We establish a novel method for the induction and collection of mesenchymal stem cells using a typical cell surface marker, CD105, through adipogenesis from mouse ES cells. ES cells were cultured in a medium for adipogenesis. Mesenchymal stem cells from mouse ES cells were easily identified by the expression of CD105, and were isolated and differentiated into multiple mesenchymal cell types. Mesenchymal stem cells showed remarkable telomerase activity and sustained their growth for a long time with a high potential for differentiation involving skeletal myogenesis in vitro. When mesenchymal stem cells were transplanted into the injured tibialis anterior muscles, they differentiated into skeletal muscle cells in vivo. In addition, they improved the vascular formation, but never formed teratoma for longer than 6 months. Gene expression profiles revealed that mesenchymal stem cells lost pluripotency, while they acquired high potential to differentiate into mesenchymal cell lines. They thus indicate a promising new source of cell-based therapy without teratoma formation. Copyright © 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  1. [Methionine metabolism regulates maintenance and differentiation of human ES/iPS cells].

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    Shiraki, Nobuaki; Kume, Shoen

    2015-05-01

    Embryonic stem (ES) and induced pluripotent stem (iPS) cells are pluripotent and can give rise to all cell types. ES/iPS cells have a unique transcriptional circuit that sustains the pluripotent state. These cells also possess a characteristically high rate of proliferation as well as an abbreviated G1 phase. These unique molecular properties distinguish ES and iPS cells from somatic cells. Mouse ES/iPS cells are in a high-flux metabolic state, with a high dependence on threonine catabolism. However, little is known about amino acid metabolism in human ES/iPS cells. Recently, we reported that human ES/iPS cells require high amounts of methionine (Met) and express high levels of Met metabolism enzymes (Shriaki N, et al: Cell Metabolism, 2014). Met deprivation results in a rapid decrease in intracellular S-adenosyl-methionine (SAM), triggering the activation of p53 signaling, reducing pluripotent marker gene NANOG expression, and poising human ES/iPS cells for differentiation, follow by potentiated differentiation into all three germ layers. However, when exposed to prolonged Met deprivation, the cells went to apoptosis. In this review, we explain the importance of SAM in Met metabolism and its relationship with pluripotency, cell survival, and differentiation of human ES/iPS cells.

  2. Mouse ES cell-derived cardiac precursor cells are multipotent and facilitate identification of novel cardiac genes

    National Research Council Canada - National Science Library

    Christoforou, Nicolas; Miller, Ronald A; Hill, Christine M; Jie, Chunfa C; McCallion, Andrew S; Gearhart, John D

    2008-01-01

    Although the differentiation of ES cells to cardiomyocytes has been firmly established, the extent to which corresponding cardiac precursor cells can contribute to other cardiac populations remains unclear...

  3. Matrix Metalloproteinase-3 in Odontoblastic Cells Derived from Ips Cells: Unique Proliferation Response as Odontoblastic Cells Derived from ES Cells

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    Hiyama, Taiki; Ozeki, Nobuaki; Mogi, Makio; Yamaguchi, Hideyuki; Kawai, Rie; Nakata, Kazuhiko; Kondo, Ayami; Nakamura, Hiroshi

    2013-01-01

    We previously reported that matrix metalloproteinase (MMP)-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL)-1β and interferon-γ) induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES) cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS) and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (PiPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (PiPS and ES cells. PMID:24358294

  4. ELECTROPORATION OF CONFLUENT HM-1 ES CELLS LEADS TO HIGHER AMOUNT COLONIES

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    A.ALENA BENCSIK

    2008-05-01

    Full Text Available Electroporation is used to induce homologous recombination in the genome of the murine ES (embryonic stem cells. Routinelly subconfluent ES cells are recommended to be used in such experiments. Electroporation of immunoglobulin specific targeting vectors with different length of homology leads to reduced number of selected colonies. The enrichment of double selected colonies is high and thus the amount of HM-1 ES cell colonies for the analysis is very low. Here we show that the electroporation of confluent HM-1 ES cells leads to an increased amount of simple and double selected colonies.

  5. Matrix metalloproteinase-3 in odontoblastic cells derived from ips cells: unique proliferation response as odontoblastic cells derived from ES cells.

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    Taiki Hiyama

    Full Text Available We previously reported that matrix metalloproteinase (MMP-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL-1β and interferon-γ induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (P<0.05. Exogenous MMP-3 was found to induce cell proliferation in odontoblast-like cells derived from iPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P<0

  6. Sox7 is dispensable for primitive endoderm differentiation from mouse ES cells.

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    Kinoshita, Masaki; Shimosato, Daisuke; Yamane, Mariko; Niwa, Hitoshi

    2015-10-16

    Primitive endoderm is a cell lineage segregated from the epiblast in the blastocyst and gives rise to parietal and visceral endoderm. Sox7 is a member of the SoxF gene family that is specifically expressed in primitive endoderm in the late blastocyst, although its function in this cell lineage remains unclear. Here we characterize the function of Sox7 in primitive endoderm differentiation using mouse embryonic stem (ES) cells as a model system. We show that ectopic expression of Sox7 in ES cells has a marginal effect on triggering differentiation into primitive endoderm-like cells. We also show that targeted disruption of Sox7 in ES cells does not affect differentiation into primitive endoderm cells in embryoid body formation as well as by forced expression of Gata6. These data indicate that Sox7 function is supplementary and not essential for this differentiation from ES cells.

  7. MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

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    STANCA CLAUDIA

    2007-01-01

    Full Text Available In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989, but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass, it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1 mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP, because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air. The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females.

  8. Abrogation of Gap Junctional Communication in ES Cells Results in a Disruption of Primitive Endoderm Formation in Embryoid Bodies.

    Science.gov (United States)

    Wörsdörfer, Philipp; Bosen, Felicitas; Gebhardt, Martina; Russ, Nicole; Zimmermann, Katrin; Komla Kessie, David; Sekaran, Thileepan; Egert, Angela; Ergün, Süleyman; Schorle, Hubert; Pfeifer, Alexander; Edenhofer, Frank; Willecke, Klaus

    2017-04-01

    Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre-loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies (EBs) without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in EB cultures, representing the inductive key step of further differentiation events. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC-incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx-mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well-described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells. Stem Cells 2017;35:859-871. © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  9. Competence of an artificial bent DNA as a transcriptional activator in mouse ES cells.

    Science.gov (United States)

    Tanase, Jun-ichi; Mitani, Tasuku; Udagawa, Koji; Nishikawa, Jun-ichi; Ohyama, Takashi

    2011-01-01

    Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. However, their potency in transgene activation in embryonic stem (ES) cells has not been examined. T20 is an artificial curved DNA of 180 bp that serves as a transcriptional activator. We investigated the effect of T20 on transcription in mouse ES cell lines or hepatocytes differentiated from them. We established 10 sets of cell lines each harboring a single copy of the reporter construct. Each set comprised a T20-harboring cell line and a T20-less control cell line. Analyses showed that in ES cells and in hepatocytes originating from these cells, T20 both activated and repressed transcription in a manner that was dependent on the locus of reporter. The present and previous studies strongly suggest that in cells that have a strict gene regulation system, transcriptional activation by T20 occurs only in a transcriptionally active locus in the genome.

  10. Effect of skin fibroblast-derived allogeneic feeder cells on porcine ES-like cell establishment.

    Science.gov (United States)

    Panasophonkul, Sasithorn; Tharasanit, Theerawat; Techakumphu, Mongkol

    2012-10-01

    In the present study, the effect of two types of allogeneic-derived feeder cells [porcine ear and tail skin fibroblasts (PESF, PTSF)] and three types of xenogeneic-derived feeder cells [human foreskin fibroblasts (HFK), mouse embryonic fibroblasts (MEF) and immortalized mouse embryonic fibroblasts (STO)] on the isolation and cultivation of putative porcine embryonic stem cells (pESCs) was evaluated. In vivo derived zona pellucida (ZP)-free blastocysts were cultured on different mitotically inactivated feeder layers. The rates of ICM outgrowth and primary colony formation were observed, and further passage onto new feeders was performed. The characteristics of pESCs, including alkaline phosphatase (AP) activity, and pluripotent-related markers (OCT3/4, NANOG, SSEA-4) and genes were examined. Attached blastocysts cultured on HFK and STO feeders showed a higher percentage of ICM outgrowths than those cultured on PESF (76.7, 72.9 and 38.9%, respectively; P<0.05). The rates of primary ES-like colony formation and the number of putative ESC lines were significantly decreased when ICM outgrowths were cultured on PESF, compared with those cultured on HFK (30.6 vs. 76.7%, respectively; P<0.05). Only ES-like colonies from one (25%) and three (50%) cell lines developed on PTSF and STO feeders, respectively, were further maintained in an undifferentiated morphology associated with the presence of all ES characteristics; however, these characteristics disappeared when colonies were continued to the 8th and 6th passages, respectively. The present study indicated that feeder cell types affect the success of pESC establishment and maintenance of their pluripotency.

  11. Regulation of Pax6 by CTCF during induction of mouse ES cell differentiation.

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    Jie Gao

    Full Text Available Pax6 plays an important role in embryonic cell (ES differentiation during embryonic development. Expression of Pax6 undergoes from a low level to high levels following ES cell differentiation to neural stem cells, and then fades away in most of the differentiated cell types. There is a limited knowledge concerning how Pax6 is regulated in ES cell differentiation. We report that Pax6 expression in mouse ES cells was controlled by CCCTC binding factor (CTCF through a promoter repression mechanism. Pax6 expression was significantly enhanced while CTCF activity was kept in the constant during ES cell differentiation to radial glial cells. Instead, the interaction of CTCF with Pax6 gene was regulated by decreased CTCF occupancy in its binding motifs upstream from Pax6 P0 promoter following the course of ES cell differentiation. Reduced occupancy of CTCF in the binding motif region upstream from the P0 promoter was due to increased DNA methylations in the CpG sites identified in the region. Furthermore, changes in DNA methylation levels in vitro and in vivo effectively altered methylation status of these identified CpG sites, which affected ability of CTCF to interact with the P0 promoter, resulting in increases in Pax6 expression. We conclude that there is an epigenetic mechanism involving regulations of Pax6 gene during ES cell differentiation to neural stem cells, which is through increases or decreases in methylation levels of Pax6 gene to effectively alter the ability of CTCF in control of Pax6 expression, respectively.

  12. Teaching Aerobic Cell Respiration Using the 5Es

    Science.gov (United States)

    Patro, Edward T.

    2008-01-01

    The 5E teaching model provides a five step method for teaching science. While the sequence of the model is strictly linear, it does provide opportunities for the teacher to "revisit" prior learning before moving on. The 5E method is described as it relates to the teaching of aerobic cell respiration.

  13. Mast Cell Subsets and Their Functional Modulation by the Acanthocheilonema viteae Product ES-62

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    Dimity H. Ball

    2013-01-01

    Full Text Available ES-62, an immunomodulator secreted by filarial nematodes, exhibits therapeutic potential in mouse models of allergic inflammation, at least in part by inducing the desensitisation of FcεRI-mediated mast cell responses. However, in addition to their pathogenic roles in allergic and autoimmune diseases, mast cells are important in fighting infection, wound healing, and resolving inflammation, reflecting that mast cells exhibit a phenotypic and functional plasticity. We have therefore characterised the differential functional responses to antigen (via FcεRI and LPS and their modulation by ES-62 of the mature peritoneal-derived mast cells (PDMC; serosal and those of the connective tissue-like mast cells (CTMC and the mucosal-like mast cells derived from bone marrow progenitors (BMMC as a first step to produce disease tissue-targeted therapeutics based on ES-62 action. All three mast cell populations were rendered hyporesponsive by ES-62 and whilst the mechanisms underlying such desensitisation have not been fully delineated, they reflect a downregulation of calcium and PKCα signalling. ES-62 also downregulated MyD88 and PKCδ in mucosal-type BMMC but not PDMC, the additional signals targeted in mucosal-type BMMC likely reflecting that these cells respond to antigen and LPS by degranulation and cytokine secretion whereas PDMC predominantly respond in a degranulation-based manner.

  14. Human embryonic stem cell (hES derived dendritic cells are functionally normal and are susceptible to HIV-1 infection

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    Bandi Sriram

    2008-01-01

    Full Text Available Abstract Background Human embryonic stem (hES cells hold considerable promise for cell replacement and gene therapies. Their remarkable properties of pluripotency, self-renewal, and tractability for genetic modification potentially allows for the production of sizeable quantities of therapeutic cells of the hematopoietic lineage. Dendritic cells (DC arise from CD34+ hematopoietic progenitor cells (HPCs and are important in many innate and adaptive immune functions. With respect to HIV-1 infection, DCs play an important role in the efficient capture and transfer of the virus to susceptible cells. With an aim of generating DCs from a renewable source for HIV-1 studies, here we evaluated the capacity of hES cell derived CD34+ cells to give rise to DCs which can support HIV-1 infection. Results Undifferentiated hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34+ HPCs which were subsequently grown in specific cytokine differentiation media to promote the development of DCs. The hES derived DCs (hES-DC were subjected to phenotypic and functional analyses and compared with DCs derived from fetal liver CD34+ HPC (FL-DC. The mature hES-DCs displayed typical DC morphology consisting of veiled stellate cells. The hES-DCs also displayed characteristic phenotypic surface markers CD1a, HLA-DR, B7.1, B7.2, and DC-SIGN. The hES-DCs were found to be capable of antigen uptake and stimulating naïve allogeneic CD4+ T cells in a mixed leukocyte reaction assay. Furthermore, the hES-DCs supported productive HIV-1 viral infection akin to standard DCs. Conclusion Phenotypically normal and functionally competent DCs that support HIV-1 infection can be derived from hES cells. hES-DCs can now be exploited in applied immunology and HIV-1 infection studies. Using gene therapy approaches, it is now possible to generate HIV-1 resistant DCs from anti-HIV gene transduced hES-CD34+ hematopoietic progenitor cells.

  15. Transposon-mediated BAC transgenesis in human ES cells.

    Science.gov (United States)

    Rostovskaya, Maria; Fu, Jun; Obst, Mandy; Baer, Isabell; Weidlich, Stefanie; Wang, Hailong; Smith, Andrew J H; Anastassiadis, Konstantinos; Stewart, A Francis

    2012-10-01

    Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs.

  16. Generation of mouse ES cell lines engineered for the forced induction of transcription factors.

    Science.gov (United States)

    Correa-Cerro, Lina S; Piao, Yulan; Sharov, Alexei A; Nishiyama, Akira; Cadet, Jean S; Yu, Hong; Sharova, Lioudmila V; Xin, Li; Hoang, Hien G; Thomas, Marshall; Qian, Yong; Dudekula, Dawood B; Meyers, Emily; Binder, Bernard Y; Mowrer, Gregory; Bassey, Uwem; Longo, Dan L; Schlessinger, David; Ko, Minoru S H

    2011-01-01

    Here we report the generation and characterization of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7-10% of all TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phenotypes. These cell lines and microarray data provide building blocks for a variety of future biomedical research applications as a community resource.

  17. Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission.

    Science.gov (United States)

    Liu, X; Wu, H; Loring, J; Hormuzdi, S; Disteche, C M; Bornstein, P; Jaenisch, R

    1997-05-01

    The ability to contribute to the germ line is the most important experimental feature of embryonic stem (ES) cells. Using ES cells, it is possible to introduce targeted mutations into any gene and to derive the corresponding mutant mice. A common problem with this technology is that the ES cells often lack or have only a low efficiency of germ line transmission. To address this issue, we examined the relationship between the growth rate and karyotype of ES cells, and their ability to contribute to the germ line. We found that chromosomal abnormalities occurred rather frequently in ES cells. Cells having an abnormal number of chromosomes, in particular trisomy 8, were found in three independently derived ES cell lines, and this abnormality conferred a selective growth advantage on these cells. Selection of abnormal cells led to depletion and eventual loss of normal ES cells during consecutive passages. In comparison with parental ES cells, ES cells with trisomy 8 contributed rarely to the germ line. This realization allowed us to select, based upon ES cell clone morphology, those clones with the highest probability of contributing to the germ line. This insight is of practical value for any given gene targeting experiment as it permits optimization of the rate of success without having to rely on more elaborate tests such as karyotyping individual clones prior to blastocyst injection.

  18. Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Shirai, Hidenori; Fujimori, Hiroaki [Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Gunji, Akemi [Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Maeda, Daisuke [Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); ADP-Ribosylation in Oncology Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Hirai, Takahisa [Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Department of Radiation Oncology, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Poetsch, Anna R. [ADP-Ribosylation in Oncology Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Harada, Hiromi [Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Yoshida, Tomoko [Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minatoku, Tokyo 105-8512 (Japan); Sasai, Keisuke [Department of Radiation Oncology, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Okayasu, Ryuichi [International Open Laboratory, National Institute of Radiological Science, 4-9-1 Anagawa, Inage, Chiba 263-8555 (Japan); Masutani, Mitsuko, E-mail: mmasutan@ncc.go.jp [Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); ADP-Ribosylation in Oncology Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2013-05-24

    Highlights: •Parg{sup −/−} ES cells were more sensitive to γ-irradiation than Parp-1{sup −/−} ES cells. •Parg{sup −/−} cells were more sensitive to carbon-ion irradiation than Parp-1{sup −/−} cells. •Parg{sup −/−} cells showed defects in DSB repair after carbon-ion irradiation. •PAR accumulation was enhanced after carbon-ion irradiation compared to γ-irradiation. -- Abstract: Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg{sup −/−} and poly(ADP-ribose) polymerase-1 deficient (Parp-1{sup −/−}) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg{sup −/−} cells were more sensitive to γ-irradiation than Parp-1{sup −/−} cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg{sup −/−} cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg{sup −/−} ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1{sup −/−} cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg{sup −/−} ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/μm) and Fe-ion irradiation (200 keV/μm) were also examined. Parg{sup −/−} cells were more sensitive to LET 70 keV/μm carbon-ion irradiation than Parp-1{sup −/−} cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24 h. The induction level of p53 phophorylation at ser18 was

  19. p53 gene targeting by homologous recombination in fish ES cells.

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    Yan Yan

    Full Text Available BACKGROUND: Gene targeting (GT provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR events in the fish medaka (Oryzias latipes. METHODOLOGY AND PRINCIPAL FINDINGS: Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53. We show that all the three medaka ES cell lines, MES1∼MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS procedure enhanced HR by ∼12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7% were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5% were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo. CONCLUSIONS: Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.

  20. Neural induction from ES cells portrays default commitment but instructive maturation.

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    Nibedita Lenka

    Full Text Available The neural induction has remained a debatable issue pertaining to whether it is a mere default process or it involves precise instructive cues. We have chosen the embryonic stem (ES cell model to address this issue. In a devised monoculture strategy, the cell-cell interaction availed through optimum cell plating density could define the niche for the attainment of efficient in vitro neurogenesis from the ES cells. The medium plating density was found ideal in generating optimum number of progenitors and also yielded about 80% mature neurons in a serum free culture set up barring any exogenous inducers. We could also demarcate and quantify the neural stem cells/progenitors among the heterogeneous cell population of differentiating ES cells using nestin intron II driven EGFP expression as a tool. The one week post-plating was determined to be the critical time window for optimum neural progenitor generation from ES cells that helped us further in purifying these cells and in demonstrating their proliferation and multipotent differentiation potential. Seeding cells at varying densities, we could decipher an interesting paradoxical scenario that interlinked both commitment and maturation with the initial plating density having a vital influence on neuronal maturation but not specification and the secretory factors were apparently playing a key role during this process. Thus it was comprehended that, the neural specification was a default process independent of exogenous factors and cellular interaction. Conversely, a defined number of cells at the specification stage itself seemed critical to provide an auto-/paracrine means of signaling threshold for the maturation process to materialize.

  1. BMP4 and FGF strongly induce differentiation of mouse ES cells into oral ectoderm

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    Hiroshi Ochiai

    2015-09-01

    Full Text Available During embryonic development, oral ectoderm differentiates into the adenohypophysis, dental epithelia, salivary glands, and nasal pit. Few reports exist concerning the induction of oral ectoderm from embryonic stem (ES cells. Generally, any lot differences in fetal bovine serum (FBS and serum replacer may affect the induction of ES cell-differentiation. Using a previously established culture strategy for differentiation, the proportion of cell aggregates containing Pitx1+ oral ectoderm varied widely between 9–36% when several different lots of FBS or serum replacer were used. We therefore tried to enhance the differentiation method. We found that bone morphogenetic protein (BMP 4 and fibroblast growth factor (FGF treatments improved oral ectoderm induction. Such treatment also improved the differentiation of oral ectoderm into the adenohypophysis. Furthermore, increased BMP4 treatment induced dental epithelium and mesenchyme. Such differentiation suggests that the Pitx1+ layer displays similar properties to oral ectoderm, as found in vivo. Differentiation of ES cells into oral ectoderm using different lots of FBS and serum replacer increased 78–90% after treatment with BMP4 and FGF. In summary, we have established a robust strategy for the induction of oral ectoderm differentiation from mouse ES cells.

  2. Gene expression profiles of HLA-G1 overexpressed in hES cells.

    Science.gov (United States)

    Zhu, Yajing; Zhao, Sanjun; Zhao, Hongxi; Yao, Yuanqing

    2012-10-01

    The goals of this study were to analyze the change in the global gene expression profile of exogenous human leukocyte antigen-G1 (HLA-G1) overexpressed in human embryonic stem (hES) cells and to explore the molecular mechanism by which the overexpression of HLA-G1 modifies immunologic pathways. Microarray and quantitative real-time PCR analyses were performed to quantify the differential expression pattern of HLA-G1 + H1 hES cells. The results showed that HLA-G1 differentially regulated the expression of 425 genes with at least a twofold increase or decrease. These differentially expressed genes were classified into 13 functional groups, including cellular components, biological processes, and molecular functions. The pathways of focal adhesion, the TGF-β signaling pathway, and the immune response were the most predominantly affected. The synergism of these genes could explain the mechanism of the immunosuppression of HLA-G1 + H1 hES cells. Thus, the expression pattern reflected a broad spectrum of roles of HLA-G1 in hES cells.

  3. A simple improvement of the conventional cryopreservation for human ES and iPS cells

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Midori Ozawa, Yutaka Ozawa, Masashi Iemura, Arihiro Kohara, Kana Yanagihara & Miho K Furue ### Abstract In this study, a simple method for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is proposed. It is based on the conventional slow-freezing method with 10% DMSO and modified mainly in a thawing protocol without specific equipment or reagents. Recovery rate of the cells cryopreserved by this method was equally high, which is c...

  4. Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

    Science.gov (United States)

    Wakayama, Teruhiko

    2007-02-01

    Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

  5. miRNA regulatory circuits in ES cells differentiation: a chemical kinetics modeling approach.

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    Zijun Luo

    Full Text Available MicroRNAs (miRNAs play an important role in gene regulation for Embryonic Stem cells (ES cells, where they either down-regulate target mRNA genes by degradation or repress protein expression of these mRNA genes by inhibiting translation. Well known tables TargetScan and miRanda may predict quite long lists of potential miRNAs inhibitors for each mRNA gene, and one of our goals was to strongly narrow down the list of mRNA targets potentially repressed by a known large list of 400 miRNAs. Our paper focuses on algorithmic analysis of ES cells microarray data to reliably detect repressive interactions between miRNAs and mRNAs. We model, by chemical kinetics equations, the interaction architectures implementing the two basic silencing processes of miRNAs, namely "direct degradation" or "translation inhibition" of targeted mRNAs. For each pair (M,G of potentially interacting miRMA gene M and mRNA gene G, we parameterize our associated kinetic equations by optimizing their fit with microarray data. When this fit is high enough, we validate the pair (M,G as a highly probable repressive interaction. This approach leads to the computation of a highly selective and drastically reduced list of repressive pairs (M,G involved in ES cells differentiation.

  6. Generation of fertile and fecund F0 XY female mice from XY ES cells.

    Science.gov (United States)

    Kuno, Junko; Poueymirou, William T; Gong, Guochun; Siao, Chia-Jen; Clarke, Georgia; Esau, Lakeisha; Kojak, Nada; Posca, Julita; Atanasio, Amanda; Strein, John; Yancopoulos, George D; Lai, Ka-Man Venus; DeChiara, Thomas M; Frendewey, David; Auerbach, Wojtek; Valenzuela, David M

    2015-02-01

    Known examples of male to female sex reversal in mice are caused by either strain incompatibilities or mutations in genes required for male sex determination. The resultant XY females are often sterile or exhibit very poor fertility. We describe here embryonic stem (ES) cell growth conditions that promote the production of healthy, anatomically normal fertile and fecund female F0 generation mice completely derived from gene-targeted XY male ES cells. The sex reversal is a transient trait that is not transmitted to the F1 progeny. Growth media with low osmolality and reduced sodium bicarbonate, maintained throughout the gene targeting process, enhance the yield of XY females. As a practical application of the induced sex reversal, we demonstrate the generation of homozygous mutant mice ready for phenotypic studies by the breeding of F0 XY females with their isogenic XY male clonal siblings, thereby eliminating one generation of breeding and the associated costs.

  7. Derivation of normal macrophages from human embryonic stem (hES cells for applications in HIV gene therapy

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    Kaufman Dan S

    2006-04-01

    Full Text Available Abstract Background Many novel studies and therapies are possible with the use of human embryonic stem cells (hES cells and their differentiated cell progeny. The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications. Here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for AIDS. Results Undifferentiated normal and lentiviral vector transduced hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34 hematopoietic progenitor cells. The differentiated CD34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages. Phenotypic and functional analyses were carried out to compare these with that of fetal liver CD34 cell derived macrophages. As assessed by FACS analysis, the hES-CD34 cell derived macrophages displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR suggesting a normal phenotype. Tests evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation showed that these macrophages are also functionally normal. When infected with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells expressing the transgene GFP were evaluated similarly like above. The transgenic hES cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation. Conclusion Phenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells. Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies. Based on these results, it is also now feasible to

  8. ES cell differentiation system recapitulates the establishment of imprinted gene expression in a cell-type-specific manner.

    Science.gov (United States)

    Kohama, Chihiro; Kato, Hidemasa; Numata, Koji; Hirose, Michiko; Takemasa, Tohru; Ogura, Atsuo; Kiyosawa, Hidenori

    2012-03-15

    Genomic imprinting is a phenomenon whereby monoallelic gene expression occurs in a parent-of-origin-specific manner. A subset of imprinted genes acquires a tissue-specific imprinted status during the course of tissue development, and this process can be analyzed by means of an in vitro differentiation system utilizing embryonic stem (ES) cells. In neurons, the gene Ube3a is expressed from the maternal allele only, and a paternally expressed non-coding, antisense RNA has been implicated in the imprinting process in mice and humans. Here, to study the genomic imprinting mechanism, we established F1 hybrid ES cells derived from two sub-species of Mus musculus and established an in vitro neuronal differentiation system in which neuron-specific imprinting of Ube3a was recapitulated. With this system, we revealed that the switch from biallelic expression to maternal, monoallelic expression of Ube3a occurs late in neuronal development, during the neurite outgrowth period, and that the expression of endogenous antisense transcript from the Ube3a locus is up-regulated several hundred-fold during the same period. Our results suggest that evaluation of the quality of ES cells by studying their differentiation in vitro should include evaluation of epigenetic aspects, such as a comparison with the genomic imprinting status found in tissues in vivo, in addition to the evaluation of differentiation gene markers and morphology. Our F1 hybrid ES cells and in vitro differentiation system will allow researchers to investigate complex end-points such as neuron-specific genomic imprinting, and our F1 hybrid ES cells are a useful resource for other tissue-specific genomic imprinting and epigenetic analyses.

  9. Early-stage mantle cell lymphoma

    DEFF Research Database (Denmark)

    Dabaja, B S; Zelenetz, A D; Ng, A K

    2017-01-01

    Background: Mantle cell lymphoma (MCL) rarely presents as early-stage disease, but clinical observations suggest that patients who present with early-stage disease may have better outcomes than those with advanced-stage disease. Patients and methods: In this 13-institution study, we examined...

  10. The loss-of-allele assay for ES cell screening and mouse genotyping.

    Science.gov (United States)

    Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M

    2010-01-01

    Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction

  11. Early recognition of basal cell naevus syndrome

    NARCIS (Netherlands)

    Veenstra-Knol, HE; Scheewe, JH; van der Vlist, GJ; van Doorn, ME; Ausems, MGEM

    The basal cell naevus syndrome is an autosomal dominant syndrome characterised by major manifestations such as basal cell carcinomas, jaw cysts, palmar or plantar pits, and intracranial calcifications. Early recognition is important in order to reduce morbidity due to cutaneous and cerebral

  12. An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

    Directory of Open Access Journals (Sweden)

    David A. Turner

    2014-06-01

    Full Text Available Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a “race for fates” in which the neuroectodermal fate has an advantage.

  13. An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

    Science.gov (United States)

    Turner, David A.; Trott, Jamie; Hayward, Penelope; Rué, Pau; Martinez Arias, Alfonso

    2014-01-01

    ABSTRACT Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a “race for fates” in which the neuroectodermal fate has an advantage. PMID:24950969

  14. Mouse ES cells over-expressing the transcription factor NeuroD1 show increased differentiation towards endocrine lineages and insulin-expressing cells.

    Science.gov (United States)

    Marchand, Mélanie; Schroeder, Insa S; Markossian, Suzy; Skoudy, Anouchka; Nègre, Didier; Cosset, François-Loic; Real, Paco; Kaiser, Christian; Wobus, Anna M; Savatier, Pierre

    2009-01-01

    Embryonic stem (ES) cells which constitutively express the Pdx-1, Ngn-3, NeuroD1, Nkx2.2, and Nkx6.1 transcription factors were engineered by means of lentiviral vectors, following a multi-step infection procedure to successively generate ES cell lines expressing one, two, and three factors, respectively. Each ES cell line was allowed to differentiate into nestin+/Isl-1+ endocrine precursors, then into more mature pancreatic cells, and subsequently analysed for expression of Glc, Ins, and Sst, markers of alpha, beta and delta cells, respectively. Each ES cell line generated displayed a unique pattern of gene expression. The ES cell line expressing NeuroD1 displayed vastly elevated levels of Glc, Ins-1, Ins-2 and Sst, and showed an increase in Pdx-1, Pax-4, Nkx6.1, Isl-1, Glut-2 and GK transcript levels. Furthermore, immunofluorescence analysis revealed that differentiation of NeuroD1-expressing ES cells in nestin+/Isl-1+ multilineage progenitors, followed by the formation of C-peptide+/insulin+ clusters, was accelerated. Together, these results indicate that stable expression of NeuroD1 in ES cells facilitates differentiation into endocrine and insulin-producing cells.

  15. High glucose suppresses embryonic stem cell differentiation into cardiomyocytes : High glucose inhibits ES cell cardiogenesis.

    Science.gov (United States)

    Yang, Penghua; Chen, Xi; Kaushal, Sunjay; Reece, E Albert; Yang, Peixin

    2016-12-09

    Babies born to mothers with pregestational diabetes have a high risk for congenital heart defects (CHD). Embryonic stem cells (ESCs) are excellent in vitro models for studying the effect of high glucose on cardiac lineage specification because ESCs can be differentiated into cardiomyocytes. ESC maintenance and differentiation are currently performed under high glucose conditions, whose adverse effects have never been clarified. We investigated the effect of high glucose on cardiomyocyte differentiation from a well-characterized ESC line, E14, derived from mouse blastocysts. E14 cells maintained under high glucose (25 mM) failed to generate any beating cardiomyocytes using the hanging-drop embryonic body method. We created a glucose-responsive E14 cell line (GR-E14) through a graduated low glucose adaptation. The expression of stem cell markers was similar in the parent E14 cells and the GR-E14 cells. Glucose transporter 2 gene was increased in GR-E14 cells. When GR-E14 cells were differentiated into cardiomyocytes under low (5 mM) or high (25 mM) glucose conditions, high glucose significantly delayed the appearance and reduced the number of TNNT2 (Troponin T Type 2)-positive contracting cardiomyocytes. High glucose suppressed the expression of precardiac mesoderm markers, cardiac transcription factors, mature cardiomyocyte markers, and potassium channel proteins. High glucose impaired the functionality of ESC-derived cardiomyocytes by suppressing the frequencies of Ca 2+ wave and contraction. Our findings suggest that high glucose inhibits ESC cardiogenesis by suppressing key developmental genes essential for the cardiac program.

  16. Rapamycin Conditioning of Dendritic Cells Differentiated from Human ES Cells Promotes a Tolerogenic Phenotype

    Directory of Open Access Journals (Sweden)

    Kathryn M. Silk

    2012-01-01

    Full Text Available While human embryonic stem cells (hESCs may one day facilitate the treatment of degenerative diseases requiring cell replacement therapy, the success of regenerative medicine is predicated on overcoming the rejection of replacement tissues. Given the role played by dendritic cells (DCs in the establishment of immunological tolerance, we have proposed that DC, rendered tolerogenic during their differentiation from hESC, might predispose recipients to accept replacement tissues. As a first step towards this goal, we demonstrate that DC differentiated from H1 hESCs (H1-DCs are particularly responsive to the immunosuppressive agent rapamycin compared to monocyte-derived DC (moDC. While rapamycin had only modest impact on the phenotype and function of moDC, H1-DC failed to upregulate CD40 upon maturation and displayed reduced immunostimulatory capacity. Furthermore, coculture of naïve allogeneic T cells with rapamycin-treated H1-DC promoted an increased appearance of CD25hi Foxp3+ regulatory T cells, compared to moDC. Our findings suggest that conditioning of hESC-derived DC with rapamycin favours a tolerogenic phenotype.

  17. Differential T-cell recognition of native and recombinant Mycobacterium tuberculosis GroES

    DEFF Research Database (Denmark)

    Rosenkrands, I; Weldingh, K; Ravn, P

    1999-01-01

    Mycobacterium tuberculosis GroES was purified from culture filtrate, and its identity was confirmed by immunoblot analysis and N-terminal sequencing. Comparing the immunological recognition of native and recombinant GroES, we found that whereas native GroES elicited a strong proliferative response...

  18. Adhesion patterns in early cell spreading

    Energy Technology Data Exchange (ETDEWEB)

    Ryzhkov, Pavel; Prass, Marcus; Gummich, Meike; Kuehn, Jac-Simon; Oettmeier, Christina; Doebereiner, Hans-Guenther, E-mail: hgd@uni-bremen.d [Institut fuer Biophysik, Universitaet Bremen, D-28334 Bremen (Germany)

    2010-05-19

    Mouse embryonic fibroblasts explore the chemical suitability before spreading on a given substrate. We find this early phase of cell spreading to be characterized by transient adhesion patches with a typical mean size of (1.0 {+-} 0.4) {mu}m and a lifetime of (33 {+-} 12) s. Eventually, these patches fuse to initiate extensive spreading of the cell. We monitor cell adhesion using reflection interference contrast and total internal reflection fluorescence microscopy. Digital time lapse movies are analysed employing spatio-temporal correlation functions of adhesion patterns. Correlation length and time can be scaled to obtain a master curve at the fusion point.

  19. A Cbx8-containing polycomb complex facilitates the transition to gene activation during ES cell differentiation.

    Directory of Open Access Journals (Sweden)

    Catherine Creppe

    2014-12-01

    Full Text Available Polycomb proteins play an essential role in maintaining the repression of developmental genes in self-renewing embryonic stem cells. The exact mechanism allowing the derepression of polycomb target genes during cell differentiation remains unclear. Our project aimed to identify Cbx8 binding sites in differentiating mouse embryonic stem cells. Therefore, we used a genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-Seq. Our analysis identified 171 high confidence peaks. By crossing our data with previously published microarray analysis, we show that several differentiation genes transiently recruit Cbx8 during their early activation. Depletion of Cbx8 partially impairs the transcriptional activation of these genes. Both interaction analysis, as well as chromatin immunoprecipitation experiments support the idea that activating Cbx8 acts in the context of an intact PRC1 complex. Prolonged gene activation results in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination. The composition of PRC1 is highly modular and changes when embryonic stem cells commit to differentiation. We further demonstrate that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes. Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state. As this affects several key regulatory differentiation genes this mechanism is likely to contribute to the robust execution of differentiation programs.

  20. Progress report - fuel cell system of the L2ES 2000-2004; Rapport d'activites - systeme pile a combustible du L2ES 2000-2004

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2005-07-01

    Fuel cell activities started in L2ES in 2000. Among all the definitions of the fuel cell system, the laboratory considers as the fuel cell generator, the stack, the ancillaries (air and hydrogen or more generally fuel supply, valves, humidifier or gas treatment, cooling), power electronics, buffer storage and control. The research topics at L2ES are: - fuel cell system modelling - fuel cell system architecture and its optimization - diagnosis and life time. All these topics are described into details. The perspectives and prospects of the laboratory at middle term are specified. (O.M.)

  1. Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos

    Directory of Open Access Journals (Sweden)

    Hadjantonakis Anna-Katerina

    2009-09-01

    Full Text Available Abstract Background The use of genetically-encoded fluorescent proteins has revolutionized the fields of cell and developmental biology and in doing so redefined our understanding of the dynamic morphogenetic processes that shape the embryo. With the advent of more accessible and sophisticated imaging technologies as well as an abundance of fluorescent proteins with different spectral characteristics, the dynamic processes taking place in situ in living cells and tissues can now be probed. Photomodulatable fluorescent proteins are one of the emerging classes of genetically-encoded fluorescent proteins. Results We have compared PA-GFP, PS-CFP2, Kaede and KikGR four readily available and commonly used photomodulatable fluorescent proteins for use in ES cells and mice. Our results suggest that the green-to-red photoconvertible fluorescent protein, Kikume Green-Red (KikGR, is most suitable for cell labeling and lineage studies in ES cells and mice because it is developmentally neutral, bright and undergoes rapid and complete photoconversion. We have generated transgenic ES cell lines and strains of mice exhibiting robust widespread expression of KikGR. By efficient photoconversion of KikGR we labeled subpopulations of ES cells in culture, and groups of cells within ex utero cultured mouse embryos. Red fluorescent photoconverted cells and their progeny could be followed for extended periods of time. Conclusion Transgenic ES cells and mice exhibiting widespread readily detectable expression of KikGR are indistinguishable from their wild type counterparts and are amenable to efficient photoconversion. They represent novel tools for non-invasive selective labeling specific cell populations and live imaging cell dynamics and cell fate. Genetically-encoded photomodulatable proteins such as KikGR represent emergent attractive alternatives to commonly used vital dyes, tissue grafts and genetic methods for investigating dynamic behaviors of individual cells

  2. CD166(pos) subpopulation from differentiated human ES and iPS cells support repair of acute lung injury.

    Science.gov (United States)

    Soh, Boon Seng; Zheng, Dahai; Li Yeo, Julie Su; Yang, Henry He; Ng, Shi Yan; Wong, Lan Hiong; Zhang, Wencai; Li, Pin; Nichane, Massimo; Asmat, Atasha; Wong, Poo Sing; Wong, Peng Cheang; Su, Lin Lin; Mantalaris, Sakis A; Lu, Jia; Xian, Wa; McKeon, Frank; Chen, Jianzhu; Lim, Elaine Hsuen; Lim, Bing

    2012-12-01

    Previous efforts to derive lung progenitor cells from human embryonic stem (hES) cells using embryoid body formation or stromal feeder cocultures had been limited by low efficiencies. Here, we report a step-wise differentiation method to drive both hES and induced pluripotent stem (iPS) cells toward the lung lineage. Our data demonstrated a 30% efficiency in generating lung epithelial cells (LECs) that expresses various distal lung markers. Further enrichment of lung progenitor cells using a stem cell marker, CD166 before transplantation into bleomycin-injured NOD/SCID mice resulted in enhanced survivability of mice and improved lung pulmonary functions. Immunohistochemistry of lung sections from surviving mice further confirmed the specific engraftment of transplanted cells in the damaged lung. These cells were shown to express surfactant protein C, a specific marker for distal lung progenitor in the alveoli. Our study has therefore demonstrated the proof-of-concept of using iPS cells for the repair of acute lung injury, demonstrating the potential usefulness of using patient's own iPS cells to prevent immune rejection which arise from allogenic transplantation.

  3. CD166pos Subpopulation From Differentiated Human ES and iPS Cells Support Repair of Acute Lung Injury

    Science.gov (United States)

    Soh, Boon Seng; Zheng, Dahai; Li Yeo, Julie Su; Yang, Henry He; Ng, Shi Yan; Wong, Lan Hiong; Zhang, Wencai; Li, Pin; Nichane, Massimo; Asmat, Atasha; Wong, Poo Sing; Wong, Peng Cheang; Su, Lin Lin; Mantalaris, Sakis A; Lu, Jia; Xian, Wa; McKeon, Frank; Chen, Jianzhu; Lim, Elaine Hsuen; Lim, Bing

    2012-01-01

    Previous efforts to derive lung progenitor cells from human embryonic stem (hES) cells using embryoid body formation or stromal feeder cocultures had been limited by low efficiencies. Here, we report a step-wise differentiation method to drive both hES and induced pluripotent stem (iPS) cells toward the lung lineage. Our data demonstrated a 30% efficiency in generating lung epithelial cells (LECs) that expresses various distal lung markers. Further enrichment of lung progenitor cells using a stem cell marker, CD166 before transplantation into bleomycin-injured NOD/SCID mice resulted in enhanced survivability of mice and improved lung pulmonary functions. Immunohistochemistry of lung sections from surviving mice further confirmed the specific engraftment of transplanted cells in the damaged lung. These cells were shown to express surfactant protein C, a specific marker for distal lung progenitor in the alveoli. Our study has therefore demonstrated the proof-of-concept of using iPS cells for the repair of acute lung injury, demonstrating the potential usefulness of using patient's own iPS cells to prevent immune rejection which arise from allogenic transplantation. PMID:22968480

  4. Early Bunyavirus-Host Cell Interactions

    Directory of Open Access Journals (Sweden)

    Amelina Albornoz

    2016-05-01

    Full Text Available The Bunyaviridae is the largest family of RNA viruses, with over 350 members worldwide. Several of these viruses cause severe diseases in livestock and humans. With an increasing number and frequency of outbreaks, bunyaviruses represent a growing threat to public health and agricultural productivity globally. Yet, the receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely uncharacterized. The focus of this review is on the early steps of bunyavirus infection, from virus binding to penetration from endosomes. We address current knowledge and advances for members from each genus in the Bunyaviridae family regarding virus receptors, uptake, intracellular trafficking and fusion.

  5. Profiling of microRNA in human and mouse ES and iPS cells reveals overlapping but distinct microRNA expression patterns.

    Science.gov (United States)

    Razak, Siti Razila Abdul; Ueno, Kazuko; Takayama, Naoya; Nariai, Naoki; Nagasaki, Masao; Saito, Rika; Koso, Hideto; Lai, Chen-Yi; Murakami, Miyako; Tsuji, Koichiro; Michiue, Tatsuo; Nakauchi, Hiromitsu; Otsu, Makoto; Watanabe, Sumiko

    2013-01-01

    Using quantitative PCR-based miRNA arrays, we comprehensively analyzed the expression profiles of miRNAs in human and mouse embryonic stem (ES), induced pluripotent stem (iPS), and somatic cells. Immature pluripotent cells were purified using SSEA-1 or SSEA-4 and were used for miRNA profiling. Hierarchical clustering and consensus clustering by nonnegative matrix factorization showed two major clusters, human ES/iPS cells and other cell groups, as previously reported. Principal components analysis (PCA) to identify miRNAs that segregate in these two groups identified miR-187, 299-3p, 499-5p, 628-5p, and 888 as new miRNAs that specifically characterize human ES/iPS cells. Detailed direct comparisons of miRNA expression levels in human ES and iPS cells showed that several miRNAs included in the chromosome 19 miRNA cluster were more strongly expressed in iPS cells than in ES cells. Similar analysis was conducted with mouse ES/iPS cells and somatic cells, and several miRNAs that had not been reported to be expressed in mouse ES/iPS cells were suggested to be ES/iPS cell-specific miRNAs by PCA. Comparison of the average expression levels of miRNAs in ES/iPS cells in humans and mice showed quite similar expression patterns of human/mouse miRNAs. However, several mouse- or human-specific miRNAs are ranked as high expressers. Time course tracing of miRNA levels during embryoid body formation revealed drastic and different patterns of changes in their levels. In summary, our miRNA expression profiling encompassing human and mouse ES and iPS cells gave various perspectives in understanding the miRNA core regulatory networks regulating pluripotent cells characteristics.

  6. Avanços na abordagem do carcinoma precoce de esôfago Advances in the management of early esophageal carcinoma

    Directory of Open Access Journals (Sweden)

    Vitor Arantes

    2012-12-01

    Full Text Available Nos países ocidentais, o carcinoma de células escamosas de esôfago (CCE geralmente é detectado em estágio avançado, quando as possibilidades de cura são remotas e o prognóstico reservado. Entretanto, nos anos recentes, ocorreu uma série de avanços na abordagem do CCE de esôfago, tais como a identificação dos grupos de risco para o surgimento desta neoplasia; o uso da endoscopia de alta resolução e cromoendoscopia com lugol favorecendo o diagnóstico do CCE em estágios iniciais; e o desenvolvimento de técnicas endoscópicas de ressecção tumoral endoluminal em monobloco denominada dissecção endoscópica de submucosa. Este progresso tem possibilitado a aplicação do tratamento endoscópico minimamente invasivo com potencial curativo em pacientes selecionados com CCE superficial de esôfago. O presente artigo de revisão, elaborado por um grupo multicêntrico internacional, tem como objetivo primário contribuir para o entendimento dos principais avanços recentes ocorridos no manejo do CCE precoce de esôfago. Como objetivo secundário, pretende propiciar uma revisão detalhada e minuciosa da estratégia técnica de DES desenvolvida pelos experts japoneses, de forma a colaborar para a difusão deste conceito e a incorporação destas tecnologias na Medicina Brasileira e Latino-americana.Esophageal squamous cell cancer (ESCC has a dismal prognosis mainly because its recognition in Western countries usually occurs in late stages, when the possibilities of cure are minimal. However, in recent years, several advances have been observed in the management of ESSC, such as the identification of high-risk patients, the use of high-resolution endoscopy and lugol chromoscopy favoring the diagnosis of early stage ESCC, and the development of endoluminal techniques of en-block tumor resection, namely endoscopic submucosal dissection (ESD. These factors have enabled the application of endoscopic minimally invasive curative interventions in

  7. Cryopreservation in situ of cell monolayers on collagen vitrigel membrane culture substrata: ready-to-use preparation of primary hepatocytes and ES cells.

    Science.gov (United States)

    Miyamoto, Yoshitaka; Enosawa, Shin; Takeuchi, Tomoyo; Takezawa, Toshiaki

    2009-01-01

    Cryopreservation is generally performed on cells in suspension. In the case of adherent cells such as hepatocytes, a loss of their ability to attach is a more serious problem than a decreased viability after cryopreservation. We herein report a novel technology of direct in situ cryopreservation of cells cultured on collagen vitrigel membranes, which have excellent mechanical strength and can be easily handled by tweezers even when coated with cultured cells. Rat primary hepatocytes, mitomycin C-treated mouse fibroblasts (feeder cells for ES cells), and mouse ES cells on the feeder cells were cultured on collagen vitrigel membranes for 1 day. The membranes with cells attached were then plucked up from the dish, soaked in cryopreservation medium containing 10% dimethyl sulfoxide, frozen using a controlled-rate freezer, and transferred to liquid nitrogen. The cells cultured on plastic cell culture dishes were also frozen as controls. After storage in liquid nitrogen for periods from 1 week to 3 months, the cryopreserved membranes with the cells still attached were thawed by adding warmed culture medium. Cell viability estimated by morphology and functional staining with calcein showed significant improvement in comparison to cells cryopreserved without the collagen vitrigel membrane. The recoveries of living cells after cryopreservation were 26.7%, 76.2%, and 58.6% for rat hepatocytes, mitomycin C-treated mouse fibroblasts, and mouse ES cells on collagen vitrigel membranes, respectively. In contrast, essentially no cells at all remained on the plastic cell culture dishes after thawing. Because adherent cell storage under these conditions is very convenient, the use of this technique employing collagen vitrigel membranes should be generally applicable to the cryopreservation of adherent cells that are otherwise problematic to store as frozen stocks.

  8. Expression analysis of the endogenous Zscan4 locus and its coding proteins in mouse ES cells and preimplantation embryos.

    Science.gov (United States)

    Ishiguro, Kei-Ichiro; Nakatake, Yuhki; Chikazawa-Nohtomi, Nana; Kimura, Hiromi; Akiyama, Tomohiko; Oda, Mayumi; Ko, Shigeru B H; Ko, Minoru S H

    2017-02-01

    Mouse Zinc finger and SCAN domain containing 4 (Zscan4) is encoded in multiple copies of Zscan4 genes, which are expressed in late two-cell stage preimplantation embryos and in 1-5% of the embryonic stem (ES) cell population at a given time. Due to the highly identical nucleotide sequences of multiple copies of Zscan4 paralogs and pseudogenes in the mouse Zscan4 genomic cluster, previous analyses have been done using exogenous transgenes under the regulation of Zscan4c promoter. In this manuscript, we generated knock-in mouse ES cell lines and mouse lines, in which the expression of endogenous Zscan4c, one of the Zscan4 genes, can be specifically monitored with a green fluorescent protein variant, Emerald. Interestingly, we found that only ∼30% of Zscan4-immunopositive ES cells were Emerald positive, suggesting that even when the Zscan4 locus is active, not all Zscan4 genes are expressed synchronously. We also carried out mass spectrometry of protein complexes associated with endogenous Zscan4 proteins. Taken together, our genetic engineering at an endogenous Zscan4c gene provides the first clue for the expression and function of each gene copy of Zscan4 locus in a physiological context.

  9. Measuring Students' Emotions in the Early Years: The Achievement Emotions Questionnaire-Elementary School (AEQ-ES)

    Science.gov (United States)

    Lichtenfeld, Stephanie; Pekrun, Reinhard; Stupnisky, Robert H.; Reiss, Kristina; Murayama, Kou

    2012-01-01

    This article reports about the development and validation of a measurement instrument assessing elementary school students' achievement emotions (Achievement Emotions Questionnaire-Elementary School, AEQ-ES). Specifically, the instrument assesses students' enjoyment, anxiety, and boredom pertaining to three types of academic settings (i.e.,…

  10. Antibody-directed lentiviral gene transduction for live-cell monitoring and selection of human iPS and hES cells.

    Directory of Open Access Journals (Sweden)

    Dai-tze Wu

    Full Text Available The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology--in particular, in the identification of induced pluripotent stem (iPS cells during the reprogramming process. Based on the selective expression of stem cell surface markers, a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts, allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells, but that specific genes, including positive and negative selection markers, regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies.

  11. Use of the ES-D3 cell differentiation assay, combined with the BeWo transport model, to predict relative in vivo developmental toxicity of antifungal compounds.

    Science.gov (United States)

    Li, Hequn; Rietjens, Ivonne M C M; Louisse, Jochem; Blok, Martine; Wang, Xinyi; Snijders, Linda; van Ravenzwaay, Bennard

    2015-03-01

    We investigated the applicability of the ES-D3 cell differentiation assay combined with the in vitro BeWo transport model to predict the relative in vivo developmental toxicity potencies. To this purpose, the in vitro developmental toxicity of five antifungal compounds was investigated by characterizing their inhibitory effect on the differentiation of ES-D3 cells into cardiomyocytes. The BeWo transport model, consisting of BeWo b30 cells grown on transwell inserts and mimicking the placental barrier, was used to determine the relative placental transport velocity. The ES-D3 cell differentiation data were first compared to benchmark doses (BMDs) for in vivo developmental toxicity as derived from data reported in the literature. Correlation between the benchmark concentration for 50% effect (BMCd50) values, obtained in the ES-D3 cell differentiation assay, with in vivo BMD10 values showed a reasonable correlation (R(2)=0.57). When the ES-D3 cell differentiation data were combined with the relative transport rates obtained from the BeWo model, the correlation with the in vivo data increased (R(2)=0.95). In conclusion, we show that the ES-D3 cell differentiation assay is able to better predict the in vivo developmental toxicity ranking of antifungal compounds when combined with the BeWo transport model, than as a stand-alone assay. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system.

    Science.gov (United States)

    Takayama, Kazuo; Igai, Keisuke; Hagihara, Yasuko; Hashimoto, Rina; Hanawa, Morifumi; Sakuma, Tetsushi; Tachibana, Masashi; Sakurai, Fuminori; Yamamoto, Takashi; Mizuguchi, Hiroyuki

    2017-05-19

    Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency of biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that in transcriptionally active genes. To enhance the biallelic homologous recombination efficiency in human ES/iPS cells, we performed screenings of accessorial genes and compounds. We found that RAD51 overexpression and valproic acid treatment enhanced biallelic-targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus. Importantly, RAD51 overexpression and valproic acid treatment synergistically increased the biallelic homologous recombination efficiency. Our findings would facilitate genome editing study using human ES/iPS cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system

    Science.gov (United States)

    Takayama, Kazuo; Igai, Keisuke; Hagihara, Yasuko; Hashimoto, Rina; Hanawa, Morifumi; Sakuma, Tetsushi; Tachibana, Masashi; Sakurai, Fuminori; Yamamoto, Takashi

    2017-01-01

    Abstract Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency of biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that in transcriptionally active genes. To enhance the biallelic homologous recombination efficiency in human ES/iPS cells, we performed screenings of accessorial genes and compounds. We found that RAD51 overexpression and valproic acid treatment enhanced biallelic-targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus. Importantly, RAD51 overexpression and valproic acid treatment synergistically increased the biallelic homologous recombination efficiency. Our findings would facilitate genome editing study using human ES/iPS cells. PMID:28334759

  14. Investigation for the differentiation process of mouse ES cells by Raman spectroscopy

    Science.gov (United States)

    Yamaguchi, Yoshinori; El-Hagrasy, Maha A.; Shimizu, Eiichi; Saito, Masato; Tamiya, Eiichi

    2012-03-01

    The arrangement of differentiated pluripotent embryonic stem cells into three-dimensional aggregates, which are known as embryonic bodies, is a main step for progressing the embryonic stem cells differentiation. In this work, embryonic stem cells that were directly produced from the hanging drop step as a three-dimensional structure with no further twodimensional differentiation were diagnosed with Raman spectroscopy as a non-invasive and label-free technique. Raman spectroscopy was employed to discriminate between mouse embryonic bodies of different degrees of maturation. EBs were prepared applying the hanging drop method. The Raman scattering measurements were obtained in vitro with a Nanophoton RAMAN-11 micro-spectrometer (Japan: URL: www.nanophoton.jp equipped with an Olympus XLUM Plan FLN 20X/NA= 1.0 objective lens. Spectral data were smoothed, baseline corrected and normalized to the a welldefined intense 1003 cm-1 band (phenylalanine) which is insensitive to changes in conformation or environment. The differentiation process of embryonic stem cells is initiated by the removal of LIF from culture medium. 1, 7 and 17-dayold embryonic stem cells were collected and investigated by Raman spectroscopy. The main differences involve bands which decreased with maturation such as: 784 cm-1 (U, T, C ring br DNA/RNA, O-P-O str); 1177 cm-1 (cytosine, guanine) and 1578 cm-1 (G, A). It was found that with the progress of differentiation the protein content was amplified. The increase of protein to nucleic acid ratio was also previously observed with the progress of the differentiation process. Raman spectroscopy has the potential to distinguish between the Raman signatures of live embryonic stem cells with different degrees of maturation.

  15. Growth inhibition of mouse embryonic stem (ES) cells on the feeders ...

    African Journals Online (AJOL)

    Mouse embryonic stem cells (mESCs) can be propagated in vitro on the feeders of mouse embryonic fibroblasts. In this study, we found growth inhibition of mESCs cultured on embryonic fibroblast feeders derived from different livestock animals. Under the same condition, mESCs derived from mouse embryonic fibroblast ...

  16. Iron deficiency in sickle cell anaemia patients in Dar es Salaam ...

    African Journals Online (AJOL)

    A cross sectional descriptive study was done to determine the prevalence of iron deficiency and possible contributing factors in sickle cell anaemic patients. One hundred haemoglobin -SS children aged between six months to ten years inclusive were recruited in the study.Patients were selected using a simple random ...

  17. In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell Dissemination

    Science.gov (United States)

    2015-12-01

    AWARD NUMBER: W81XWH-13-1-0184 TITLE: In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell Dissemination PRINCIPAL...Sep 2013 - 14 Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell...understand the early events that accompany invasive behavior in vivo , we proposed to develop a lineage-labeling system to detect and isolate cells of lung

  18. HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells

    Directory of Open Access Journals (Sweden)

    Sandeep R. Varma

    2013-01-01

    Full Text Available HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA at concentrations ranging from 125 to 1000 μg/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380 μg/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections.

  19. HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells.

    Science.gov (United States)

    Varma, Sandeep R; Sundaram, R; Gopumadhavan, S; Vidyashankar, Satyakumar; Patki, Pralhad S

    2013-01-01

    HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000  μ g/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380  μ g/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections.

  20. Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity

    Science.gov (United States)

    2011-01-01

    Background In addition to determining static states of gene expression (high vs. low), it is important to characterize their dynamic status. For example, genes with H3K27me3 chromatin marks are not only suppressed but also poised for activation. However, the responsiveness of genes to perturbations has never been studied systematically. To distinguish gene responses to specific factors from responsiveness in general, it is necessary to analyze gene expression profiles of cells responding to a large variety of disturbances, and such databases did not exist before. Results We estimated the responsiveness of all genes in mouse ES cells using our recently published database on expression change after controlled induction of 53 transcription factors (TFs) and other genes. Responsive genes (N = 4746), which were readily upregulated or downregulated depending on the kind of perturbation, mostly have regulatory functions and a propensity to become tissue-specific upon differentiation. Tissue-specific expression was evaluated on the basis of published (GNF) and our new data for 15 organs and tissues. Non-responsive genes (N = 9562), which did not change their expression much following any perturbation, were enriched in housekeeping functions. We found that TF-responsiveness in ES cells is the best predictor known for tissue-specificity in gene expression. Among genes with CpG islands, high responsiveness is associated with H3K27me3 chromatin marks, and low responsiveness is associated with H3K36me3 chromatin, stronger tri-methylation of H3K4, binding of E2F1, and GABP binding motifs in promoters. Conclusions We thus propose the responsiveness of expression to perturbations as a new way to define the dynamic status of genes, which brings new insights into mechanisms of regulation of gene expression and tissue specificity. PMID:21306619

  1. JARID2 regulates binding of the Polycomb repressive complex 2 to target genes in ES cells

    DEFF Research Database (Denmark)

    Pasini, Diego; Cloos, Paul A C; Walfridsson, Julian

    2010-01-01

    The Polycomb group (PcG) proteins have an important role in controlling the expression of genes essential for development, differentiation and maintenance of cell fates. The Polycomb repressive complex 2 (PRC2) is believed to regulate transcriptional repression by catalysing the di- and tri......-methylation of lysine 27 on histone H3 (H3K27me2/3). At present, it is unknown how the PcG proteins are recruited to their target promoters in mammalian cells. Here we show that PRC2 forms a stable complex with the Jumonji- and ARID-domain-containing protein, JARID2 (ref. 4). Using genome-wide location analysis, we...... show that JARID2 binds to more than 90% of previously mapped PcG target genes. Notably, we show that JARID2 is sufficient to recruit PcG proteins to a heterologous promoter, and that inhibition of JARID2 expression leads to a major loss of PcG binding and to a reduction of H3K27me3 levels on target...

  2. DNA breaks early in replication in B cell cancers

    Science.gov (United States)

    Research by scientists at the NCI has identified a new class of DNA sites in cells that break early in the replication process. They found that these break sites correlate with damage often seen in B cell cancers, such as diffuse large B cell lymphoma.

  3. Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

    Directory of Open Access Journals (Sweden)

    Muyrers Joep PP

    2003-01-01

    Full Text Available Abstract Background The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined. Results Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα but only requires a phage annealing protein (RecT or Redβ. Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein. Conclusion Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

  4. Oxidative stress tolerance of early stage diabetic endothelial progenitor cell

    Directory of Open Access Journals (Sweden)

    Dewi Sukmawati

    2015-06-01

    Conclusions: Primitive BM-EPCs showed vasculogenic dysfunction in early diabetes. However the oxidative stress is not denoted as the major initiating factor of its cause. Our results suggest that primitive BM-KSL cell has the ability to compensate oxidative stress levels in early diabetes by increasing the expression of anti-oxidative enzymes.

  5. Early cell death detection with digital holographic microscopy.

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    Nicolas Pavillon

    Full Text Available BACKGROUND: Digital holography provides a non-invasive measurement of the quantitative phase shifts induced by cells in culture, which can be related to cell volume changes. It has been shown previously that regulation of cell volume, in particular as it relates to ionic homeostasis, is crucially involved in the activation/inactivation of the cell death processes. We thus present here an application of digital holographic microscopy (DHM dedicated to early and label-free detection of cell death. METHODS AND FINDINGS: We provide quantitative measurements of phase signal obtained on mouse cortical neurons, and caused by early neuronal cell volume regulation triggered by excitotoxic concentrations of L-glutamate. We show that the efficiency of this early regulation of cell volume detected by DHM, is correlated with the occurrence of subsequent neuronal death assessed with the widely accepted trypan blue method for detection of cell viability. CONCLUSIONS: The determination of the phase signal by DHM provides a simple and rapid optical method for the early detection of cell death.

  6. Mechanisms of epigenetic and cell-type specific regulation of Hey target genes in ES cells and cardiomyocytes.

    Science.gov (United States)

    Weber, David; Heisig, Julia; Kneitz, Susanne; Wolf, Elmar; Eilers, Martin; Gessler, Manfred

    2015-02-01

    Hey bHLH transcription factors are critical effectors of Notch signaling. During mammalian heart development they are expressed in atrial and ventricular cardiomyocytes and in the developing endocardium. Hey knockout mice suffer from lethal cardiac defects, such as ventricular septum defects, valve defects and cardiomyopathy. Despite this functional relevance, little is known about the regulation of downstream targets in relevant cell types. The objective of this study was to elucidate the regulatory mechanisms by which Hey proteins affect gene expression in a cell type specific manner. We used an in vitro cardiomyocyte differentiation system with inducible Hey1 or Hey2 expression to study target gene regulation in cardiomyocytes (CM) generated from murine embryonic stem cells (ESC). The effects of Hey1 and Hey2 are largely redundant, but cell type specific. The number of regulated genes is comparable between ESC and CM, but the total number of binding sites is much higher, especially in ESC, targeting mainly genes involved in transcriptional regulation and developmental processes. Repression by Hey proteins generally correlates with the extent of Hey-binding to target promoters, Hdac recruitment and lower histone acetylation. Functionally, treatment with the Hdac inhibitor TSA abolished Hey target gene regulation. However, in CM the repressive effect of Hey-binding is lost for a subset of genes. These also lack Hey-dependent histone deacetylation in CM and are enriched for binding sites of cardiac specific activators like Srf, Nkx2-5, and Gata4. Ectopic Nkx2-5 overexpression in ESC blocks Hey-mediated repression of these genes. Thus, Hey proteins mechanistically repress target genes via Hdac recruitment and histone deacetylation. In CM Hey-repression is counteracted by cardiac activators, which recruit histone acetylases and prevent Hey mediated deacetylation and subsequent repression for a subset of genes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Ethanol diverts early neuronal differentiation trajectory of embryonic stem cells by disrupting the balance of lineage specifiers.

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    Rosa Sánchez-Alvarez

    Full Text Available Ethanol is a toxin responsible for the neurodevelopmental deficits of Fetal Alcohol Spectrum Disorders (FASD. Recent evidence suggests that ethanol modulates the protein expression of lineage specifier transcription factors Oct4 (Pou5f1 and Sox2 in early stages of mouse embryonic stem (ES cell differentiation. We hypothesized that ethanol induced an imbalance in the expression of Oct4 and Sox2 in early differentiation, that dysregulated the expression of associated and target genes and signaling molecules and diverted cells from neuroectodermal (NE formation.We showed modulation by ethanol of 33 genes during ES cell differentiation, using high throughput microfluidic dynamic array chips measuring 2,304 real time quantitative PCR assays. Based on the overall gene expression dynamics, ethanol drove cells along a differentiation trajectory away from NE fate. These ethanol-induced gene expression changes were observed as early as within 2 days of differentiation, and were independent of cell proliferation or apoptosis. Gene expression changes were correlated with fewer βIII-tubulin positive cells of an immature neural progenitor phenotype, as well as a disrupted actin cytoskeleton were observed. Moreover, Tuba1a and Gapdh housekeeping genes were modulated by ethanol during differentiation and were replaced by a set of ribosomal genes with stable expression.These findings provided an ethanol-response gene signature and pointed to the transcriptional dynamics underlying lineage imbalance that may be relevant to FASD phenotype.

  8. Extended evaluation on the ES-D3 cell differentiation assay combined with the BeWo transport model, to predict relative developmental toxicity of triazole compounds.

    Science.gov (United States)

    Li, Hequn; Flick, Burkhard; Rietjens, Ivonne M C M; Louisse, Jochem; Schneider, Steffen; van Ravenzwaay, Bennard

    2016-05-01

    The mouse embryonic stem D3 (ES-D3) cell differentiation assay is based on the morphometric measurement of cardiomyocyte differentiation and is a promising tool to detect developmental toxicity of compounds. The BeWo transport model, consisting of BeWo b30 cells grown on transwell inserts and mimicking the placental barrier, is useful to determine relative placental transport velocities of compounds. We have previously demonstrated the usefulness of the ES-D3 cell differentiation assay in combination with the in vitro BeWo transport model to predict the relative in vivo developmental toxicity potencies of a set of reference azole compounds. To further evaluate this combined in vitro toxicokinetic and toxicodynamic approach, we combined ES-D3 cell differentiation data of six novel triazoles with relative transport rates obtained from the BeWo model and compared the obtained ranking to the developmental toxicity ranking as derived from in vivo data. The data show that the combined in vitro approach provided a correct prediction for in vivo developmental toxicity, whereas the ES-D3 cell differentiation assay as stand-alone did not. In conclusion, we have validated the combined in vitro approach for developmental toxicity, which we have previously developed with a set of reference azoles, for a set of six novel triazoles. We suggest that this combined model, which takes both toxicodynamic and toxicokinetic aspects into account, should be further validated for other chemical classes of developmental toxicants.

  9. T-cell activation and early gene response in dogs.

    Science.gov (United States)

    Mortlock, Sally-Anne; Wei, Jerry; Williamson, Peter

    2015-01-01

    T-cells play a crucial role in canine immunoregulation and defence against invading pathogens. Proliferation is fundamental to T-cell differentiation, homeostasis and immune response. Initiation of proliferation following receptor mediated stimuli requires a temporally programmed gene response that can be identified as immediate-early, mid- and late phases. The immediate-early response genes in T-cell activation engage the cell cycle machinery and promote subsequent gene activation events. Genes involved in this immediate-early response in dogs are yet to be identified. The present study was undertaken to characterise the early T-cell gene response in dogs to improve understanding of the genetic mechanisms regulating immune function. Gene expression profiles were characterised using canine gene expression microarrays and quantitative reverse transcription PCR (qRT-PCR), and paired samples from eleven dogs. Significant functional annotation clusters were identified following stimulation with phytohemagluttinin (PHA) (5μg/ml), including the Toll-like receptor signaling pathway and phosphorylation pathways. Using strict statistical criteria, 13 individual genes were found to be differentially expressed, nine of which have ontologies that relate to proliferation and cell cycle control. These included, prostaglandin-endoperoxide synthase 2 (PTGS2/COX2), early growth response 1 (EGR1), growth arrest and DNA damage-inducible gene (GADD45B), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), V-FOS FBJ murine osteosarcoma viral oncogene homolog (FOS), early growth response 2 (EGR2), hemogen (HEMGN), polo-like kinase 2 (PLK2) and polo-like kinase 3 (PLK3). Differential gene expression was re-examined using qRT-PCR, which confirmed that EGR1, EGR2, PMAIP1, PTGS2, FOS and GADD45B were significantly upregulated in stimulated cells and ALAS2 downregulated. PTGS2 and EGR1 showed the highest levels of response in these dogs. Both of these genes are involved in cell cycle

  10. T-cell activation and early gene response in dogs.

    Directory of Open Access Journals (Sweden)

    Sally-Anne Mortlock

    Full Text Available T-cells play a crucial role in canine immunoregulation and defence against invading pathogens. Proliferation is fundamental to T-cell differentiation, homeostasis and immune response. Initiation of proliferation following receptor mediated stimuli requires a temporally programmed gene response that can be identified as immediate-early, mid- and late phases. The immediate-early response genes in T-cell activation engage the cell cycle machinery and promote subsequent gene activation events. Genes involved in this immediate-early response in dogs are yet to be identified. The present study was undertaken to characterise the early T-cell gene response in dogs to improve understanding of the genetic mechanisms regulating immune function. Gene expression profiles were characterised using canine gene expression microarrays and quantitative reverse transcription PCR (qRT-PCR, and paired samples from eleven dogs. Significant functional annotation clusters were identified following stimulation with phytohemagluttinin (PHA (5μg/ml, including the Toll-like receptor signaling pathway and phosphorylation pathways. Using strict statistical criteria, 13 individual genes were found to be differentially expressed, nine of which have ontologies that relate to proliferation and cell cycle control. These included, prostaglandin-endoperoxide synthase 2 (PTGS2/COX2, early growth response 1 (EGR1, growth arrest and DNA damage-inducible gene (GADD45B, phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, V-FOS FBJ murine osteosarcoma viral oncogene homolog (FOS, early growth response 2 (EGR2, hemogen (HEMGN, polo-like kinase 2 (PLK2 and polo-like kinase 3 (PLK3. Differential gene expression was re-examined using qRT-PCR, which confirmed that EGR1, EGR2, PMAIP1, PTGS2, FOS and GADD45B were significantly upregulated in stimulated cells and ALAS2 downregulated. PTGS2 and EGR1 showed the highest levels of response in these dogs. Both of these genes are involved in

  11. Human and mouse ZFP57 proteins are functionally interchangeable in maintaining genomic imprinting at multiple imprinted regions in mouse ES cells.

    Science.gov (United States)

    Takikawa, Sachiko; Wang, Xin; Ray, Chelsea; Vakulenko, Max; Bell, Fong T; Li, Xiajun

    2013-12-01

    Genomic imprinting is a common epigenetic phenomenon in mammals. Dysregulation of genomic imprinting has been implicated in a variety of human diseases. ZFP57 is a master regulator in genomic imprinting. Loss of ZFP57 causes loss of DNA methylation imprint at multiple imprinted regions in mouse embryos, as well as in embryonic stem (ES) cells. Similarly, mutations in human ZFP57 result in hypomethylation at many imprinted regions and are associated with transient neonatal diabetes and other human diseases. Mouse and human Zfp57 genes are located in the same syntenic block. However, mouse and human ZFP57 proteins only display about 50% sequence identity with different number of zinc fingers. It is not clear if they share similar mechanisms in maintaining genomic imprinting. Here we report that mouse and human ZFP57 proteins are functionally interchangeable. Expression of exogenous wild-type human ZFP57 could maintain DNA methylation imprint at three imprinted regions in mouse ES cells in the absence of endogenous mouse ZFP57. However, mutant human ZFP57 proteins containing the mutations found in human patients could not substitute for endogenous mouse ZFP57 in maintaining genomic imprinting in ES cells. Like mouse ZFP57, human ZFP57 and its mutant proteins could bind to mouse KAP1, the universal cofactor for KRAB zinc finger proteins, in mouse ES cells. Thus, we conclude that mouse and human ZFP57 are orthologs despite relatively low sequence identity and mouse ES cell system that we had established before is a valuable system for functional analyses of wild-type and mutant human ZFP57 proteins.

  12. Telmisartan inhibits human urological cancer cell growth through early apoptosis

    Science.gov (United States)

    MATSUYAMA, MASAHIDE; FUNAO, KIYOAKI; KURATSUKURI, KATSUYUKI; TANAKA, TOMOAKI; KAWAHITO, YUTAKA; SANO, HAJIME; CHARGUI, JAMEL; TOURAINE, JEAN-LOUIS; YOSHIMURA, NORIO; YOSHIMURA, RIKIO

    2010-01-01

    Angiotensin II receptor blockers (ARBs) are widely used as hypertensive therapeutic agents. In addition, studies have provided evidence that ARBs have the potential to inhibit the growth of several types of cancer cells. It was reported that telmisartan (a type of ARB) has peroxisome proliferator-activated receptor (PPAR)-γ activation activity. We previously reported that the PPAR-γ ligand induces growth arrest in human urological cancer cells through apoptosis. In this study, we evaluated the effects of telmisartan and other ARBs on cell proliferation in renal cell carcinoma (RCC), bladder cancer (BC), prostate cancer (PC) and testicular cancer (TC) cell lines. The inhibitory effects of telmisartan and other ARBs (candesartan, valsartan, irbesartan and losartan) on the growth of the RCC, BC, PC and TC cell lines was investigated using an MTT assay. Flow cytometry and Hoechst staining were used to determine whether the ARBs induced apoptosis. Telmisartan caused marked growth inhibition in the urological cancer cells in a dose- and time-dependent manner. Urological cancer cells treated with 100 μM telmisartan underwent early apoptosis and DNA fragmentation. However, the other ARBs had no effect on cell proliferation in any of the urological cancer cell lines. Telmisartan may mediate potent anti-proliferative effects in urological cancer cells through PPAR-γ. Thus, telmisartan is a potent target for the prevention and treatment of human urological cancer. PMID:22993542

  13. New insights into human primordial germ cells and early embryonic development from single-cell analysis.

    Science.gov (United States)

    Otte, Jörg; Wruck, Wasco; Adjaye, James

    2017-08-01

    Human preimplantation developmental studies are difficult to accomplish due to associated ethical and moral issues. Preimplantation cells are rare and exist only in transient cell states. From a single cell, it is very challenging to analyse the origination of the heterogeneity and complexity inherent to the human body. However, recent advances in single-cell technology and data analysis have provided new insights into the process of early human development and germ cell specification. In this Review, we examine the latest single-cell datasets of human preimplantation embryos and germ cell development, compare them to bulk cell analyses, and interpret their biological implications. © 2017 Federation of European Biochemical Societies.

  14. Dataset of transcriptional landscape of B cell early activation

    Directory of Open Access Journals (Sweden)

    Alexander S. Garruss

    2015-09-01

    Full Text Available Signaling via B cell receptors (BCR and Toll-like receptors (TLRs result in activation of B cells with distinct physiological outcomes, but transcriptional regulatory mechanisms that drive activation and distinguish these pathways remain unknown. At early time points after BCR and TLR ligand exposure, 0.5 and 2 h, RNA-seq was performed allowing observations on rapid transcriptional changes. At 2 h, ChIP-seq was performed to allow observations on important regulatory mechanisms potentially driving transcriptional change. The dataset includes RNA-seq, ChIP-seq of control (Input, RNA Pol II, H3K4me3, H3K27me3, and a separate RNA-seq for miRNA expression, which can be found at Gene Expression Omnibus Dataset GSE61608. Here, we provide details on the experimental and analysis methods used to obtain and analyze this dataset and to examine the transcriptional landscape of B cell early activation.

  15. The evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in mouse ES cells.

    Science.gov (United States)

    Niwa, Hitoshi; Nakamura, Akira; Urata, Makoto; Shirae-Kurabayashi, Maki; Kuraku, Shigehiro; Russell, Steven; Ohtsuka, Satoshi

    2016-08-31

    In mouse ES cells, the function of Sox2 is essential for the maintenance of pluripotency. Since the Sox-family of transcription factors are well conserved in the animal kingdom, addressing the evolutionary origin of Sox2 function in pluripotent stem cells is intriguing from the perspective of understanding the origin of pluripotency. Here we approach this question using a functional complementation assay in inducible Sox2-null ES cells. Assaying mouse Sox proteins from different Groups, we found that only Group B1 and Group G proteins were able to support pluripotency. Interestingly, invertebrate homologs of mammalian Group B1 Sox proteins were able to replace the pluripotency-associated function of mouse Sox2. Moreover, the mouse ES cells rescued by the Drosophila SoxNeuro protein are able to contribute to chimeric embryos. These data indicate that the function of mouse Sox2 supporting pluripotency is based on an evolutionally conserved activity of the Group B1 Sox family. Since pluripotent stem cell population in developmental process could be regarded as the evolutional novelty in vertebrates, it could be regarded as a co-optional use of their evolutionally conserved function.

  16. Early human prostate adenocarcinomas harbor androgen-independent cancer cells.

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    Rita R Fiñones

    Full Text Available Although blockade of androgen receptor (AR signaling represents the main treatment for advanced prostate cancer (PrCa, many patients progress to a lethal phenotype of "Castration-Resistant" prostate cancer (CR-PrCa. With the hypothesis that early PrCa may harbor a population of androgen-unresponsive cancer cells as precursors to CR-recurrent disease, we undertook the propagation of androgen-independent cells from PrCa-prostatectomy samples of early, localized (Stage-I cases. A collection of 120 surgical specimens from prostatectomy cases was established, among which 54 were adenocarcinomas. Hormone-free cell culture conditions were developed allowing routine propagation of cells expressing prostate basal cell markers and stem/progenitor cell markers, and which proliferated as spheres/spheroids in suspension cultures. Colonies of androgen-independent epithelial cells grew out from 30/43 (70% of the adenocarcinoma cases studied in detail. Fluorescence microscopy and flow cytometry showed that CR-PrCa cells were positive for CD44, CD133, CK5/14, c-kit, integrin α2β1, SSEA4, E-Cadherin and Aldehyde Dehydrogenase (ALDH. All 30 CR-PrCa cell cultures were also TERT-positive, but negative for TMPRSS2-ERG. Additionally, a subset of 22 of these CR-PrCa cell cultures was examined by orthotopic xenografting in intact and castrated SCID mice, generating histologically typical locally-invasive human PrCa or undifferentiated cancers, respectively, in 6-8 weeks. Cultured PrCa cells and orthotopically-induced in vivo cancers lacked PSA expression. We report here the propagation of Cancer Initiating Cells (CIC directly from Stage I human PrCa tissue without selection or genetic manipulation. The propagation of stem/progenitor-like CR-PrCa cells derived from early human prostate carcinomas suggests the existence of a subpopulation of cells resistant to androgen-deprivation therapy and which may drive the subsequent emergence of disseminated CR-PrCa.

  17. Proinflammatory T Cell Status Associated with Early Life Adversity.

    Science.gov (United States)

    Elwenspoek, Martha M C; Hengesch, Xenia; Leenen, Fleur A D; Schritz, Anna; Sias, Krystel; Schaan, Violetta K; Mériaux, Sophie B; Schmitz, Stephanie; Bonnemberger, Fanny; Schächinger, Hartmut; Vögele, Claus; Turner, Jonathan D; Muller, Claude P

    2017-12-15

    Early life adversity (ELA) has been associated with an increased risk for diseases in which the immune system plays a critical role. The ELA immune phenotype is characterized by inflammation, impaired cellular immunity, and immunosenescence. However, data on cell-specific immune effects are largely absent. Additionally, stress systems and health behaviors are altered in ELA, which may contribute to the generation of the ELA immune phenotype. The present investigation tested cell-specific immune differences in relationship to the ELA immune phenotype, altered stress parameters, and health behaviors in individuals with ELA (n = 42) and those without a history of ELA (control, n = 73). Relative number and activation status (CD25, CD69, HLA-DR, CD11a, CD11b) of monocytes, NK cells, B cells, T cells, and their main subsets were assessed by flow cytometry. ELA was associated with significantly reduced numbers of CD69+CD8+ T cells (p = 0.022), increased numbers of HLA-DR+ CD4 and HLA-DR+ CD8 T cells (p ELA also showed a trend toward higher numbers of CCR4+CXCR3-CCR6+ CD4 T cells. Taken together, our data suggest an elevated state of immune activation in ELA, in which particularly T cells are affected. Although several aspects of the ELA immune phenotype were related to increased activation markers, neither stress nor health-risk behaviors explained the observed group differences. Thus, the state of immune activation in ELA does not seem to be secondary to alterations in the stress system or health-risk behaviors, but rather a primary effect of early life programming on immune cells. Copyright © 2017 by The American Association of Immunologists, Inc.

  18. Hydrogen Storage Needs for Early Motive Fuel Cell Markets

    Energy Technology Data Exchange (ETDEWEB)

    Kurtz, J.; Ainscough, C.; Simpson, L.; Caton, M.

    2012-11-01

    The National Renewable Energy Laboratory's (NREL) objective for this project is to identify performance needs for onboard energy storage of early motive fuel cell markets by working with end users, manufacturers, and experts. The performance needs analysis is combined with a hydrogen storage technology gap analysis to provide the U.S. Department of Energy (DOE) Fuel Cell Technologies Program with information about the needs and gaps that can be used to focus research and development activities that are capable of supporting market growth.

  19. Transcriptome analysis of mouse stem cells and early embryos.

    Directory of Open Access Journals (Sweden)

    Alexei A Sharov

    2003-12-01

    Full Text Available Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

  20. Protection against collagen-induced arthritis in mice afforded by the parasitic worm product, ES-62, is associated with restoration of the levels of interleukin-10-producing B cells and reduced plasma cell infiltration of the joints.

    Science.gov (United States)

    Rodgers, David T; Pineda, Miguel A; McGrath, Mairi A; Al-Riyami, Lamyaa; Harnett, William; Harnett, Margaret M

    2014-03-01

    We have previously reported that ES-62, a molecule secreted by the parasitic filarial nematode Acanthocheilonema viteae, protects mice from developing collagen-induced arthritis (CIA). Together with increasing evidence that worm infection may protect against autoimmune conditions, this raises the possibility that ES-62 may have therapeutic potential in rheumatoid arthritis and hence, it is important to fully understand its mechanism of action. To this end, we have established to date that ES-62 protection in CIA is associated with suppressed T helper type 1 (Th1)/Th17 responses, reduced collagen-specific IgG2a antibodies and increased interleukin-10 (IL-10) production by splenocytes. IL-10-producing regulatory B cells have been proposed to suppress pathogenic Th1/Th17 responses in CIA: interestingly therefore, although the levels of IL-10-producing B cells were decreased in the spleens of mice with CIA, ES-62 was found to restore these to the levels found in naive mice. In addition, exposure to ES-62 decreased effector B-cell, particularly plasma cell, infiltration of the joints, and such infiltrating B cells showed dramatically reduced levels of Toll-like receptor 4 and the activation markers, CD80 and CD86. Collectively, this induction of hyporesponsiveness of effector B-cell responses, in the context of the resetting of the levels of IL-10-producing B cells, is suggestive of a modulation of the balance between effector and regulatory B-cell responses that may contribute to ES-62-mediated suppression of CIA-associated inflammation and inhibition of production of pathogenic collagen-specific IgG2a antibodies. © 2013 The Authors. Immunology published by John Wiley & Sons Ltd.

  1. Tratamento endoscópico do câncer epidermóide do esôfago Endoscopic treatment of squamous cell esophageal cancer

    Directory of Open Access Journals (Sweden)

    Fauze Maluf-Filho

    2006-06-01

    Full Text Available OBJETIVOS: Procurou-se avaliar o papel atual dos procedimentos terapêuticos endoscópicos no manejo do pacientes com carcinoma epidermóide do esôfago. LEVANTAMENTO DE DADOS: Utilizando o banco de dados do PubMed (U.S. National Library of Medicine, analisaram-se as publicações sobre o tema nos últimos 10 anos, cotejando-as com a experiência desenvolvida no Serviço de Endoscopia Gastrointestinal do Departamento de Gastroenterologia da Faculdade de Medicina da Universidade de São Paulo. SÍNTESE DOS DADOS: Neste campo, destacam-se a ressecção endoscópica do câncer esofágico precoce e a tunelização do tumor avançado daquele órgão. A ressecção endoscópica da mucosa do câncer epidermóide precoce do esôfago é indicada quando a lesão é confinada ao epitélio (m1 ou à lamina própria (m2. A taxa de sobrevida conhecida de 5 anos após a ressecção endoscópica da mucosa do tumor epidermóide intramucoso do esôfago aproxima-se de 95%. CONCLUSÕES: Baseado nas evidências disponíveis, parece razoável indicar a ressecção endoscópica da mucosa como tratamento de primeira escolha para pacientes com carcinoma esofágico epidermóide intramucoso. Existem vários métodos endoscópicos paliativos para o alívio da disfagia em câncer esofágico avançado. A escolha variará de acordo com as características anatômicas e a localização do tumor, as preferências do paciente, a disponibilidade e a capacitação do centro assistencial. A taxa de sucesso técnico da colocação de próteses metálicas auto-expansíveis em estenose maligna praticamente atinge 100%. A taxa de efeito paliativo em longo prazo da disfagia aproxima-se de 80%, o que faz com que esta opção seja, até o momento, o tratamento paliativo de escolha para os sintomas de obstrução causados pelo câncer esofágico de células escamosas.OBJECTIVE: In this article, it was evaluated the role of endoscopic procedures for the management of squamous cell

  2. Early Trypanosoma cruzi Infection Reprograms Human Epithelial Cells

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    María Laura Chiribao

    2014-01-01

    Full Text Available Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of prophylaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion is important for the understanding of disease pathogenesis. In Chagas disease, most of the focus is on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions, respectively. In the present work, we studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response, a great number of transcription factors (including the majority of NFκB family members, and host metabolism (cholesterol, fatty acids, and phospholipids. These results raise the possibility that early host cell reprogramming is exploited by the parasite to establish the initial infection and posterior systemic dissemination.

  3. Phosphorylation site dynamics of early T-cell receptor signaling

    DEFF Research Database (Denmark)

    Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn

    2014-01-01

    that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites......In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein-protein interactions and phosphorylation events have been studied extensively, we lack...... a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...

  4. The dynamic changes of X chromosome inactivation during early culture of human embryonic stem cells.

    Science.gov (United States)

    Xie, Pingyuan; Ouyang, Qi; Leng, Lizhi; Hu, Liang; Cheng, Dehua; Tan, Yueqiu; Lu, Guangxiu; Lin, Ge

    2016-07-01

    X chromosome inactivation (XCI) is required for dosage compensation of X-linked genes in human female cells. Several previous reports have described the promiscuous XCI status in long-term cultured female human embryonic stem cells (hESCs), and the majority of them exhibit non-random XCI. However, when and how such female hESCs acquire the aberrant XCI states during culture is unknown. Herein, through comparing the XCI states in 18 paired hES cell lines throughout early culture, we revealed a uniform dynamic change during this culture period under a widely used culture condition. The female initial hESCs (ihESCs, P4-P9) expressed XIST RNA, H3K27me3 punctate enrichment and displayed random XCI pattern. By further culturing, the female early hESCs (ehESCs, P20-P30) lost the expression of XIST RNA, H3K27me3 punctate enrichment and exhibited a completely skewed XCI pattern. Importantly, a subset of X-linked genes was up-regulated in ehESCs, including some cancer-related genes. At last, we found 5% physiological oxygen was beneficial for the expression of XIST and H3K27me3 punctate enrichment, but not for the XCI pattern. We conclude that the XCI dynamic change is a frequent epigenetic instability event during early culture, which is accompanied by the up-regulation of some X-linked genes. Furthermore, we emphasize that physiological oxygen is beneficial for XCI fidelity. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Natural killer cells promote early CD8 T cell responses against cytomegalovirus.

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    Scott H Robbins

    2007-08-01

    Full Text Available Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-alpha/beta production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs for cytokine production, preserves the conventional dendritic cell (cDC compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-alpha administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate

  6. Revised roles of ISL1 in a hES cell-based model of human heart chamber specification

    NARCIS (Netherlands)

    Quaranta, Roberto; Fell, Jakob; Rühle, Frank; Rao, Jyoti; Piccini, Ilaria; Araúzo-Bravo, Marcos J.; Verkerk, Arie O.; Stoll, Monika; Greber, Boris

    2018-01-01

    The transcription factor ISL1 is thought to be key for conveying the multipotent and proliferative properties of cardiac precursor cells. Here, we investigate its function upon cardiac induction of human embryonic stem cells. We find that ISL1 does not stabilize the transient cardiac precursor cell

  7. The Spatial Predilection for Early Esophageal Squamous Cell Neoplasia

    Science.gov (United States)

    Wang, Wen-Lun; Chang, I.-Wei; Chen, Chien-Chuan; Chang, Chi-Yang; Lin, Jaw-Town; Mo, Lein-Ray; Wang, Hsiu-Po; Lee, Ching-Tai

    2016-01-01

    Abstract Early esophageal squamous cell neoplasias (ESCNs) are easily missed with conventional white-light endoscopy. This study aimed to assess whether early ESCNs have a spatial predilection and the patterns of recurrence after endoscopic treatment. We analyzed the circumferential and longitudinal location of early ESCNs, as well as their correlations with exposure to carcinogens in a cohort of 162 subjects with 248 early ESCNs; 219 of which were identified by screening and 29 by surveillance endoscopy. The circumferential location was identified using a clock-face orientation, and the longitudinal location was identified according to the distance from the incisor. The most common circumferential and longitudinal distributions of the early ESCNs were found in the 6 to 9 o’clock quadrant (38.5%) and at 26 to 30 cm from the incisor (41.3%), respectively. A total of 163 lesions (75%) were located in the lower hemisphere arc, and 149 (68.4%) were located at 26 to 35 cm from the incisor. One hundred eleven (51%) early ESCNs were centered within the “hot zone” (i.e., lower hemisphere arc of the esophagus at 26 to 35 cm from the incisor), which comprised 20% of the esophageal area. Exposure to alcohol, betel nut, or cigarette was risk factors for the development of early ESCNs in the lower hemisphere. After complete endoscopic treatment, the mean annual incidence of metachronous tumors was 10%. In addition, 43% of the metachronous recurrent neoplasias developed within the “hot zone.” Cox regression analysis revealed that the index tumor within the hot zone (hazard ratio [HR]: 3.19; 95% confidence interval [CI]: 1.17–8.68; P = 0.02) and the presence of numerous Lugol-voiding lesions in the esophageal background mucosa were independent predictors for metachronous recurrence (HR: 4.61; 95% CI: 1.36–15.56; P = 0.01). We identified a hot zone that may be used to enhance the detection of early ESCNs during endoscopic screening and surveillance

  8. A Christianson syndrome-linked deletion mutation (∆(287)ES(288)) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death.

    Science.gov (United States)

    Ilie, Alina; Gao, Andy Y L; Reid, Jonathan; Boucher, Annie; McEwan, Cassandra; Barrière, Hervé; Lukacs, Gergely L; McKinney, R Anne; Orlowski, John

    2016-09-02

    Christianson Syndrome, a recently identified X-linked neurodevelopmental disorder, is caused by mutations in the human gene SLC9A6 encoding the recycling endosomal alkali cation/proton exchanger NHE6. The patients have pronounced limitations in cognitive ability, motor skills and adaptive behaviour. However, the mechanistic basis for this disorder is poorly understood as few of the more than 20 mutations identified thus far have been studied in detail. Here, we examined the molecular and cellular consequences of a 6 base-pair deletion of amino acids Glu(287) and Ser(288) (∆ES) in the predicted seventh transmembrane helix of human NHE6 expressed in established cell lines (CHO/AP-1, HeLa and neuroblastoma SH-SY5Y) and primary cultures of mouse hippocampal neurons by measuring levels of protein expression, stability, membrane trafficking, endosomal function and cell viability. In the cell lines, immunoblot analyses showed that the nascent mutant protein was properly synthesized and assembled as a homodimer, but its oligosaccharide maturation and half-life were markedly reduced compared to wild-type (WT) and correlated with enhanced ubiquitination leading to both proteasomal and lysosomal degradation. Despite this instability, a measurable fraction of the transporter was correctly sorted to the plasma membrane. However, the rates of clathrin-mediated endocytosis of the ∆ES mutant as well as uptake of companion vesicular cargo, such as the ligand-bound transferrin receptor, were significantly reduced and correlated with excessive endosomal acidification. Notably, ectopic expression of ∆ES but not WT induced apoptosis when examined in AP-1 cells. Similarly, in transfected primary cultures of mouse hippocampal neurons, membrane trafficking of the ∆ES mutant was impaired and elicited marked reductions in total dendritic length, area and arborization, and triggered apoptotic cell death. These results suggest that loss-of-function mutations in NHE6 disrupt recycling

  9. Early gene regulation of osteogenesis in embryonic stem cells

    KAUST Repository

    Kirkham, Glen R.

    2012-01-01

    The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2 in mouse embryonic stem cells were monitored over a 48 hour period after exposure to the growth factors BMP2 and TGFβ1. Candidate GRNs of early osteogenesis were constructed based on published experimental findings and simulation results of Boolean and ordinary differential equation models were compared with our experimental data in order to test the validity of these models. Three gene regulatory networks were found to be consistent with the data, one of these networks exhibited sustained oscillation, a behaviour which is consistent with the general view of embryonic stem cell plasticity. The work cycle presented in this paper illustrates how mathematical modelling can be used to elucidate from gene expression profiles GRNs that are consistent with experimental data. © 2012 The Royal Society of Chemistry.

  10. Early Clostridium difficile infection during allogeneic hematopoietic stem cell transplantation.

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    Melissa A Kinnebrew

    Full Text Available Clostridium difficile infection (CDI is frequently diagnosed in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT. We characterized early-transplant CDI and its associations, and analyzed serially-collected feces to determine intestinal carriage of toxigenic C. difficile. Fecal specimens were collected longitudinally from 94 patients during allo-HSCT hospitalization, from the start of pre-transplant conditioning until up to 35 days after stem cell infusion. Presence of C. difficile 16S rRNA and tcdB genes was determined. Clinical variables and specimen data were analyzed for association with development of CDI. Historical data from an additional 1144 allo-HSCT patients was also used. Fecal specimens from 37 patients (39% were found to harbor C. difficile. Early-transplant CDI was diagnosed in 16 of 94 (17% patients undergoing allo-HSCT; cases were generally mild and resembled non-CDI diarrhea associated with transplant conditioning. CDI was associated with preceding colonization with tcdB-positive C. difficile and conditioning regimen intensity. We found no associations between early-transplant CDI and graft-versus-host disease or CDI later in transplant. CDI occurs with high frequency during the early phase of allo-HSCT, where recipients are pre-colonized with toxigenic C. difficile. During this time, CDI incidence peaks during pre-transplant conditioning, and is correlated to intensity of the treatment. In this unique setting, high rates of CDI may be explained by prior colonization and chemotherapy; however, cases were generally mild and resembled non-infectious diarrhea due to conditioning, raising concerns of misdiagnosis. Further study of this unique population with more discriminating CDI diagnostic tests are warranted.

  11. Early NK Cell Reconstitution Predicts Overall Survival in T-Cell Replete Allogeneic Hematopoietic Stem Cell Transplantation

    DEFF Research Database (Denmark)

    Minculescu, Lia; Marquart, Hanne Vibeke; Friis, Lone Smidstrups

    2016-01-01

    Early immune reconstitution plays a critical role in clinical outcome after allogeneic hematopoietic stem cell transplantation (HSCT). Natural killer (NK) cells are the first lymphocytes to recover after transplantation and are considered powerful effector cells in HSCT. We aimed to evaluate...... the clinical impact of early NK cell recovery in T-cell replete transplant recipients. Immune reconstitution was studied in 298 adult patients undergoing HSCT for acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and myelodysplastic syndrome (MDS) from 2005 to 2013. In multivariate analysis NK...... cell numbers day 30 (NK30) >150cells/µL were independently associated with superior overall survival (hazard ratio 0.79, 95% confidence interval 0.66-0.95, p=0.01). Cumulative incidence analyses showed that patients with NK30 >150cells/µL had significantly less transplant related mortality (TRM), p=0...

  12. Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo

    Directory of Open Access Journals (Sweden)

    Santos Joana

    2010-01-01

    Full Text Available Abstract Background During early mouse development, two extra-embryonic lineages form alongside the future embryo: the trophectoderm (TE and the primitive endoderm (PrE. Epigenetic changes known to take place during these early stages include changes in DNA methylation and modified histones, as well as dynamic changes in gene expression. Results In order to understand the role and extent of chromatin-based changes for lineage commitment within the embryo, we examined the epigenetic profiles of mouse embryonic stem (ES, trophectoderm stem (TS and extra-embryonic endoderm (XEN stem cell lines that were derived from the inner cell mass (ICM, TE and PrE, respectively. As an initial indicator of the chromatin state, we assessed the replication timing of a cohort of genes in each cell type, based on data that expressed genes and acetylated chromatin domains, generally, replicate early in S-phase, whereas some silent genes, hypoacetylated or condensed chromatin tend to replicate later. We found that many lineage-specific genes replicate early in ES, TS and XEN cells, which was consistent with a broadly 'accessible' chromatin that was reported previously for multiple ES cell lines. Close inspection of these profiles revealed differences between ES, TS and XEN cells that were consistent with their differing lineage affiliations and developmental potential. A comparative analysis of modified histones at the promoters of individual genes showed that in TS and ES cells many lineage-specific regulator genes are co-marked with modifications associated with active (H4ac, H3K4me2, H3K9ac and repressive (H3K27me3 chromatin. However, in XEN cells several of these genes were marked solely by repressive modifications (such as H3K27me3, H4K20me3. Consistent with TS and XEN having a restricted developmental potential, we show that these cells selectively reprogramme somatic cells to induce the de novo expression of genes associated with extraembryonic differentiation

  13. PHENOTYPIC FEATURES OF T REGULATORY CELLS IN EARLY RHEUMATOID ARTHRITIS

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    A. S. Avdeeva

    2016-01-01

    Full Text Available Objective: to investigate the count and characteristics of the phenotype of T regulatory cells (Treg in the peripheral blood of healthy donors and patients with early rheumatoid arthritis (RA, by using multicolor flow cytometry.Subjects and methods. The investigation enrolled 39 patients with early RA. The percentage and absolute count of Treg (FoxP3+CD25+, surface CD152+, intracellular CD152+, FoxP3+CD127, CD25+CD127, FoxP3+ICOS+, FoxP3+CD154+; and FoxP3+CD274+ was determined by multicolor flow-cytometry. A control group consisted of 20 healthy donors matched for sex and age with the examined patients.Results and discussion. In the patients included in the study, the median [25th; 75th percentiles] DAS28 was 5.01 [4.2; 5.8]; high, moderate, and low activity showed 22 (48.9%, 20 (44.4%, and 3 (6.7% patients, respectively. The patients with early RA had a lower percentage of FoxP3+CD25+ cells and a lower percentage and absolute count of FoxP3+ICOS+, FoxP3+CD154+, and FoxP3+CD274+ T cells than the healthy donors (p<0.05 in all cases. There was a negative correlation of the percentage of FoxP3+CD25+ cells with C-reactive protein (CRP (r = -0.4, that of intracellular CD152+ with DAS28 (r = -0.35, erythrocyte sedimentation rate (ESR (r = -0.46, and CRP (r=-0.54; that of FoxP3+CD127 with CRP (r = -0.42; that of CD25+CD127 with DAS28 (r = -0.38, Simplified Disease Activity Index (r = -0.41, Clinical Disease Activity Index (r = -0.36, ESR (r = -0.39, and CRP (r = -0.47 (p < 0.05 in all cases.Conclusion. The findings suggest that the functional activity of Treg is impaired in early RA, which has an impact on the activity of the inflammatory process.

  14. Dendritic Cell Lineage Potential in Human Early Hematopoietic Progenitors

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    Julie Helft

    2017-07-01

    Full Text Available Conventional dendritic cells (cDCs are thought to descend from a DC precursor downstream of the common myeloid progenitor (CMP. However, a mouse lymphoid-primed multipotent progenitor has been shown to generate cDCs following a DC-specific developmental pathway independent of monocyte and granulocyte poiesis. Similarly, here we show that, in humans, a large fraction of multipotent lymphoid early progenitors (MLPs gives rise to cDCs, in particular the subset known as cDC1, identified by co-expression of DNGR-1 (CLEC9A and CD141 (BDCA-3. Single-cell analysis indicates that over one-third of MLPs have the potential to efficiently generate cDCs. cDC1s generated from CMPs or MLPs do not exhibit differences in transcriptome or phenotype. These results demonstrate an early imprinting of the cDC lineage in human hematopoiesis and highlight the plasticity of developmental pathways giving rise to human DCs.

  15. Factors affecting daughter cells' arrangement during the early bacterial divisions.

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    Pin-Tzu Su

    Full Text Available On agar plates, daughter cells of Escherichia coli mutually slide and align side-by-side in parallel during the first round of binary fission. This phenomenon has been previously attributed to an elastic material that restricts apparently separated bacteria from being in string. We hypothesize that the interaction between bacteria and the underneath substratum may affect the arrangement of the daughter bacteria. To test this hypothesis, bacterial division on hyaluronic acid (HA gel, as an alternative substratum, was examined. Consistent with our proposition, the HA gel differs from agar by suppressing the typical side-by-side alignments to a rare population. Examination of bacterial surface molecules that may contribute to the daughter cells' arrangement yielded an observation that, with disrupted lpp, the E. coli daughter cells increasingly formed non-typical patterns, i.e. neither sliding side-by-side in parallel nor forming elongated strings. Therefore, our results suggest strongly that the early cell patterning is affected by multiple interaction factors. With oscillatory optical tweezers, we further demonstrated that the interaction force decreased in bacteria without Lpp, a result substantiating our notion that the side-by-side sliding phenomenon directly reflects the strength of in-situ interaction between bacteria and substratum.

  16. Gene function in early mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  17. Early evolution of animal cell signaling and adhesion genes.

    Science.gov (United States)

    Nichols, Scott A; Dirks, William; Pearse, John S; King, Nicole

    2006-08-15

    In stark contrast to the rapid morphological radiation of eumetazoans during the Cambrian explosion, the simple body plan of sponges (Phylum Porifera) emerged from the Cambrian relatively unchanged. Although the genetic and developmental underpinnings of these disparate evolutionary outcomes are unknown, comparisons between modern sponges and eumetazoans promise to reveal the extent to which critical genetic factors were present in their common ancestors. Two particularly interesting classes of genes in this respect are those involved in cell signaling and adhesion. These genes help guide development and morphogenesis in modern eumetazoans, but the timing and sequence of their origins is unknown. Here, we demonstrate that the sponge Oscarella carmela, one of the earliest branching animals, expresses core components of the Wnt, transforming growth factor beta, receptor tyrosine kinase, Notch, Hedgehog, and Jak/Stat signaling pathways. Furthermore, we identify sponge homologs of nearly every major eumetazoan cell-adhesion gene family, including those that encode cell-surface receptors, cytoplasmic linkers, and extracellular-matrix proteins. From these data, we infer that key signaling and adhesion genes were in place early in animal evolution, before the divergence of sponge and eumetazoan lineages.

  18. EmTIP, a T-Cell immunomodulatory protein secreted by the tapeworm Echinococcus multilocularis is important for early metacestode development.

    Directory of Open Access Journals (Sweden)

    Justin Komguep Nono

    Full Text Available BACKGROUND: Alveolar echinococcosis (AE, caused by the metacestode of the tapeworm Echinococcus multilocularis, is a lethal zoonosis associated with host immunomodulation. T helper cells are instrumental to control the disease in the host. Whereas Th1 cells can restrict parasite proliferation, Th2 immune responses are associated with parasite proliferation. Although the early phase of host colonization by E. multilocularis is dominated by a potentially parasitocidal Th1 immune response, the molecular basis of this response is unknown. PRINCIPAL FINDINGS: We describe EmTIP, an E. multilocularis homologue of the human T-cell immunomodulatory protein, TIP. By immunohistochemistry we show EmTIP localization to the intercellular space within parasite larvae. Immunoprecipitation and Western blot experiments revealed the presence of EmTIP in the excretory/secretory (E/S products of parasite primary cell cultures, representing the early developing metacestode, but not in those of mature metacestode vesicles. Using an in vitro T-cell stimulation assay, we found that primary cell E/S products promoted interferon (IFN-γ release by murine CD4+ T-cells, whereas metacestode E/S products did not. IFN-γ release by T-cells exposed to parasite products was abrogated by an anti-EmTIP antibody. When recombinantly expressed, EmTIP promoted IFN-γ release by CD4+ T-cells in vitro. After incubation with anti-EmTIP antibody, primary cells showed an impaired ability to proliferate and to form metacestode vesicles in vitro. CONCLUSIONS: We provide for the first time a possible explanation for the early Th1 response observed during E. multilocularis infections. Our data indicate that parasite primary cells release a T-cell immunomodulatory protein, EmTIP, capable of promoting IFN-γ release by CD4+ T-cells, which is probably driving or supporting the onset of the early Th1 response during AE. The impairment of primary cell proliferation and the inhibition of metacestode

  19. Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential

    Directory of Open Access Journals (Sweden)

    Kristi A. Hohenstein Elliott

    2012-01-01

    Full Text Available Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

  20. [Application of exfoliated cells in early diagnosis of oral cancer].

    Science.gov (United States)

    Liu, T; Zhang, X Y; Sun, Z

    2017-03-09

    Exfoliative cytology is a simple and non-invasive examination method that is easily accepted by the patient. A number of new techniques are used to further increase the accuracy of sample collecting. It is widely used in the detection of cervical, oral cavity and various coelom exfoliated cells. This article reviews the development of exfoliative cytology in oral cancer diagnosis. It is realized that the qualitative and quantitative analysis of cancer and precancerous lesions, through DNA quantitative analysis to calculate DNA index (DI value), multiple parameter analysis and statistical modeling calculation to evaluate oral cancer risk index (OCRI) of the patient has great significance in cancer screening, early diagnosis and prognosis review, especially in the field of oral cancer.

  1. Hydrolyzed infant formula and early β-cell autoimmunity

    DEFF Research Database (Denmark)

    Knip, Mikael; Åkerblom, Hans K; Becker, Dorothy

    2014-01-01

    IMPORTANCE: The disease process leading to clinical type 1 diabetes often starts during the first years of life. Early exposure to complex dietary proteins may increase the risk of β-cell autoimmunity in children at genetic risk for type 1 diabetes. Extensively hydrolyzed formulas do not contain...... intact proteins. OBJECTIVE: To test the hypothesis that weaning to an extensively hydrolyzed formula decreases the cumulative incidence of diabetes-associated autoantibodies in young children. DESIGN, SETTING, AND PARTICIPANTS: A double-blind randomized clinical trial of 2159 infants with HLA......-conferred disease susceptibility and a first-degree relative with type 1 diabetes recruited from May 2002 to January 2007 in 78 study centers in 15 countries; 1078 were randomized to be weaned to the extensively hydrolyzed casein formula and 1081 were randomized to be weaned to a conventional cows' milk...

  2. Esophageal squamous cell carcinoma - precursor lesions and early diagnosis

    Science.gov (United States)

    Lopes, Antonio Barros; Fagundes, Renato Borges

    2012-01-01

    Squamous cell carcinoma of the esophagus (SCCE) carries a poor prognosis due to late diagnosis. Early detection is highly desirable, since surgical and endoscopic resection offers the only possible cure for esophageal cancer. Population screening should be undertaken in high risk areas, and in low or moderate risk areas for people with risk factors (alcoholics, smokers, mate drinkers, history of head and neck cancer, achalasia and lye stricture of the esophagus). Esophageal balloon cytology is an easy and inexpensive sampling technique, but the current methods are insufficient for primary screening due to sampling errors. Conventional endoscopy with biopsy remains the standard procedure for the identification of pre-malignant and early malignant changes in esophageal mucosa and endoscopic detection. It may be enhanced by several techniques such as dye and optic chromoendoscopy, magnifying endoscopy, and optical-based spectroscopic and imaging modalities. Since more than 80% of SCCE deaths occur in developing countries, where expensive techniques such as narrow band imaging (NBI) and autofluorescence imaging are unavailable, the most cost-effective tool for targeting biopsies may be Lugol dye chromoendoscopy, since it is easy, accurate, inexpensive and available worldwide. In ideal conditions, or in developed countries, is it reasonable to think that optimal detection will require a combination of techniques, such as the combination of Lugol’s chromoendoscopy and NBI to identify esophageal areas that require further characterization by a high resolution technique. The efficacy and cost-effectiveness will determine whether these modalities will become part of standard endoscopy practice. PMID:22267978

  3. Early Onset Squamous Cell Carcinoma In A Case Of Lichen Planus

    Directory of Open Access Journals (Sweden)

    Singh Shri Nath

    1998-01-01

    Full Text Available Lichen planus, which is a very common condition, is being presented. However, the uncommon feature in this cases is its early onset and equally early development of squamous cell carcinoma on a lesion on the right thigh.

  4. Current treatment of early breast cancer: adjuvant and neoadjuvant therapy [v1; ref status: indexed, http://f1000r.es/3l2

    Directory of Open Access Journals (Sweden)

    Elizabeth Miller

    2014-08-01

    Full Text Available Breast cancer is the most commonly diagnosed cancer in women. The latest world cancer statistics calculated by the International Agency for Research on Cancer (IARC revealed that 1,677,000 women were diagnosed with breast cancer in 2012 and 577,000 died. The TNM classification of malignant tumor (TNM is the most commonly used staging system for breast cancer. Breast cancer is a group of very heterogeneous diseases. The molecular subtype of breast cancer carries important predictive and prognostic values, and thus has been incorporated in the basic initial process of breast cancer assessment/diagnosis. Molecular subtypes of breast cancers are divided into human epidermal growth factor receptor 2 positive (HER2 +, hormone receptor positive (estrogen or progesterone +, both positive, and triple negative breast cancer. By virtue of early detection via mammogram, the majority of breast cancers in developed parts of world are diagnosed in the early stage of the disease. Early stage breast cancers can be completely resected by surgery. Over time however, the disease may come back even after complete resection, which has prompted the development of an adjuvant therapy. Surgery followed by adjuvant treatment has been the gold standard for breast cancer treatment for a long time. More recently, neoadjuvant treatment has been recognized as an important strategy in biomarker and target evaluation. It is clinically indicated for patients with large tumor size, high nodal involvement, an inflammatory component, or for those wish to preserve remnant breast tissue. Here we review the most up to date conventional and developing treatments for different subtypes of early stage breast cancer.

  5. Fish Stem Cell Cultures

    OpenAIRE

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is th...

  6. Blood based cell biopsy for early detection of cancer

    Science.gov (United States)

    Tang, Cha-Mei; Adams, Daniel; Adams, Diane; Alpaugh, R. Katherine; Cristofanilli, Massimo; Martin, Stuart; Chumsri, Saranya; Marks, Jeffrey

    Early detection (ED) of cancer holds the promise for less aggressive treatments and better outcome. However, there are few accepted methods for ED. We report on a previously unknown blood cell found specifically in the peripheral blood of many solid tumors. They are defined as Cancer Associated Macrophage-Like cells (CAMLs) and are characterized by large size (25-300 μm) and expression of cancer markers. CAMLs were isolated on precision filters during blood filtration. We conducted prospective studies in breast cancer (BC) to ascertain CAML prevalence, specificity and sensitivity in relation to disease status at clinical presentation. We report on two related but separate studies: 1) the isolation of CAMLs from patients with known invasive BC, compared to healthy volunteers and, 2) a double blind study conducted on women undergoing core needle biopsy to evaluate suspicious breast masses. The studies show that CAMLs are found in all stages of BC and suggest that detection of CAMLs can differentiate patients with BC from those with benign breast conditions and healthy individuals. This non-invasive blood test can be potentially used for ED of BC and other malignancies after validation studies with the advantage of a minimally invasive procedure and longitudinal monitoring. This work was supported by Grants from Maryland TEDCO MTTCF, R01-CA154624 from NIH, KG100240 from Susan G. Komen Foundation, Era of Hope Scholar award from DoD (BC100675), and U01-CA084955 from NCI EDRN.

  7. Evaluation of Hematopoietic Stem Cell Mobilization Rates with Early Plerixafor Administration for Adult Stem Cell Transplantation.

    Science.gov (United States)

    Stover, Jessica T; Shaw, J Ryan; Kuchibhatla, Maragatha; Horwitz, Mitchell E; Engemann, Ashley M

    2017-08-01

    The addition of plerixafor to high-dose colony-stimulating growth factor has been shown to improve stem cell mobilization rates in autologous transplant patients with multiple myeloma and non-Hodgkin lymphoma. This study evaluates the change in administration time of plerixafor to determine if cell mobilization rates are similar between the US Food and Drug Administration-approved administration time of 11 hours before apheresis and an earlier administration time of 16 hours before apheresis. Medical records of patients age ≥ 18 years undergoing autologous stem cell transplantation requiring the use of plerixafor after at least 4 days of granulocyte colony-stimulating factor therapy to complete stem cell mobilization from January 1, 2010 through September 30, 2014 were retrospectively reviewed. The primary outcome was CD34 + cell mobilization success rates when plerixafor was administered 11 ± 2 hours (standard administration group) compared with 16 ± 2 hours before cell apheresis (early administration group), as defined as collection of  ≥2 × 10 6 CD34 + cells/kg. Secondary outcomes included the number of plerixafor therapy days required to collect a total of ≥2 × 10 6 CD34 + cells/kg, the number of apheresis cycles required to achieve ≥2 × 10 6 CD34 + cells/kg, the median CD34 + cells/kg collected in each apheresis session, and the rates of reported adverse events that occurred in the standard administration time group compared with the early administration time group. Of the 197 patients included, 114 patients received plerixafor 11 ± 2 hours before apheresis and 83 patients received plerixafor 16 hours ± 2 hours before apheresis. Ninety-four percent of patients in the early administration group achieved successful stem cell mobilization compared with 81.6% in the standard administration group (P = .0111). The median number of plerixafor days to reach the collection goal of  ≥2 × 10 6 CD34 + cells/kg was 1 day for

  8. Identification of a novel gene signature of ES cells self-renewal fluctuation through system-wide analysis.

    Directory of Open Access Journals (Sweden)

    Luigi Cerulo

    Full Text Available Embryonic Stem cells (ESCs can be differentiated into ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. In regular culture conditions, ESCs' self-renewal is maintained through molecules that inhibit spontaneous differentiation enabling long-term cellular expansion. This undifferentiating condition is characterized by multiple metastable states that fluctuate between self-renewal and differentiation balance. Here, we aim to characterize the high-pluripotent ESC metastate marked by the expression of Zscan4 through a supervised machine learning framework based on an ensemble of support vector machine (SVM classifiers. Our study revealed a leukaemia inhibitor factor (Lif dependent not-canonical pluripotency signature (AF067063, BC061212, Dub1, Eif1a, Gm12794, Gm13871, Gm4340, Gm4850, Tcstv1/3, and Zfp352, that specifically marks Zscan4 ESCs' fluctuation. This novel ESC metastate is enhanced by high-pluripotency culture conditions obtained through Extracellular signal Regulated-Kinase (ERK and Glycogen synthase kinase-3 (Gsk-3 signaling inhibition (2i. Significantly, we reported that the conditional ablation of the novel ESC metastate marked by the expression of Gm12794 is required for ESCs self-renewal maintenance. In conclusion, we extend the comprehension of ESCs biology through the identification of a novel molecular signature associated to pluripotency programming.

  9. Early B-lymphocyte precursor cells in mouse bone marrow: Subosteal localization of B220+ cells during postirradiation regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Jacobsen, K.; Tepper, J.; Osmond, D.G. (McGill Univ., Montreal, Quebec (Canada))

    1990-05-01

    The localization of early B-lymphocyte precursor cells in the bone marrow of young mice has been studied during recovery from sublethal whole body gamma-irradiation (150 rad). Initial studies by double immunofluorescence labeling of the B-lineage-associated cell surface glycoprotein, B220, and of mu heavy chains in bone marrow cell suspensions, demonstrated a sequential wave of regeneration of early B precursor cells, pre-B cells, and B cells. Early B precursor cells expressing B220 but not mu chains were enriched at 1-3 days following irradiation. After in vivo administration of 125I-labeled monoclonal antibody 14.8 to detect B220+ cells in situ, light and electron microscope radioautography of femoral bone marrow sections revealed concentrations of labeled B220+ cells located peripherally near the cortical bone at 1-3 days following irradiation, increasing in numbers in more central areas by 5-7 days. Proliferative B220+ precursor cells were found within layers of bone-lining cells and in a subosteal area characterized by a prominent electron-dense extracellular matrix, often associated with stromal reticular cells. The results demonstrate that the precursor cells that are active in the bone marrow early in the recovery of B lymphopoiesis after gamma-irradiation are located both within and near the endosteum of the surrounding bone. The distinctive extracellular matrix and stromal cell associations noted in this region may contribute to a supportive local microenvironment for early hemopoietic progenitor cells.

  10. Early Diagnosis and Monitoring of Neurodegenerative Langerhans Cell Histiocytosis.

    Directory of Open Access Journals (Sweden)

    Elena Sieni

    Full Text Available Neurodegenerative Langerhans Cell Histiocytosis (ND-LCH is a rare, unpredictable consequence that may devastate the quality of life of patients cured from LCH. We prospectively applied a multidisciplinary diagnostic work-up to early identify and follow-up patients with ND-LCH, with the ultimate goal of better determining the appropriate time for starting therapy.We studied 27 children and young adults with either ND-LCH verified by structural magnetic resonance imaging (MRI (group 1 or specific risk factors for (diabetes insipidus, craniofacial bone lesions, but no evidence of, neurodegenerative MRI changes (group 2. All patients underwent clinical, neurophysiological and MRI studies.Seventeen patients had MRI alterations typical for ND-LCH. Nine showed neurological impairment but only three were symptomatic; 11 had abnormal somatosensory evoked potentials (SEPs, and five had abnormal brainstem auditory evoked potentials (BAEPs. MR spectroscopy (MRS showed reduced cerebellar NAA/Cr ratio in nine patients. SEPs showed sensitivity, specificity, positive predictive value (PPV and negative predictive value (NPV for predicting ND-LCH of 70.6% (95%CI, 44.0%-89.7%, 100% (69.2%-100%, 100% (73.5%-100%, and 66.7% (38.4%-88.2%, respectively. Repeated investigations in group 1 revealed increasingly abnormal EP parameters, or neurological examination, or both, in nine of fifteen patients while MRI remained unchanged in all but one patient.A targeted MRI study should be performed in all patients with risk factors for ND-LCH for early identification of demyelination. The combined use of SEPs and careful neurological evaluation may represent a valuable, low-cost, well-tolerated and easily available methodology to monitor patients from pre-symptomatic to symptomatic stages. We suggest a multidisciplinary protocol including clinical, MRS, and neurophysiological investigations to identify a population target for future therapeutic trials.

  11. A tool box for operational mosquito larval control: preliminary results and early lessons from the Urban Malaria Control Programme in Dar es Salaam, Tanzania

    Directory of Open Access Journals (Sweden)

    Govella Nico J

    2008-01-01

    Full Text Available Abstract Background As the population of Africa rapidly urbanizes, large populations could be protected from malaria by controlling aquatic stages of mosquitoes if cost-effective and scalable implementation systems can be designed. Methods A recently initiated Urban Malaria Control Programme in Dar es Salaam delegates responsibility for routine mosquito control and surveillance to modestly-paid community members, known as Community-Owned Resource Persons (CORPs. New vector surveillance, larviciding and management systems were designed and evaluated in 15 city wards to allow timely collection, interpretation and reaction to entomologic monitoring data using practical procedures that rely on minimal technology. After one year of baseline data collection, operational larviciding with Bacillus thuringiensis var. israelensis commenced in March 2006 in three selected wards. Results The procedures and staff management systems described greatly improved standards of larval surveillance relative to that reported at the outset of this programme. In the first year of the programme, over 65,000 potential Anopheles habitats were surveyed by 90 CORPs on a weekly basis. Reaction times to vector surveillance at observations were one day, week and month at ward, municipal and city levels, respectively. One year of community-based larviciding reduced transmission by the primary malaria vector, Anopheles gambiae s.l., by 31% (95% C.I. = 21.6–37.6%; p = 0.04. Conclusion This novel management, monitoring and evaluation system for implementing routine larviciding of malaria vectors in African cities has shown considerable potential for sustained, rapidly responsive, data-driven and affordable application. Nevertheless, the true programmatic value of larviciding in urban Africa can only be established through longer-term programmes which are stably financed and allow the operational teams and management infrastructures to mature by learning from experience.

  12. CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.

    Science.gov (United States)

    Oji, Asami; Noda, Taichi; Fujihara, Yoshitaka; Miyata, Haruhiko; Kim, Yeon Joo; Muto, Masanaga; Nozawa, Kaori; Matsumura, Takafumi; Isotani, Ayako; Ikawa, Masahito

    2016-08-17

    Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene, Dnajb13, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that Dnajb13 is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.

  13. Radiofrequency Ablation Treatment for Renal Cell Carcinoma: Early Clinical Experience

    Energy Technology Data Exchange (ETDEWEB)

    Park, Seong Hoon; Yoon, Seong Kuk; Cho, Jin Han; Oh, Jong Young; Nam, Kyung Jin; Kwon, Hee Jin; Kim, Su Yeon; Kang, Myong Jin; Choi, Sun Seob; Sung, Gyung Tak [Dong-A University College of Medicine, Busan (Korea, Republic of)

    2008-08-15

    To evaluate the early clinical experience associated with radiofrequency (RF) ablation in patients with renal cell carcinoma (RCC). The RF ablation treatment was performed on 17 tumors from 16 patients (mean age, 60.5 years; range, 43 73 years) with RCC. The treatment indications were localized, solid renal mass, comorbidities, high operation risk, and refusal to perform surgery. All tumors were treated by a percutaneous CT (n = 10), followed by an US-guided (n = 2), laparoscopy-assisted US (n = 2), and an open (n = 2) RF ablation. Furthermore, patients underwent a follow- up CT at one day, one week, one month, three and six months, and then every six months from the onset of treatment. We evaluated the technical success, technical effectiveness, ablation zone, benign periablation enhancement, irregular peripheral enhancement, and complications. All 17 exophytic tumors (mean size, 2.2 cm; range, 1.1 5.0 cm) were completely ablated. Technical success and effectiveness was achieved in all cases and the mean follow-up period was 23.8 months (range, 17 33 months). A local recurrence was not detected in any of the cases; however, five patients developed complications as a result of treatment, including hematuria (n = 2), mild thermal injury of the psoas muscle (n = 1), mild hydronephrosis (n = 1), and fistula formation (n = 1). The RF ablation is an alternative treatment for exophytic RCCs and represents a promising treatment for some patients with small RCCs.

  14. Early death during chemotherapy in patients with small-cell lung cancer

    DEFF Research Database (Denmark)

    Lassen, U N; Osterlind, K; Hirsch, F R

    1999-01-01

    Based on an increased frequency of early death (death within the first treatment cycle) in our two latest randomized trials of combination chemotherapy in small-cell lung cancer (SCLC), we wanted to identify patients at risk of early non-toxic death (ENTD) and early toxic death (ETD). Data were...

  15. Early Life Processes, Endocrine Mediators and Number of Susceptible Cells in Relation to Breast Cancer Risk

    Science.gov (United States)

    2007-04-01

    Early life processes, endocrine mediators and number of susceptible cells in relation 5a. CONTRACT NUMBER to breast cancer ... cancer risk. Method: Five interlinked component projects covering the spectrum from endometrial to adult life . Progress report: Component projects...Analyses are pending and no findings can be reported yet. 15. SUBJECT TERMS Breast cancer , early life , mammary gland specific stem cells, hormones 16

  16. Innate Immune Landscape in Early Lung Adenocarcinoma by Paired Single-Cell Analyses.

    Science.gov (United States)

    Lavin, Yonit; Kobayashi, Soma; Leader, Andrew; Amir, El-Ad David; Elefant, Naama; Bigenwald, Camille; Remark, Romain; Sweeney, Robert; Becker, Christian D; Levine, Jacob H; Meinhof, Klaus; Chow, Andrew; Kim-Shulze, Seunghee; Wolf, Andrea; Medaglia, Chiara; Li, Hanjie; Rytlewski, Julie A; Emerson, Ryan O; Solovyov, Alexander; Greenbaum, Benjamin D; Sanders, Catherine; Vignali, Marissa; Beasley, Mary Beth; Flores, Raja; Gnjatic, Sacha; Pe'er, Dana; Rahman, Adeeb; Amit, Ido; Merad, Miriam

    2017-05-04

    To guide the design of immunotherapy strategies for patients with early stage lung tumors, we developed a multiscale immune profiling strategy to map the immune landscape of early lung adenocarcinoma lesions to search for tumor-driven immune changes. Utilizing a barcoding method that allows a simultaneous single-cell analysis of the tumor, non-involved lung, and blood cells, we provide a detailed immune cell atlas of early lung tumors. We show that stage I lung adenocarcinoma lesions already harbor significantly altered T cell and NK cell compartments. Moreover, we identified changes in tumor-infiltrating myeloid cell (TIM) subsets that likely compromise anti-tumor T cell immunity. Paired single-cell analyses thus offer valuable knowledge of tumor-driven immune changes, providing a powerful tool for the rational design of immune therapies. VIDEO ABSTRACT. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Sclerocorneal limbal stem cell autograft transplantation in dogs Transplante autógeno de células tronco do limbo esclerocorneal em cães

    Directory of Open Access Journals (Sweden)

    A.T.J. Brunelli

    2007-10-01

    Full Text Available The effects of sclerocorneal limbal stem cell autograft transplantation in dogs with corneal wounds were studied. Eighteen dogs were divided in two groups (GI and GII. The animals of GI (n=12 underwent limbal transplantation 30 days after the destruction of limbal stem cells. The dogs of GII (n=6 only underwent destruction of stem cells (control group. Light microscopy examination of the right eye was performed on days 3, 7, 14, 30, 60, and 120 after limbal transplantation (GI, and on days 33, 37, 44, 60, 90, and 150 after limbal destruction (GII. Results showed a complete destruction of limbal stem cells with loss of corneal transparency. Limbal transplantation prevented conjunctivalization in grafted area. Corneal vascularization and a 360º corneal conjunctivalization were noted in the control dogs (GII. Corneal transparency was restored from day 60th after surgery. Histological examination did not distinguish the transition between the graft and the normal corneal epithelium at anytime. Goblet cells were found in control animals (GII on 33, 37, 60, and 150 days, whereas a single grafted dog (GI presented a few goblet cells on day 60th post-transplantation. Limbal autograft transplantation was effective in restoring corneal clarity with no development of ocular complications.Avaliaram-se os efeitos do transplante de células tronco autógenas do limbo esclerocórneo de cães, sobre lesões córneo-limbais. Empregaram-se 18 cães, distribuídos em dois grupos, GI e GII. Nos animais do GI (n=12, foram realizados transplantes de limbo, após 30 dias da destruição das células tronco-límbicas. Nos do GII (n=6, realizou-se apenas a destruição do limbo (controle. Aos 3, 7, 15, 30, 60 e 120 dias do transplante de limbo (GI e aos 33, 37, 45, 60, 90 e 150 dias da destruição do limbo (GII, os olhos foram coletados por enucleação subconjuntival, para estudos em microscopia de luz. A destruição do limbo resultou em completa excisão das c

  18. Single-Cell Landscape of Transcriptional Heterogeneity and Cell Fate Decisions during Mouse Early Gastrulation

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    Hisham Mohammed

    2017-08-01

    Full Text Available The mouse inner cell mass (ICM segregates into the epiblast and primitive endoderm (PrE lineages coincident with implantation of the embryo. The epiblast subsequently undergoes considerable expansion of cell numbers prior to gastrulation. To investigate underlying regulatory principles, we performed systematic single-cell RNA sequencing (seq of conceptuses from E3.5 to E6.5. The epiblast shows reactivation and subsequent inactivation of the X chromosome, with Zfp57 expression associated with reactivation and inactivation together with other candidate regulators. At E6.5, the transition from epiblast to primitive streak is linked with decreased expression of polycomb subunits, suggesting a key regulatory role. Notably, our analyses suggest elevated transcriptional noise at E3.5 and within the non-committed epiblast at E6.5, coinciding with exit from pluripotency. By contrast, E6.5 primitive streak cells became highly synchronized and exhibit a shortened G1 cell-cycle phase, consistent with accelerated proliferation. Our study systematically charts transcriptional noise and uncovers molecular processes associated with early lineage decisions.

  19. Early stages in human and mouse T-cell development

    NARCIS (Netherlands)

    Spits, H.

    1994-01-01

    One important question in lymphopoiesis is where stem cells commit to T-, B- and natural killer (NK)-cell lineages. Recent findings in human and mouse systems suggest that the thymus is seeded by a yet uncommitted progenitor cell. The earliest murine thymic progenitor cells have the capacity to

  20. Ezh2 Controls an Early Hematopoietic Program and Growth and Survival Signaling in Early T Cell Precursor Acute Lymphoblastic Leukemia

    Directory of Open Access Journals (Sweden)

    Etienne Danis

    2016-03-01

    Full Text Available Earlycell precursor acute lymphoblastic leukemia (ETP-ALL is an aggressive subtype of ALL distinguished by stem-cell-associated and myeloid transcriptional programs. Inactivating alterations of Polycomb repressive complex 2 components are frequent in human ETP-ALL, but their functional role is largely undefined. We have studied the involvement of Ezh2 in a murine model of NRASQ61K-driven leukemia that recapitulates phenotypic and transcriptional features of ETP-ALL. Homozygous inactivation of Ezh2 cooperated with oncogenic NRASQ61K to accelerate leukemia onset. Inactivation of Ezh2 accentuated expression of genes highly expressed in human ETP-ALL and in normal murine early thymic progenitors. Moreover, we found that Ezh2 contributes to the silencing of stem-cell- and early-progenitor-cell-associated genes. Loss of Ezh2 also resulted in increased activation of STAT3 by tyrosine 705 phosphorylation. Our data mechanistically link Ezh2 inactivation to stem-cell-associated transcriptional programs and increased growth/survival signaling, features that convey an adverse prognosis in patients.

  1. Probing early heart development to instruct stem cell differentiation strategies

    National Research Council Canada - National Science Library

    Calderon, Damelys; Bardot, Evan; Dubois, Nicole

    2016-01-01

    .... These discoveries are driven by the need to answer long‐standing questions regarding the origin of the earliest cells specified to the cardiac lineage, the differentiation potential of distinct cardiac progenitor cells, and, very importantly, the molecular...

  2. Brugia malayi Antigen (BmA Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells.

    Directory of Open Access Journals (Sweden)

    Emily E I M Mouser

    Full Text Available One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA and excretory-secretory product 62 (ES-62 from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs.

  3. A Cell-Based Approach to Early Pancreatic Cancer Detection

    Science.gov (United States)

    2016-10-01

    disease would be improved by the ability to non- invasively – through a blood test – determine the status of the tumor. Our research is therefore aimed at...quite high when cells were spiked into whole blood. In particular, recovery rates of 90% were seen in these experiments even when low cell numbers... nanoparticle labeled cultured tumor cells positive for YFP were separated from mouse leukocytes using TEMPO, and quantified by flow cytometry a

  4. Fuel cell added value for early market applications

    Science.gov (United States)

    Hardman, Scott; Chandan, Amrit; Steinberger-Wilckens, Robert

    2015-08-01

    Fuel Cells are often considered in the market place as just power providers. Whilst fuel cells do provide power, there are additional beneficial characteristics that should be highlighted to consumers. Due to the high price premiums associated with fuel cells, added value features need to be exploited in order to make them more appealing and increase unit sales and market penetration. This paper looks at the approach taken by two companies to sell high value fuel cells to niche markets. The first, SFC Energy, has a proven track record selling fuel cell power providers. The second, Bloom Energy, is making significant progress in the US by having sold its Energy Server to more than 40 corporations including Wal-Mart, Staples, Google, eBay and Apple. Further to these current markets, two prospective added value applications for fuel cells are discussed. These are fuel cells for aircraft APUs and fuel cells for fire prevention. These two existing markets and two future markets highlight that fuel cells are not just power providers. Rather, they can be used as solutions to many needs, thus being more cost effective by replacing a number of incumbent systems at the same time.

  5. Epithelial cell migration as a potential therapeutic target in early lung cancer

    Directory of Open Access Journals (Sweden)

    Fraser R. Millar

    2017-02-01

    Full Text Available Lung cancer is the most lethal cancer type worldwide, with the majority of patients presenting with advanced stage disease. Targeting early stage disease pathogenesis would allow dramatic improvements in lung cancer patient survival. Recently, cell migration has been shown to be an integral process in early lung cancer ontogeny, with preinvasive lung cancer cells shown to migrate across normal epithelium prior to developing into invasive disease. TP53 mutations are the most abundant mutations in human nonsmall cell lung cancers and have been shown to increase cell migration via regulation of Rho-GTPase protein activity. In this review, we explore the possibility of targeting TP53-mediated Rho-GTPase activity in early lung cancer and the opportunities for translating this preclinical research into effective therapies for early stage lung cancer patients.

  6. Understanding the Molecular Circuitry of Cell Lineage Specification in the Early Mouse Embryo

    Science.gov (United States)

    Bergsmedh, Anna; Donohoe, Mary E.; Hughes, Rebecca-Ayme; Hadjantonakis, Anna-Katerina

    2011-01-01

    Pluripotent stem cells hold great promise for cell-based therapies in regenerative medicine. However, critical to understanding and exploiting mechanisms of cell lineage specification, epigenetic reprogramming, and the optimal environment for maintaining and differentiating pluripotent stem cells is a fundamental knowledge of how these events occur in normal embryogenesis. The early mouse embryo has provided an excellent model to interrogate events crucial in cell lineage commitment and plasticity, as well as for embryo-derived lineage-specific stem cells and induced pluripotent stem (iPS) cells. Here we provide an overview of cell lineage specification in the early (preimplantation) mouse embryo focusing on the transcriptional circuitry and epigenetic marks necessary for successive differentiation events leading to the formation of the blastocyst. PMID:24710206

  7. Low doses of gamma-irradiation induce an early bystander effect in zebrafish cells which is sufficient to radioprotect cells.

    Science.gov (United States)

    Pereira, Sandrine; Malard, Véronique; Ravanat, Jean-Luc; Davin, Anne-Hélène; Armengaud, Jean; Foray, Nicolas; Adam-Guillermin, Christelle

    2014-01-01

    The term "bystander effect" is used to describe an effect in which cells that have not been exposed to radiation are affected by irradiated cells though various intracellular signaling mechanisms. In this study we analyzed the kinetics and mechanisms of bystander effect and radioadaptation in embryonic zebrafish cells (ZF4) exposed to chronic low dose of gamma rays. ZF4 cells were irradiated for 4 hours with total doses of gamma irradiation ranging from 0.01-0.1 Gy. In two experimental conditions, the transfer of irradiated cells or culture medium from irradiated cells results in the occurrence of DNA double strand breaks in non-irradiated cells (assessed by the number of γ-H2AX foci) that are repaired at 24 hours post-irradiation whatever the dose. At low total irradiation doses the bystander effect observed does not affect DNA repair mechanisms in targeted and bystander cells. An increase in global methylation of ZF4 cells was observed in irradiated cells and bystander cells compared to control cells. We observed that pre-irradiated cells which are then irradiated for a second time with the same doses contained significantly less γ-H2AX foci than in 24 h gamma-irradiated control cells. We also showed that bystander cells that have been in contact with the pre-irradiated cells and then irradiated alone present less γ-H2AX foci compared to the control cells. This radioadaptation effect is significantly more pronounced at the highest doses. To determine the factors involved in the early events of the bystander effect, we performed an extensive comparative proteomic study of the ZF4 secretomes upon irradiation. In the experimental conditions assayed here, we showed that the early events of bystander effect are probably not due to the secretion of specific proteins neither the oxidation of these secreted proteins. These results suggest that early bystander effect may be due probably to a combination of multiple factors.

  8. Usefulness of computed tomography and ultrasonography for early detection of renal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Horii, Yasuhiro; Tsumatani, Kenichi; Natsume, Osamu

    1987-07-01

    Eighty-three cases of renal cell carcinomas admitted to Nara Medical University and its related hospitals from August, 1962 through July, 1984 were reviewed. We have been using computed tomography (CT) and ultrasonography (US) for early detection of renal cell carcinomas since 1980. Thereafter the number of patients with low stage renal cell carcinoma was significantly increased. Furthermore 6 carcinomas were incidentally detected by CT and/or US examination for checkup of other diseases. We believe that CT and US may be valuable as a screening modality for early detection of renal cell carcinoma.

  9. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Van Hoof, Dennis; Muñoz, Javier; Braam, Stefan R

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during...

  10. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during

  11. DAR ES SALAAM, TANZANIA

    African Journals Online (AJOL)

    Othman-Specialion of Cadmium, Copper , Lead and Zinc in Msimbazi river ... Samples of water were taken from river Msimbazi that runs through the city of Dar es ... immediately subjected to laboratory analysis and when necessary stored in a.

  12. Diatomées

    African Journals Online (AJOL)

    Impact environnemental et évaluation de la qualité des eaux par des méthodes chimiques et biologiques « Diatomées » ... possèdent un caractère alcaliphyle prononcé avec une abondance des espèces résistantes à la pollution organique. Mots clés : Diatomées, qualité de l'eau, bio-indicateur, IDL, IPO, Merja Fouarat.

  13. Fluoxetine Targets Early Progenitor Cells in the Adult Brain

    National Research Council Canada - National Science Library

    Juan M. Encinas; Anne Vaahtokari; Grigori Enikolopov

    2006-01-01

    .... We use this line to demonstrate that the selective serotonin reuptake inhibitor antidepressant fluoxetine does not affect division of stem-like cells in the dentate gyrus but increases symmetric...

  14. B cells: from early development to regulating allergic diseases.

    Science.gov (United States)

    Samitas, Konstantinos; Lötvall, Jan; Bossios, Apostolos

    2010-06-01

    B lymphocytes are characterized by a unique and highly specialized developmental pathway that is responsible for their vast phenotypic and function diversity. B cell development is strictly regulated to ensure sufficient specific humoral immunity while at the same time avoiding any errors that would compromise B cell functionality. The generation and maintenance of mature B cells from the constant flux of bone marrow progenitors is a complex process that is generally poorly understood, although great progress has been made in recent years. B cells have for long been considered mainly as antibody-producing cells and therefore believed to play an important role in the pathophysiology of allergic diseases, primarily through their ability to produce IgE antibodies. However, recent findings have revealed new aspects of their role in immune responses that place them again under the spotlight as important immune regulators, independent of antibody production. This review focuses on the developmental processes responsible for the numerous phenotypes and functions of the B-lymphocyte pool and the different aspects of effector B cell functionality in the context of allergy.

  15. Polyamines affect histamine synthesis during early stages of IL-3-induced bone marrow cell differentiation.

    Science.gov (United States)

    García-Faroldi, Gianni; Correa-Fiz, Florencia; Abrighach, Hicham; Berdasco, María; Fraga, Mario F; Esteller, Manel; Urdiales, José L; Sánchez-Jiménez, Francisca; Fajardo, Ignacio

    2009-09-01

    Mast cells synthesize and store histamine, a key immunomodulatory mediator. Polyamines are essential for every living cell. Previously, we detected an antagonistic relationship between the metabolisms of these amines in established mast cell and basophilic cell lines. Here, we used the IL-3-driven mouse bone marrow-derived mast cell (BMMC) culture system to further investigate this antagonism in a mast cell model of deeper physiological significance. Polyamines and histamine levels followed opposite profiles along the bone marrow cell cultures leading to BMMCs. alpha-Difluoromethylornithine (DFMO)-induced polyamine depletion resulted in an upregulation of histidine decarboxylase (HDC, the histamine-synthesizing enzyme) expression and activity, accompanied by increased histamine levels, specifically during early stages of these cell cultures, where an active histamine synthesis process occurs. In contrast, DFMO did not induce any effect in either HDC activity or histamine levels of differentiated BMMCs or C57.1 mast cells, that exhibit a nearly inactive histamine synthesis rate. Sequence-specific DNA methylation analysis revealed that the DFMO-induced HDC mRNA upregulation observed in early bone marrow cell cultures is not attributable to a demethylation of the gene promoter caused by the pharmacological polyamine depletion. Taken together, the results support an inverse relationship between histamine and polyamine metabolisms during the bone marrow cell cultures leading to BMMCs and, moreover, suggest that the regulation of the histamine synthesis occurring during the early stages of these cultures depends on the concentrations of polyamines. (c) 2009 Wiley-Liss, Inc.

  16. Carcinoma oat-cell de esófago: presentación de dos casos y actualización bibliográfica Oat-cell carcinoma of the esophagus: Presentation of two cases and literature review

    Directory of Open Access Journals (Sweden)

    A. Liñán Padilla

    2007-07-01

    Full Text Available Objetivo: nuestro objetivo es aportar una visión actualizada sobre una patología tan infrecuente como el carcinoma oat-cell esofágico, mediante revisión de la bibliografía y la exposición de dos casos de reciente aparición en nuestro servicio. Material y método: se realizó un estudio retrospectivo con revisión de las historias clínicas de los pacientes ingresados en nuestro servicio con el diagnóstico de neoplasia esofágica en los últimos 6 años (enero de 2000 a diciembre de 2006. Resultados: se estudiaron 249 casos de neoplasias de esófago en el curso de este periodo de 6 años, de ellas 106 fueron de estirpe epidermoide (42,6%, 141 adenocarcinomas (56,6% y 2 oat-cell (0,8%. Del total de estos tumores sólo en 45 (18% de ellos se pudo realizar resección quirúrgica, en 23 (9,3% cirugía paliativa (no se incluyen prótesis endoscópicas, gastrostomías ni yeyunostomías y en el resto (181 casos, 72,7% cirugía derivativa o ninguna opción quirúrgica. Conclusiones: podemos afirmar que esta neoplasia es altamente agresiva, presentando en prácticamente la totalidad de los casos diseminación a otras localizaciones. Se trata de un cáncer muy poco frecuente que afecta mayoritariamente a varones y cuya clínica es similar al resto de procesos neoplásicos que afectan al esófago.Objective: our objective is to contribute an updated view on a condition as rare as oat-cell carcinoma of the esophagus by reviewing the literature and reporting two recent patients seen in our department. Material and method: a retrospective study with a review of all medical records of patients seen in our ward and diagnosed with esophageal neoplasm for 6 years (January 2000 to December 2006. Results: 249 cases of esophageal neoplasms were found, of them 106 were of squamous ancestry (42.6%, 141 were adenocarcinomas (56.6%, and 2 were oat-cell carcinomas (0.8%. Only in 45 (18% was surgical resection feasible, 23 underwent palliative surgery

  17. Prevalence of epithelial ovarian cancer stem cells correlates with recurrence in early-stage ovarian cancer

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Alvero, Ayesha B; Yang, Yingkui

    2011-01-01

    Epithelial ovarian cancer stem cells (EOC stem cells) have been associated with recurrence and chemoresistance. CD44 and CK18 are highly expressed in cancer stem cells and function as tools for their identification and characterization. We investigated the association between the number of CD44......+ EOC stem cells in ovarian cancer tumors and progression-free survival. EOC stem cells exist as clusters located close to the stroma forming the cancer stem cell "niche". 17.1% of the samples reveled high number of CD44+ EOC stem cells (>20% positive cells). In addition, the number of CD44+ EOC stem...... cells was significantly higher in patients with early-stage ovarian cancer (FIGO I/II), and it was associated with shorter progression-free survival (P = 0.026). This study suggests that quantification of the number of EOC stem cells in the tumor can be used as a predictor of disease and could...

  18. Circulating endothelial cells are an early predictor in renal cell carcinoma for tumor response to sunitinib

    Directory of Open Access Journals (Sweden)

    Ivanyi Philipp

    2010-12-01

    Full Text Available Abstract Background Tyrosine kinase inhibitors (TKI have enriched the therapeutic options in patients with renal cell carcinoma (RCC, which frequently induce morphological changes in tumors. However, only little is known about the biological activity of TKI. Circulating endothelial cells (CEC have been associated with endothelial damage and, hence, may serve as a putative marker for the biological activity of TKI. The main objective of our study was to evaluate the predictive value of CEC, monocytes, and soluble vascular endothelial growth factor receptor (sVEGFR-2 in RCC patients receiving sunitinib treatment. Methods Analyses of CEC, monocytes, and sVEGFR-2 were accomplished for twenty-six consecutive patients with metastatic RCC who received treatment with sunitinib (50 mg, 4 wks on 2 wks off schedule at our institution in 2005 and 2006. Results In RCC patients CEC are elevated to 49 ± 44/ml (control 8 ± 8/ml; P = 0.0001. Treatment with sunitinib is associated with an increase in CEC within 28 days of treatment in patients with a Progression free survival (PFS above the median to 111 ± 61 (P = 0.0109, whereas changes in patients with a PFS below the median remain insignificant 69 ± 61/ml (P = 0.1848. Monocytes and sVEGFR2 are frequently altered upon sunitinib treatment, but fail to correlate with clinical response, defined by PFS above or below the median. Conclusions Sunitinib treatment is associated with an early increase of CEC in responding patients, suggesting superior endothelial cell damage in these patients as a putative predictive biomarker.

  19. Distrator articular no diagnóstico radiográfico precoce da displasia coxofemoral em cães Articular distractor in the early radiographic diagnosis of canine hip dysplasia

    Directory of Open Access Journals (Sweden)

    R.C.S. Tôrres

    2005-02-01

    Full Text Available Foram estudadas radiograficamente as articulações coxofemorais de 60 cães de diferentes raças (32 fêmeas e 28 machos, com o objetivo de se estabelecer o diagnóstico precoce da displasia coxofemoral (DCF. Os exames foram feitos em idade jovem (7,2± 1,2 meses e repetidos em idade adulta (14,4± 1,6 meses. Utilizaram-se os métodos radiográfico convencional (MRC e radiográfico por distração (MRD, feito com o auxílio de um dispositivo, especialmente idealizado e confeccionado para o experimento. Observou-se correlação (PAiming the canine hip dysplasia (CHD early diagnosis, 60 dogs of both sexes (32 females and 28 males and of different breeds had their hip joints radiographically studied. The X ray examinations were taken in early age (7.2± 1.2 months and repeated at adult age (14.4± 1.6 months using the conventional radiographic method (CRM and the radiographic distraction method (RDM performed, by its turn, with a new device, specially designed or this experiment. In order to quantify the relationship between the femoral head and the acetabulus the Norberg Angle (NA was measured at CRM and the distraction index (DI was calculated at RDM. There was a significant statistical correlation (P<0.001 between DI of the first examination and NA of the second examination. The cut off for sick and normal subjects for DI was 0.35. The dogs with DI values greater than 0,35 presented radiographic signals of degenerative joint disease (DJD. Interestingly, the highest was the DI; the greatest were the radiographic signals of DJD. Four animals considered normal when CRM was performed at second examination had DI superior than 0.35 at first examination. This fact shows that false negative results can occur when only the conventional method is used. Two of three sick dogs that had DI values bellow 0.35 were classified as borderline by CRM. This confirms the indefinite of this class. The association of CRM and the proposed method showed to be

  20. Distribution of LGR5+ cells and associated implications during the early stage of gastric tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Bo Gun Jang

    Full Text Available Lgr5 was identified as a promising gastrointestinal tract stem cell marker in mice. Lineage tracing indicates that Lgr5(+ cells may not only be the cells responsible for the origin of tumors; they may also be the so-called cancer stem cells. In the present study, we investigated the presence of Lgr5(+ cells and their biological significance in normal human gastric mucosa and gastric tumors. RNAscope, a newly developed RNA in situ hybridization technique, specifically labeled Lgr5(+ cells at the basal glands of the gastric antrum. Notably, the number of Lgr5(+ cells was remarkably increased in intestinal metaplasia. In total, 76% of gastric adenomas and 43% of early gastric carcinomas were positive for LGR5. Lgr5(+ cells were found more frequently in low-grade tumors with active Wnt signaling and an intestinal gland type, suggesting that LGR5 is likely involved in the very early stages of Wnt-driven tumorigenesis in the stomach. Interestingly, similar to stem cells in normal tissues, Lgr5(+ cells were often restricted to the base of the tumor glands, and such Lgr5(+ restriction was associated with high levels of intestinal stem cell markers such as EPHB2, OLFM4, and ASCL2. Thus, our findings show that Lgr5(+ cells are present at the base of the antral glands in the human stomach and that this cell population significantly expands in intestinal metaplasias. Furthermore, Lgr5(+ cells are seen in a large number of gastric tumors ; their frequent basal arrangements and coexpression of ISC markers support the idea that Lgr5(+ cells act as stem cells during the early stage of intestinal-type gastric tumorigenesis.

  1. Early reversal cells in adult human bone remodeling

    DEFF Research Database (Denmark)

    Abdelgawad, Mohamed Essameldin; Delaissé, Jean-Marie; Hinge, Maja

    2016-01-01

    The mechanism coupling bone resorption and formation is a burning question that remains incompletely answered through the current investigations on osteoclasts and osteoblasts. An attractive hypothesis is that the reversal cells are likely mediators of this coupling. Their nature is a big matter...... of debate. The present study performed on human cancellous bone is the first one combining in situ hybridization and immunohistochemistry to demonstrate their osteoblastic nature. It shows that the Runx2 and CD56 immunoreactive reversal cells appear to take up TRAcP released by neighboring osteoclasts....... Earlier preclinical studies indicate that reversal cells degrade the organic matrix left behind by the osteoclasts and that this degradation is crucial for the initiation of the subsequent bone formation. To our knowledge, this study is the first addressing these catabolic activities in adult human bone...

  2. Intraoperative Margin Assessment in Early Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Chiosea, Simion I

    2017-03-01

    The surgical method of margin sampling affects local control, pathologists' approach to margin sampling, and clarity of pathology reports. Studies have shown that exclusive reliance on tumor bed margins is associated with worse local control and should be avoided. En bloc resections and margins obtained from the resection specimen remain the "gold standard." Successful surgical treatment of early carcinomas of the oral cavity relies on close cooperation between surgeons and pathologists on issues of specimen orientation and margin sampling. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. The role of natural killer cells in the early period of infection in murine cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    M.D. Laurenti

    1999-03-01

    Full Text Available In order to study the role of natural killer (NK cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania amazonensis (HSJD-1 strain. 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 106 spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 106 spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection.

  4. NADAR ES LA CLAVE

    Directory of Open Access Journals (Sweden)

    MANUEL RUIZ C.

    2015-03-01

    Full Text Available Claro, la clave es nadar, nadar para crecer y trascender.  Pues, es el agua, el fluido mágico en el cual se dinamiza  nuestro cuerpo en forma sensorial, y en donde el espíritu  se recrea, mientras la mente se complace al comprender,  que es la natación, la que compromete al ser en su totalidad  y lo funde en una perfecta unidad funcional;  cuerpo y mente, soma y psiquis, sensaciones y sentimientos.

  5. Signaling Proteins and Transcription Factors in Normal and Malignant Early B Cell Development

    Directory of Open Access Journals (Sweden)

    Patricia Pérez-Vera

    2011-01-01

    Full Text Available B cell development starts in bone marrow with the commitment of hematopoietic progenitors to the B cell lineage. In murine models, the IL-7 and preBCR receptors, and the signaling pathways and transcription factors that they regulate, control commitment and maintenance along the B cell pathway. E2A, EBF1, PAX5, and Ikaros are among the most important transcription factors controlling early development and thereby conditioning mice homeostatic B cell lymphopoiesis. Importantly, their gain or loss of function often results in malignant development in humans, supporting conserved roles for these transcription factors. B cell acute lymphoblastic leukemia is the most common cause of pediatric cancer, and it is characterized by unpaired early B cell development resulting from genetic lesions in these critical signaling pathways and transcription factors. Fine mapping of these genetic abnormalities is allowing more specific treatments, more accurately predicting risk profiles for this disease, and improving survival rates.

  6. Signaling Proteins and Transcription Factors in Normal and Malignant Early B Cell Development

    Science.gov (United States)

    Pérez-Vera, Patricia; Reyes-León, Adriana; Fuentes-Pananá, Ezequiel M.

    2011-01-01

    B cell development starts in bone marrow with the commitment of hematopoietic progenitors to the B cell lineage. In murine models, the IL-7 and preBCR receptors, and the signaling pathways and transcription factors that they regulate, control commitment and maintenance along the B cell pathway. E2A, EBF1, PAX5, and Ikaros are among the most important transcription factors controlling early development and thereby conditioning mice homeostatic B cell lymphopoiesis. Importantly, their gain or loss of function often results in malignant development in humans, supporting conserved roles for these transcription factors. B cell acute lymphoblastic leukemia is the most common cause of pediatric cancer, and it is characterized by unpaired early B cell development resulting from genetic lesions in these critical signaling pathways and transcription factors. Fine mapping of these genetic abnormalities is allowing more specific treatments, more accurately predicting risk profiles for this disease, and improving survival rates. PMID:22046564

  7. Lineage-Specific Early Differentiation of Human Embryonic Stem Cells Requires a G2 Cell Cycle Pause.

    Science.gov (United States)

    Van Oudenhove, Jennifer J; Grandy, Rodrigo A; Ghule, Prachi N; Del Rio, Roxana; Lian, Jane B; Stein, Janet L; Zaidi, Sayyed K; Stein, Gary S

    2016-07-01

    Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However, precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs, and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase, which blocks entry into mitosis by phosphorylating CDK1 at Y15, was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment. Stem Cells 2016;34:1765-1775. © 2016 AlphaMed Press.

  8. Overexpression of IL-7R alpha provides a competitive advantage during early T-cell development.

    Science.gov (United States)

    Laouar, Yasmina; Crispe, I Nicholas; Flavell, Richard A

    2004-03-15

    Critical checkpoints controlling early thymic T-cell development and homeostasis are set by the proper signaling function of the interleukin 7 receptor (IL-7R) and the pre-T-cell antigen receptor. Although alpha beta T-cell development is observed in IL-7- and IL-7R alpha-deficient mice, the number of thymocytes is significantly reduced, implying a role for the IL-7R in controlling the size of the thymic T-cell compartment. Here, we report the overexpression of IL-7R alpha that occurs in the early T-cell compartment from AKR/J mice, animals that are highly susceptible to the spontaneous development of thymoma. Increased IL-7R alpha was revealed by surface staining, and increased IL-7R alpha mRNA was documented by using reverse transcriptase-polymerase chain reaction (RT-PCR). This resulted in increased survival of AKR/J early thymocytes, shown by the decreased frequency of TUNEL(+) (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate [dUTP]-fluorescein nick end labeling) cells. In an in vivo thymocyte repopulation model, AKR/J thymocytes had a selective advantage over healthy thymocytes. This advantage occurred at early stages of T-cell development. Our findings support the model that overexpression of growth factor receptors can contribute to proliferation and malignancy.

  9. Ultra-estrutura dos mastócitos de diferentes tipos histológicos de mastocitoma em cães Mast cell ultrastructure in different types of canine mast cell tumor

    Directory of Open Access Journals (Sweden)

    F.A.R. Sueiro

    2002-06-01

    Full Text Available Este trabalho teve por objetivo estudar as diferenças ultraestruturais de mastócitos neoplásicos de diferentes tipos histológicos de mastocitoma em cães, usando microscopia eletrônica de transmissão Os resultados mostraram que o núcleo e os grânulos citoplasmáticos são as estruturas mais indicadas para se avaliar o grau de anaplasia celular e o estádio de indiferenciação do tumor.The objective of this work was study the ultrastructural differences among the different histologic types of mast cell tumors in dogs collected in vivo. The ultrastructural analyses showed that the nuclei and cytoplasmic granules characteristics are the best structures to be appointed on evaluating the undifferentiation stage of this tumor.

  10. R-ES-ONANCEI

    Indian Academy of Sciences (India)

    A Tribute. H R Krishnamurthy. 70 John Maynard Smith (1920-2004). NOne of the last Grand Evolutionary Theorists of the 20th. Century'". Vidyanand Nanjundiah. BOOK REVIEWS. 93 Indian Mathematics and Astronomy - Some. Landmarks. B Sury. -6--------------------------~---------R-ES-O-N-A-N-C-E-I--N-ov-e-m-be-r-2-o--o5 ...

  11. COSIMA-ES-PORT

    DEFF Research Database (Denmark)

    Barfod, Michael Bruhn; Leleur, Steen

    2007-01-01

    -ES-PORT model showed some promising perspectives in the handling of the three pre-feasibility studies. Parameters such as cargo handling and ship related issues could with advantage be implemented in the decision model and forecasted similarly to road infrastructure impacts. Furthermore, the development...

  12. Es imposible no comunicar

    Directory of Open Access Journals (Sweden)

    Joan Costa y Solá-Segalés

    2012-04-01

    Full Text Available La confusión entre "comunicar" y "significar" proviene del hecho de que la comunicación transporta significados, pero también se producen significados fuera del proceso de comunicación. Es lo que llamamos "semiosis no comunicativa".

  13. Functional limitations of plasmacytoid dendritic cells limit type I interferon, T cell responses and virus control in early life.

    Directory of Open Access Journals (Sweden)

    Elodie Belnoue

    Full Text Available Infant mortality from viral infection remains a major global health concern: viruses causing acute infections in immunologically mature hosts often follow a more severe course in early life, with prolonged or persistent viral replication. Similarly, the WE strain of lymphocytic choriomeningitis virus (LCMV-WE causes acute self-limiting infection in adult mice but follows a protracted course in infant animals, in which LCMV-specific CD8⁺ T cells fail to expand and control infection. By disrupting type I IFNs signaling in adult mice or providing IFN-α supplementation to infant mice, we show here that the impaired early life T cell responses and viral control result from limited early type I IFN responses. We postulated that plasmacytoid dendritic cells (pDC, which have been identified as one major source of immediate-early IFN-I, may not exert adult-like function in vivo in the early life microenvironment. We tested this hypothesis by studying pDC functions in vivo during LCMV infection and identified a coordinated downregulation of infant pDC maturation, activation and function: despite an adult-like in vitro activation capacity of infant pDCs, the expression of the E2-2 pDC master regulator (and of critical downstream antiviral genes such as MyD88, TLR7/TLR9, NF-κB, IRF7 and IRF8 is downregulated in vivo at baseline and during LCMV infection. A similar pattern was observed in response to ssRNA polyU, a model ligand of the TLR7 viral sensor. This suggests that the limited T cell-mediated defense against early life viral infections is largely attributable to / regulated by infant pDC responses and provides incentives for novel strategies to supplement or stimulate immediate-early IFN-α responses.

  14. Conserved POU-binding site linked to SP1-binding site within FZD5 promoter: Transcriptional mechanisms of FZD5 in undifferentiated human ES cells, fetal liver/spleen, adult colon, pancreatic islet, and diffuse-type gastric cancer.

    Science.gov (United States)

    Katoh, Yuriko; Katoh, Masaru

    2007-03-01

    Canonical WNT signals are transduced through Frizzled (FZD) family receptor and LRP5/LRP6 co-receptor to upregulate FGF20, JAG1, DKK1, WISP1, CCND1 and MYC genes for cell-fate determination, while non-canonical WNT signals are transduced through FZD family receptor and ROR2/PTK7/RYK co-receptor to activate RHOA/RHOU/RAC/CDC42, JNK, PKC, NLK and NFAT signaling cascades for the regulation of tissue polarity, cell movement, and adhesion. We previously reported molecular cloning and characterization of human FZD5, which showed six amino-acid substitutions with human Hfz5. FZD5, functioning as WNT5A receptor, is the key molecule in the fields of oncology, regenerative medicine, cardiology, rheumatology, diabetology, and gastroenterology. Here, comparative integromics analyses on FZD5 orthologs were performed by using bioinformatics (Techint) and human intelligence (Humint). Chimpanzee FZD5 and cow Fzd5 genes were identified within NW_104292.1 and AC166656.2 genome sequences, respectively. FZD5 orthologs were seven-transmembrane proteins with extracellular Frizzled domain, leucine zipper motif around the 5th transmembrane domain, and cytoplasmic DVL- and PDZ-binding motifs. Ser523 and Ser529 around the DVL-binding motif of FZD5 orthologs were putative aPKC phosphorylation sites. POU5F1 (OCT4)-binding site linked to SP1-binding site within the 5'-promoter region of human FZD5 gene was evolutionarily conserved among mammalian FZD5 orthologs. POU5F1 was more related to POU2F and POU3F subfamily members. POU5F1 was preferentially expressed in undifferentiated human embryonic stem (ES) cells, pancreatic islet, and diffuse-type gastric cancer. POU2F1 (OCT1) was expressed in ES cells, fetal liver/spleen, adult colon, POU2F2 in ES cells, fetal liver/spleen, and POU2F3 in diffuse-type gastric cancer. Multiple SP1/KLF family members, other than KLF2 or KLF4, were expressed in undifferentiated human ES cells. Together, these facts indicate that POU5F1 and POU2F subfamily members

  15. Ultrastructural Complexity of Nuclear Components During Early Apoptotic Phases in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Christian Castelli

    2001-01-01

    Full Text Available Fractal morphometry was used to investigate the ultrastructural features of the plasma membrane, perinuclear membrane and nuclear chromatin in SK‐BR‐3 human breast cancer cells undergoing apoptosis. Cells were incubated with 1 μM calcimycin (A23187 for 24 h. Cells in the early stage of apoptosis had fractal dimension (FD values indicating that their plasma membranes were less rough (lower FD than those of control cells, while their perinuclear membranes were unaffected. Changes of the chromatin texture within the entire nucleus and in selected nuclear domains were more pronounced in treated cells. This confirms that the morphological reorganization imputable to a loss of structural complexity (reduced FD occurs in the early stage of apoptosis, is accompanied by the inhibition of distinct enzymatic events and precedes the onset of conventional cellular markers, which can only be detected during the active phases of the apoptotic process.

  16. A comparison of the tube forming potentials of early and late endothelial progenitor cells.

    Science.gov (United States)

    Mukai, Nana; Akahori, Taichi; Komaki, Motohiro; Li, Qin; Kanayasu-Toyoda, Toshie; Ishii-Watabe, Akiko; Kobayashi, Akiko; Yamaguchi, Teruhide; Abe, Mayumi; Amagasa, Teruo; Morita, Ikuo

    2008-02-01

    The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 mum wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.

  17. Socioeconomic position and surgery for early-stage non-small-cell lung cancer

    DEFF Research Database (Denmark)

    Kærgaard Starr, Laila; Osler, Merete; Steding-Jessen, Marianne

    2013-01-01

    AIM: To examine possible associations between socioeconomic position and surgical treatment of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS: In a register-based clinical cohort study, patients with early-stage (stages I-IIIa) NSCLC were identified in the Danish Lung Cancer...... in a health care system with free, equal access to health services, disadvantaged groups are less likely to receive surgery for lung cancer....

  18. The unique pathophysiology of early-onset severe preeclampsia: role of decidual T regulatory cells.

    Science.gov (United States)

    Quinn, Kristen H; Lacoursiere, D Yvette; Cui, Li; Bui, Jack; Parast, Mana M

    2011-09-01

    Immunological mechanisms play a pivotal role in the pathophysiology of preeclampsia. T regulatory cells (Treg cells, FoxP3(+)) suppress the cytotoxic T cell (CD8(+)) and natural killer (NK) cell response, thereby promoting immunological tolerance to the fetus. In peripheral blood, Treg cells are elevated during pregnancy, decrease throughout gestation, and are decreased in preeclampsia. To determine their role at the implantation site, we characterized the proportion of decidual Treg and CD8+ cells, and compared these with placental histology, villous sFlt expression, and chorionic trophoblast apoptotic index in normal and preeclamptic pregnancies. Decidua from first (n=5) and second (n=4) trimester terminations and chorioamniotic membranes, containing decidua, from term deliveries (n=14), early-onset (≤ 34 weeks) (n=12), and late-onset (>34 weeks) (n=14) severe preeclampsia were evaluated. Immunohistochemistry for CD3, CD8, and FoxP3 was performed: CD8(+) and FoxP3(+) cells were calculated as a proportion of CD3(+) cells. Placental tissue was evaluated for villous hypermaturity and sFlt staining. Chorioamniotic membranes were evaluated, via TUNEL assay, for chorionic trophoblast apoptosis. Decidual Treg cells were seen to peak in second trimester and decrease with advancing gestational age and were lower in early-onset (0.46%) compared with late-onset severe preeclampsia (3.34%) and term pregnancies (5.21%). The proportion of CD8(+) cells was higher in cases of severe preeclampsia. Early-onset severe preeclamptic cases had the highest sFlt score, placental insufficiency score, and apoptotic index. Our data suggest that early-onset severe preeclampsia has a unique pathophysiology involving defective immunoregulatory pathways, potentially causing vascular and trophoblast damage at the implantation site. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  19. Early pregnancy complicated with juvenile granulosa cell tumor.

    Science.gov (United States)

    Xu, Hongmei; Shu, Chang; Li, Na; Xia, Meihui; Li, Tingting; Zhong, Yanping; Yan, Xu; Wang, Hong; Zhang, Haipeng; He, Jin

    2011-11-01

    Granulosa cell tumors (GCTs) are extremely rare tumors and are divided into 2 types: adult (AGCT) and juvenile (JGCT). The JGCTs represent only 5% of all cases. The incidence of ovarian carcinoma diagnosed during pregnancy varies about 0.0179 to 0.11 per 1000 pregnancies. A 24-year-old woman at 12 weeks and 3 days of pregnancy was admitted to the authors' hospital due to a pelvic mass. Surgical exploration revealed a smooth, solid, mobile and well-encapsulated left ovarian mass. Histological and immunohistochemical findings led to the diagnosis of a well-differentiated JGCT. Pregnancy continued uncomplicated and she delivered a healthy baby girl at 37 weeks gestation. About 9 months after the original diagnosis, the patient showed no evidence of disease. Treatment options and a review of the literature related to JGCTs are discussed.

  20. Prevalence of Epithelial Ovarian Cancer Stem Cells Correlates with Recurrence in Early-Stage Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Karina Dahl Steffensen

    2011-01-01

    Full Text Available Epithelial ovarian cancer stem cells (EOC stem cells have been associated with recurrence and chemoresistance. CD44 and CK18 are highly expressed in cancer stem cells and function as tools for their identification and characterization. We investigated the association between the number of CD44+ EOC stem cells in ovarian cancer tumors and progression-free survival. EOC stem cells exist as clusters located close to the stroma forming the cancer stem cell “niche”. 17.1% of the samples reveled high number of CD44+ EOC stem cells (>20% positive cells. In addition, the number of CD44+ EOC stem cells was significantly higher in patients with early-stage ovarian cancer (FIGO I/II, and it was associated with shorter progression-free survival (P=0.026. This study suggests that quantification of the number of EOC stem cells in the tumor can be used as a predictor of disease and could be applied for treatment selection in early-stage ovarian cancer.

  1. Early Loss of Splenic Tfh Cells in SIV-Infected Rhesus Macaques.

    Directory of Open Access Journals (Sweden)

    Félicien Moukambi

    2015-12-01

    Full Text Available Follicular T helper cells (Tfh, a subset of CD4 T lymphocytes, provide crucial help to B cells in the production of antigen-specific antibodies. Although several studies have analyzed the dynamics of Tfh cells in peripheral blood and lymph nodes (LNs during Aids, none has yet addressed the impact of SIV infection on the dynamics of Tfh cells in the spleen, the primary organ of B cell activation. We show here a significant decrease in splenic Tfh cells in SIVmac251-infected rhesus macaques (RMs during the acute phase of infection, which persists thereafter. This profound loss is associated with lack of sustained expression of the Tfh-defining transcription factors, Bcl-6 and c-Maf but with higher expression of the repressors KLF2 and Foxo1. In this context of Tfh abortive differentiation and loss, we found decreased percentages of memory B cell subsets and lower titers of SIV-specific IgG. We further demonstrate a drastic remodeling of the lymphoid architecture of the spleen and LNs, which disrupts the crucial cell-cell interactions necessary to maintain memory B cells and Tfh cells. Finally, our data demonstrated the early infection of Tfh cells. Paradoxically, the frequencies of SIV DNA were higher in splenic Tfh cells of RMs progressing more slowly suggesting sanctuaries for SIV in the spleen. Our findings provide important information regarding the impact of HIV/SIV infection on Tfh cells, and provide new clues for future vaccine strategies.

  2. Potential role of immunoablation and hematopoietic cell transplantation in the treatment of early diabetes type 1.

    Science.gov (United States)

    Snarski, Emilian; Milczarczyk, Alicja; Franek, Edward; Jedrzejczak, Wieslaw

    2010-01-01

    Immunoablation with autologous hematopoietic cell transplantation has shown some effectiveness in the treatment of autoimmune diseases as diverse as aplastic anemia, systemic lupus erythematosus, multiple sclerosis and Crohn's disease. It has been recently shown that this treatment might prevent or delay development of diabetes type 1. The majority of more than 30 patients with early diabetes type 1 who underwent immunoablation and hematopoietic cell transplantation in various centers in the world achieved durable remission of diabetes and independence of exogenous insulin. This review summarizes advantages and risks of this treatment of early diabetes type 1.

  3. Immunohistochemical markers for corneal stem cells in the early developing human eye

    DEFF Research Database (Denmark)

    Lyngholm, Mikkel; Høyer, Poul E; Vorum, Henrik

    2008-01-01

    markers and potential markers for LSCs and early transient amplifying cells in human adults. In this study, we describe the development of the ectodermally derived LSCs and the mesodermally derived niche cells from the time at which the cornea is defined (week 6) until the formation of the early limbal...... niche (week 14) in human embryos and fetuses. The expression of SOD2 and CK15 was investigated together with other recently identified limbal proteins. Previously suggested LSC and differentiation markers (PAX6, aquaporin-1 and nestin) were also investigated. Both SOD2 and CK15 were present...

  4. The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

    DEFF Research Database (Denmark)

    Morovic, Martin; Murin, Matej; Strejcek, Frantisek

    2016-01-01

    One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription....... In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly...... influenced by the ooplasmic environment....

  5. Nuclear and cellular expression data from the whole 16-cell stage Arabidopsis thaliana embryo and a cell type-specific expression atlas of the early Arabidopsis embryo

    NARCIS (Netherlands)

    Palovaara, J.P.J.

    2017-01-01

    SuperSeries contain expression data from the nuclei of cell types involved in patterning events, with focus on root apical stem cell formation, at 16-cell stage, early globular stage and late globular stage in the early Arabidopsis embryo (atlas). Expression data comparing nuclear and cellular RNA

  6. Characterization of xylan in the early stages of secondary cell wall formation in tobacco bright yellow-2 cells.

    Science.gov (United States)

    Ishii, Tadashi; Matsuoka, Keita; Ono, Hiroshi; Ohnishi-Kameyama, Mayumi; Yaoi, Katsuro; Nakano, Yoshimi; Ohtani, Misato; Demura, Taku; Iwai, Hiroaki; Satoh, Shinobu

    2017-11-15

    The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and non-transformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays.

    Science.gov (United States)

    Giannoutsou, E; Sotiriou, P; Apostolakos, P; Galatis, B

    2013-10-01

    The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs. Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy. Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes. The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule

  8. Presentation of an immunodominant immediate-early CD8+ T cell epitope resists human cytomegalovirus immunoevasion.

    Directory of Open Access Journals (Sweden)

    Stefanie Ameres

    Full Text Available Control of human cytomegalovirus (HCMV depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1, that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy.

  9. Role for early-differentiated natural killer cells in infectious mononucleosis.

    Science.gov (United States)

    Azzi, Tarik; Lünemann, Anna; Murer, Anita; Ueda, Seigo; Béziat, Vivien; Malmberg, Karl-Johan; Staubli, Georg; Gysin, Claudine; Berger, Christoph; Münz, Christian; Chijioke, Obinna; Nadal, David

    2014-10-16

    A growing body of evidence suggests that the human natural killer (NK)-cell compartment is phenotypically and functionally heterogeneous and is composed of several differentiation stages. Moreover, NK-cell subsets have been shown to exhibit adaptive immune features during herpes virus infection in experimental mice and to expand preferentially during viral infections in humans. However, both phenotype and role of NK cells during acute symptomatic Epstein-Barr virus (EBV) infection, termed infectious mononucleosis (IM), remain unclear. Here, we longitudinally assessed the kinetics, the differentiation, and the proliferation of subsets of NK cells in pediatric IM patients. Our results indicate that acute IM is characterized by the preferential proliferation of early-differentiated CD56(dim) NKG2A(+) immunoglobulin-like receptor(-) NK cells. Moreover, this NK-cell subset exhibits features of terminal differentiation and persists at higher frequency during at least the first 6 months after acute IM. Finally, we demonstrate that this NK-cell subset preferentially degranulates and proliferates on exposure to EBV-infected B cells expressing lytic antigens. Thus, early-differentiated NK cells might play a key role in the immune control of primary infection with this persistent tumor-associated virus. © 2014 by The American Society of Hematology.

  10. Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

    Science.gov (United States)

    Duivenvoorden, Hendrika M; Rautela, Jai; Edgington-Mitchell, Laura E; Spurling, Alex; Greening, David W; Nowell, Cameron J; Molloy, Timothy J; Robbins, Elizabeth; Brockwell, Natasha K; Lee, Cheok Soon; Chen, Maoshan; Holliday, Anne; Selinger, Cristina I; Hu, Min; Britt, Kara L; Stroud, David A; Bogyo, Matthew; Möller, Andreas; Polyak, Kornelia; Sloane, Bonnie F; O'Toole, Sandra A; Parker, Belinda S

    2017-12-01

    Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  11. Early imaging findings in germ cell tumors arising from the basal ganglia

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So Mi [Seoul National University College of Medicine, Department of Radiology, 101 Daehak-ro, Jongno-gu, Seoul (Korea, Republic of); Kyungpook National University Medical Center, Department of Radiology, Daegu (Korea, Republic of); Kim, In-One; Choi, Young Hun; Cheon, Jung-Eun; Kim, Woo Sun [Seoul National University College of Medicine, Department of Radiology and Institute of Radiation Medicine, 101 Daehak-ro, Jongno-gu, Seoul (Korea, Republic of); Cho, Hyun-Hae [Seoul National University College of Medicine, Department of Radiology, 101 Daehak-ro, Jongno-gu, Seoul (Korea, Republic of); Ewha Woman' s University Mokdong Hospital, Department of Radiology, Seoul (Korea, Republic of); You, Sun Kyoung [Seoul National University College of Medicine, Department of Radiology, 101 Daehak-ro, Jongno-gu, Seoul (Korea, Republic of); Chungnam National University Hospital, Department of Radiology, Daejeon (Korea, Republic of)

    2016-05-15

    It is difficult to diagnosis early stage germ cell tumors originating in the basal ganglia, but early recognition is important for better outcome. To evaluate serial MR images of basal ganglia germ cell tumors, with emphasis on the features of early stage tumors. We retrospectively reviewed serial MR images of 15 tumors in 14 children and young adults. We categorized MR images of the tumors as follows: type I, ill-defined patchy lesions (<3 cm) without cyst; type II, small mass lesions (<3 cm) with cyst; and type III, large lesions (≥3 cm) with cyst. We also assessed temporal changes of the MR images. On the initial images, 8 of 11 (73%) type I tumors progressed to types II or III, and 3 of 4 (75%) type II tumors progressed to type III. The remaining 4 tumors did not change in type. All type II tumors (5/5, 100%) that changed from type I had a few tiny cysts. Intratumoral hemorrhage was observed even in the type I tumor. Ipsilateral hemiatrophy was observed in most of the tumors (13/15, 87%) on initial MR images. As tumors grew, cystic changes, intratumoral hemorrhage, and ipsilateral hemiatrophy became more apparent. Early stage basal ganglia germ cell tumors appear as ill-defined small patchy hyperintense lesions without cysts on T2-weighted images, are frequently associated with ipsilateral hemiatrophy, and sometimes show microhemorrhage. Tumors develop tiny cysts at a relatively early stage. (orig.)

  12. Liraglutide protects pancreatic beta cells during an early intervention in Gato-Kakizaki rats.

    Science.gov (United States)

    Luo, Xiu; Pan, Linlin; Nie, Aifang; Wang, Qidi; Gu, Yanyun; Li, Fengying; Zhang, Hongli; Li, Wenyi; Li, Xiaoying

    2013-12-01

    Glucagon-like peptide-1 (GLP-1) analogues have emerged as insulin secretagogues and are widely used in type 2 diabetic patients. GLP-1 analogues also demonstrate a promotion of beta cell proliferation and reduction of apoptosis in rodents. In the present study, we investigated the protection of pancreatic beta cells by early use (at the age of 2 weeks) of GLP-1 analogue, liraglutide in Gato-Kakizaki (GK) rats and explored the underlying mechanisms. The effects of liraglutide on glucose tolerance were evaluated by intraperitoneal glucose tolerance test (IPGTT) and insulin release tests (IRT). Ki67 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) immunostaining, Western blots and real-time polymerase chain reaction were applied to evaluate cell proliferation, apoptosis and related gene expressions. Our results demonstrated that early use of liraglutide improved glucose tolerance during liraglutide treatment in GK rats. Liraglutide increased pancreatic insulin contents and markedly reduced beta cell apoptosis. Liraglutide also downregulated pro-apoptotic gene expressions and reduced intra-islet macrophage infiltration. This experiment reported for the first time that early use of liraglutide could protect beta cell failure in pre-diabetic GK rats through reduction of beta cell apoptosis and ameliorating islet inflammation. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

  13. Nrf2 inhibition affects cell cycle progression during early mouse embryo development.

    Science.gov (United States)

    Lin, Ying; Sui, Liu-Cai; Wu, Rong-Hua; Ma, Ru-Jun; Fu, Hai-Yan; Xu, Juan-Juan; Qiu, Xu-Hua; Chen, Li

    2017-12-16

    Brusatol, a quassinoid isolated from the fruit of Bruceajavanica, has recently been shown to inhibit nuclear factor erythroid 2-related factor 2 (Nrf2) via Keap1-dependent ubiquitination and proteasomal degradation or protein synthesis. Nrf2 is a transcription factor that regulates the cellular defense response. Most studies have focused on the effects of Nrf2in tumor development. Here, the critical roles of Nrf2 in mouse early embryonic development were investigated. We found that brusatol treatment at the zygotic stage prevented the early embryo development. Most embryos stayed at the two-cell stage after 5 days of culture (P CRISPR activation plasmid. Thus, brusatol inhibited early embryo development by affecting Nrf2-related cell cycle transition from G2 to M phase that is dependent on cyclin B-CDK1 complex.

  14. Central Cell-Derived Peptides Regulate Early Embryo Patterning in Flowering Plants

    NARCIS (Netherlands)

    Costa, L.M.; Marshall, E.; Tesfaye, M.; Silverstein, K.A.T.; Mori, M.; Umetsu, Y.; Otterbach, S.L.; Papareddy, R.; Dickinson, H.G.; Boutilier, K.A.; VandenBosch, K.A.; Ohki, S.; Gutierrez-Marcos, J.F.

    2014-01-01

    Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning

  15. MITOGENIC SIGNALS CONTROL TRANSLATION OF THE EARLY GROWTH-RESPONSE GENE-1 IN MYOGENIC CELLS

    NARCIS (Netherlands)

    MAASS, A; GROHE, C; OBERDORF, S; SUKHATME, VP; VETTER, H; NEYSES, L

    1994-01-01

    Muscle is a major site of expression of the early growth reponse gene-1 (Egr-1). To investigate its role in muscle proliferation and/or differentiation we studied the effect of a variety of growth factors on cultured mouse muscle So18 cells. Three groups of responses could be distinguished: 1. AII,

  16. Different Auxin Response Machineries Control Distinct Cell Fates in the Early Plant Embryo

    NARCIS (Netherlands)

    Rademacher, E.H.; Lokerse, A.S.; Schlereth, A.; Llavata Peris, C.I.; Bayer, M.; Kientz, M.; Freire Rios, A.; Borst, J.W.; Lukowitz, W.; Juergens, G.; Weijers, D.

    2012-01-01

    The cell types of the plant root are first specified early during embryogenesis and are maintained throughout plant life. Auxin plays an essential role in embryonic root initiation, in part through the action of the ARF5/MP transcription factor and its auxin-labile inhibitor IAA12/BDL. MP and BDL

  17. Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming.

    Science.gov (United States)

    Prieto, Javier; León, Marian; Ponsoda, Xavier; Sendra, Ramón; Bort, Roque; Ferrer-Lorente, Raquel; Raya, Angel; López-García, Carlos; Torres, Josema

    2016-03-31

    During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency.

  18. Detection of early lymphocyte activation by the fluorescent cell membrane probe N-phenyl-1-naphthylamine.

    Science.gov (United States)

    Halliday, G M; Nairn, R C; Pallett, M A; Rolland, J M; Ward, H A

    1979-01-01

    N-phenyl-1-naphthylamine (NPN) becomes fluorescent after binding to hydrophobic regions of cell membranes. Rat and mouse lymphoid cell suspensions stained with NPN showed changes in fluorescence emission 30 min after stimulation with mitogen or antigen, detected by microfluorimetry. Incubation of NPN-labelled mouse and rat thymocytes with phytohaemagglutinin or concanavalin A (Con A) caused an increase in mean cell fluorescence intensity. The response to Con A was inhibited by sodium azide and alpha-methyl mannoside. Stimulation of spleen cells from mice by allogeneic cells, or from tumour-bearing rats by tumour antigen consistently resulted in decreased fluorescence. The 'mixed lymphocyte response' detected only certain genetic differences between mouse strains and was proportional to the ratio of stimulator to responder cell number. The NPN staining procedure offers a simple and rapid assay of immunoreactivity and a means of studying early subcellular changes following lymphocyte activation.

  19. Impaired Circulating Angiogenic Cells Mobilization and Metalloproteinase-9 Activity after Dynamic Exercise in Early Metabolic Syndrome

    Science.gov (United States)

    Rocha, Natalia G.; Sales, Allan R. K.; Penedo, Leticia A.; Pereira, Felipe S.; Silva, Mayra S.; Miranda, Renan L.; Silva, Jemima F. R.; Silva, Bruno M.; Santos, Aline A.; Nobrega, Antonio C. L.

    2015-01-01

    Increased levels of adhesion molecules or metalloproteinases (MMPs) may indicate endothelial dysfunction. Exercise mobilizes circulating angiogenic cells (CACs) from bone marrow in healthy subjects, improving vascular function. However, it is unclear whether this mechanism is preserved in the early stages of metabolic syndrome (early MetS). We aimed to evaluate the acute effects of exercise on adhesion molecules, angiogenic factors, MMPs, and CACs in early MetS. Fifteen subjects with early MetS and nine healthy controls underwent an exercise session and a nonexercise session, randomly. Adhesion molecules, angiogenic factors, CACs, and MMPs were evaluated before and after exercise or nonexercise sessions. At baseline, levels of sE-selectin, sICAM-1, and MMP-9 were higher in early MetS than in controls (P ≤ 0.03). After exercise, sE-selectin, sICAM-1, and MMP-9 levels were still higher in early MetS (P exercise. There was no difference between moments in nonexercise session (P > 0.05). In conclusion, subjects with early MetS already presented impaired endothelial function at rest along with a decrease in CACs and an increase in MMP-9 activity in response to exercise. PMID:26557715

  20. Quantificação de células CD34+ do sangue do cordão umbilical de cães Quantification of CD34+ hematopoietic stem cells from the umbilical cord of dogs

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    Ana Paula Massae Nakage

    2009-04-01

    Full Text Available As células-tronco promovem a reconstituição hematopoética e de outros tecidos, estando presentes no embrião, no sangue periférico, na medula óssea e no sangue do cordão umbilical (SCU. Os modelos experimentais de células-tronco do sangue periférico e da medula óssea em cães têm propiciado informações relevantes para transplantes de células-tronco em humanos. Entretanto, não existe estudo sobre as células-tronco no SCU canino no Brasil. O objetivo deste ensaio foi quantificar as células nucleadas e as células-tronco CD34+ no SCU de cães para propostas de reconstituição hematológica. No presente protocolo experimental, a coleta de SCU de 40 cães neonatos foi realizada para contagem das células nucleadas no contador automático de células sangüíneas e contagem das células-tronco CD34+ no citômetro de fluxo. O método de coleta do SCU na porção justaplacentária dos vasos umbilicais permitiu a quantificação das células-tronco CD34+. Os valores das células nucleadas do SCU foram reduzidos em relação àqueles do sangue periférico de cães sadios recém-nascidos e adultos. Apesar do volume escasso de SCU (1325µL, a quantidade de células-tronco do cordão umbilical canino (3,38±2,72 CD34+ x10(6 kg-1 é semelhante àquela reportada para medula óssea, bem como para o sangue periférico mobilizado de cães adultos, sendo coincidente com aquela preconizada para reconstituição hematopoética.Stem cells promote the reconstitution of the hematopoietic and other tissues, and are present in the embryo, peripheral blood, bone marrow and umbilical cord blood (UCB. The experimental models of bone marrow and peripheral blood stem cells in dogs provide important information for stem cells transplants in humans. However, there are no studies on the UCB cells of dogs in Brazil. This experiment aimed at quantifying nucleated and stem cells in the umbilical cord of dogs having as its purpose the hematological recuperation. In

  1. Monitoring ferumoxide-labelled neural progenitor cells and lesion evolution by magnetic resonance imaging in a model of cell transplantation in cerebral ischaemia [v1; ref status: indexed, http://f1000r.es/20l

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    Rachael A Panizzo

    2013-11-01

    Full Text Available Efficacy of neural stem/progenitor cell (NPC therapies after cerebral ischaemia could be better evaluated by monitoring in vivo migration and distribution of cells post-engraftment in parallel with analysis of lesion volume and functional recovery. Magnetic resonance imaging (MRI is ideally placed to achieve this, but still poses several challenges. We show that combining the ferumoxide MRI contrast agent Endorem with protamine sulphate (FePro improves iron oxide uptake in cells compared to Endorem alone and is non-toxic. Hence FePro complex is a better contrast agent than Endorem for monitoring NPCs. FePro complex-labelled NPCs proliferated and differentiated normally in vitro, and upon grafting into the brain 48 hours post-ischaemia they were detected in vivo by MRI. Imaging over four weeks showed the development of a confounding endogenous hypointense contrast evolution at later timepoints within the lesioned tissue. This was at least partly due to accumulation within the lesion of macrophages and endogenous iron. Neither significant NPC migration, assessed by MRI and histologically, nor a reduction in the ischaemic lesion volume was observed in NPC-grafted brains.  Crucially, while MRI provides reliable information on engrafted cell location early after an ischaemic insult, pathophysiological changes to ischaemic lesions can interfere with cellular imaging at later timepoints.

  2. Monitoring ferumoxide-labelled neural progenitor cells and lesion evolution by magnetic resonance imaging in a model of cell transplantation in cerebral ischaemia [v2; ref status: indexed, http://f1000r.es/30c

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    Rachael A Panizzo

    2014-03-01

    Full Text Available Efficacy of neural stem/progenitor cell (NPC therapies after cerebral ischaemia could be better evaluated by monitoring in vivo migration and distribution of cells post-engraftment in parallel with analysis of lesion volume and functional recovery. Magnetic resonance imaging (MRI is ideally placed to achieve this, but still poses several challenges. We show that combining the ferumoxide MRI contrast agent Endorem with protamine sulphate (FePro improves iron oxide uptake in cells compared to Endorem alone and is non-toxic. Hence FePro complex is a better contrast agent than Endorem for monitoring NPCs. FePro complex-labelled NPCs proliferated and differentiated normally in vitro, and upon grafting into the brain 48 hours post-ischaemia they were detected in vivo by MRI. Imaging over four weeks showed the development of a confounding endogenous hypointense contrast evolution at later timepoints within the lesioned tissue. This was at least partly due to accumulation within the lesion of macrophages and endogenous iron. Neither significant NPC migration, assessed by MRI and histologically, nor a reduction in the ischaemic lesion volume was observed in NPC-grafted brains.  Crucially, while MRI provides reliable information on engrafted cell location early after an ischaemic insult, pathophysiological changes to ischaemic lesions can interfere with cellular imaging at later timepoints.

  3. Innate B cells: oxymoron or validated concept? [v1; ref status: indexed, http://f1000r.es/T4CAVP

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    Carl F Ware

    2012-08-01

    Full Text Available B lymphocytes promote the initial innate interferon response to viral pathogens without the need for antigen receptor activation. B cell dependent IFN production requires the cytokine, lymphotoxin-β. The LTβ pathway is well known to regulate lymphoid organogenesis and homeostasis by differentiating stromal cells and macrophages. However, in response to viral pathogens these same B cell-regulated populations rapidly produce type 1 interferons. Thus, B cells act as innate effector cells via LTβ homeostatic pathways, which serve as innate host barriers to viral pathogens.

  4. Induction of contact sensitivity by cell-associated immunocomplexes requires activation of the early complement components.

    Science.gov (United States)

    Lio, D; Sireci, G; Gervasi, F; Dieli, F; Salerno, A

    1992-12-01

    Lymph node cells collected from CBA/J mice 4 days after painting the skin with picryl chloride behave like antigen presenting cells and induce contact sensitivity when injected into naive recipient mice. The immunizing capacity of these '4-day' cells is due to T cells which carry on their membrane hapten-IgM immunocomplexes. Incubation of the cells with complement from mouse strains that express high C4 serum levels (C4H), abolishes their immunizing capacity. This effect is related to the activation of the early components of the classical complement pathway, as supported by experiments using C3 and C4-depleted or C5 and C6-genetically deficient mouse sera. The detection of different amounts of C3b and C4b on the surface of 4-day T cells after incubation with C4L and C4H sera supports the possibility that membrane bound activated complement components could modify the immunizing capacity of these cells. Results herein reported suggest that membrane-bound C3b and C4b are not per se inhibitory but interfere with the residual complement activating capacity of 4-day T cells. The role of complement activation by 4-day T cells is pivotal as complement depletion of recipient mice by cobra venom factor (CVF) inhibits the immunizing capacity of untreated 4-day T cells, while 4-day T cells treated with complement in vitro and injected together with C4a anaphylatoxin are able to immunize recipient mice.

  5. Raman spectral dynamics of single cells in the early stages of growth factor stimulation.

    Science.gov (United States)

    Takanezawa, Sota; Morita, Shin-ichi; Ozaki, Yukihiro; Sako, Yasushi

    2015-05-05

    Cell fates change dynamically in response to various extracellular signals, including growth factors that stimulate differentiation and proliferation. The processes underlying cell-fate decisions are complex and often include large cell-to-cell variations, even within a clonal population in the same environment. To understand the origins of these cell-to-cell variations, we must detect the internal dynamics of single cells that reflect their changing chemical milieu. In this study, we used the Raman spectra of single cells to trace their internal dynamics during the early stages of growth factor stimulation. This method allows nondestructive and inclusive time-series analyses of chemical compositions of the same single cells. Applying a Gaussian mixture model to the major principal components of the single-cell Raman spectra, we detected the dynamics of the chemical states in MCF-7 cancer-derived cells in the absence and presence of differentiation and proliferation factors. The dynamics displayed characteristic variations according to the functions of the growth factors. In the differentiation pathway, the chemical composition changed directionally between multiple states, including both reversible and irreversible state transitions. In contrast, in the proliferation pathway, the chemical composition was homogenized into a single state. The differentiation factor also stimulated fluctuations in the chemical composition, whereas the proliferation factor did not. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. EL ORO ES TRISTE

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    Jose María Corella Hurtado

    2012-01-01

    Full Text Available Esas narraciones eran las mismas cantaletas de mis abuelos, de mis tíos, de los negros y los mineros, contadas bajo las torrenciales noches de lluvia o bajo los indignos soles. Mi madre también relataba de memoria, como una cotorra y con la misma exactitud mientras realizaba los oficios domésticos, bien sean los de la barraca del puerto de Barbacoas o los de la casa de Pasto. Los barbacoanos de esa lejana generación, debieron soportar los recuerdos nefastos de la historia enquistada con dolor porque crecieron coreando episodios con la misma y sorprendente precisión. Era, es y será por siempre, una carcoma dolorosa y perseverante, aquello de la draga. Es que lo sucedido en Barbacoas no debe olvidarse y menos repetirse. Esos sucesos me comprometían de alguna manera; vividos unos en carne propia y a oídas otros que mezclaban el dolor que causó a los paisanos la pobreza en que quedaron.

  7. Blastocyst axis is specified independently of early cell lineage but aligns with the ZP shape.

    Science.gov (United States)

    Kurotaki, Yoko; Hatta, Kohei; Nakao, Kazuki; Nabeshima, Yo-Ichi; Fujimori, Toshihiko

    2007-05-04

    The mechanisms controlling the establishment of the embryonic-abembryonic (E-Ab) axis of the mammalian blastocyst are controversial. We used in vitro time-lapse imaging and in vivo lineage labeling to provide evidence that the E-Ab axis of the mouse blastocyst is generated independently of early cell lineage. Rather, both the boundary between two-cell blastomeres and the E-Ab axis of the blastocyst align relative to the ellipsoidal shape of the zona pellucida (ZP), an extraembryonic structure. Lack of correlation between cell lineage and the E-Ab axis can be explained by the rotation of the embryo within the ZP.

  8. The need for early detection of neck nodal metastasis in squamous cell carcinoma of oral cavity

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    P Satish Kumaran

    2012-01-01

    Full Text Available In the Indian subcontinent, the incidence of squamous cell carcinoma of the oral cavity has been constantly increasing despite the improvement in the awareness about squamous cell carcinoma. The patients generally report to us in the period in which the tumor would have undergone metastasis. This article highlights about the grading, staging, and decision-making regarding the surgical management of squamous cell carcinoma of the oral cavity. The article also stresses upon the early detection of the lesion by the practitioner for a definitive successful surgical treatment of the patient.

  9. GM-CSF produced by nonhematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa.

    Science.gov (United States)

    Egea, Laia; McAllister, Christopher S; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Kagnoff, Martin F

    2013-02-15

    GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, as well as dendritic cell differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn's disease in humans and colitis in murine models has mainly been considered to reflect its activity on myeloid cells. We used GM-CSF-deficient (GM-CSF(-/-)) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS), at doses that resulted in little epithelial damage and mucosal ulceration in wild type mice, caused marked colon ulceration and delayed ulcer healing in GM-CSF(-/-) mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF(-/-) mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF(-/-) mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Nonhematopoietic cells, and not myeloid cells, produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury, as revealed by bone marrow chimera and dendritic cell-depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell-produced GM-CSF has a novel nonredundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium.

  10. Power law relationship between cell cycle duration and cell volume in the early embryonic development of Caenorhabditis elegans.

    Science.gov (United States)

    Arata, Yukinobu; Takagi, Hiroaki; Sako, Yasushi; Sawa, Hitoshi

    2014-01-01

    Cell size is a critical factor for cell cycle regulation. In Xenopus embryos after midblastula transition (MBT), the cell cycle duration elongates in a power law relationship with the cell radius squared. This correlation has been explained by the model that cell surface area is a candidate to determine cell cycle duration. However, it remains unknown whether this second power law is conserved in other animal embryos. Here, we found that the relationship between cell cycle duration and cell size in Caenorhabditis elegans embryos exhibited a power law distribution. Interestingly, the powers of the time-size relationship could be grouped into at least three classes: highly size-correlated, moderately size-correlated, and potentially a size-non-correlated class according to C. elegans founder cell lineages (1.2, 0.81, and relationship is conserved in Xenopus and C. elegans, while the absolute powers in C. elegans were different from that in Xenopus. Furthermore, we found that the volume ratio between the nucleus and cell exhibited a power law relationship in the size-correlated classes. The power of the volume relationship was closest to that of the time-size relationship in the highly size-correlated class. This correlation raised the possibility that the time-size relationship, at least in the highly size-correlated class, is explained by the volume ratio of nuclear size and cell size. Thus, our quantitative measurements shed a light on the possibility that early embryonic C. elegans cell cycle duration is coordinated with cell size as a result of geometric constraints between intracellular structures.

  11. Early responses of human cancer cells upon photodynamic treatment monitored by laser phase microscopy

    Science.gov (United States)

    Roelofs, Theo A.; Graschew, Georgi; Perevedentseva, Elena V.; Rakowsky, Stefan; Dressler, Cathrin; Beuthan, Juergen; Schlag, Peter M.

    2001-04-01

    Photodynamic treatment of cancer cells is known to eventually cause cell death in most cases. The precise pathways and the time course seem to vary among different cell types and modes of photodynamic treatment. In this contribution, the focus was put on the responses of human colon carcinoma cells HCT-116 within the first 15 minutes after laser irradiation in the presence of Photofrin« II (PII). To monitor the cell response in this early time period laser phase microscopic imaging was used, a method sensitive to changes in overall cell shape and intracellular structures, mediated by changes in the local refractive index. Laser irradiation of cells loaded with PII induced a significant reduction of the phase shifts, which probably reflects the induced damage to the different cellular membrane structures. The data suggest that even within the first 30 s after the onset of laser illumination, a significant reduction of the phase shifts can be detected. These results underline that laser phase microscopy is a suitable diagnostic tool for cellular research, also in the early time domain.

  12. Added effects of dexamethasone and mesenchymal stem cells on early Natural Killer cell activation

    NARCIS (Netherlands)

    Michelo, C.M.; Fasse, E.; Cranenbroek, B. van; Linda, K.; Meer, A. van der; Abdelrazik, H.; Joosten, I.

    2016-01-01

    Graft rejection and graft-versus-host disease are leading causes of transplant related mortality despite advancements in immunosuppressive therapy. Mesenchymal stem cells (MSCs) offer a promising addition to immunosuppressive drugs (ISD), while NK-cells are increasingly used as effector cells in

  13. T Cell Immunosenescence after Early Life Adversity: Association with Cytomegalovirus Infection

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    Martha M. C. Elwenspoek

    2017-10-01

    Full Text Available Early life adversity (ELA increases the risk for multiple age-related diseases, such as diabetes type 2 and cardiovascular disease. As prevalence is high, ELA poses a major and global public health problem. Immunosenescence, or aging of the immune system, has been proposed to underlie the association between ELA and long-term health consequences. However, it is unclear what drives ELA-associated immunosenescence and which cells are primarily affected. We investigated different biomarkers of immunosenescence in a healthy subset of the EpiPath cohort. Participants were either parent-reared (Ctrl, n = 59 or had experienced separation from their parents in early childhood and were subsequently adopted (ELA, n = 18. No difference was observed in telomere length or in methylation levels of age-related CpGs in whole blood, containing a heterogeneous mixture of immune cells. However, when specifically investigating T cells, we found a higher expression of senescence markers (CD57 in ELA. In addition, senescent T cells (CD57+ in ELA had an increased cytolytic potential compared to senescent cells in controls. With a mediation analysis we demonstrated that cytomegalovirus (CMV infection, which is an important driving force of immunosenescence, largely accounted for elevated CD57 expression observed in ELA. Leukocyte telomere length may obscure cell-specific immunosenescence; here, we demonstrated that the use of cell surface markers of senescence can be more informative. Our data suggest that ELA may increase the risk of CMV infection in early childhood, thereby mediating the effect of ELA on T cell-specific immunosenescence. Thus, future studies should include CMV as a confounder or selectively investigate CMV seronegative cohorts.

  14. HLA-G in human early pregnancy: Control of uterine immune cell activation and likely

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    Philippe Le Bouteiller

    2015-02-01

    Full Text Available Despite a number of controversies, the functional importance of human leukocyte antigen G (HLA-G in early human pregnancy is now sustained by a large amount of sound data. Membrane-bound and soluble HLA-G isoforms, either as β2-microglobulin-free or -associated as monomers or dimers, are expressed by different trophoblast subpopulations, the only fetal-derived cells that are directly in contact with maternal cells (maternal-fetal interfaces. Trophoblast HLA-G is the specific ligand of multiple cellular receptors present in maternal immune and non-immune cells, including CD8, leukocyte immunoglobulin-like receptor (LILR B1, LILRB2, killer cell immunoglobulin-like receptor (KIR 2DL4, and possibly CD160. Trophoblast HLA-G specific engagement of these cellular receptors triggers either inhibitory or activating signals in decidual CD8 + T cells, CD4 + T cells, natural killer (NK cells, macrophages, dendritic cells, or endothelial cells. Such HLA-G-receptor specific interactions first contribute to limit potentially harmful maternal anti-paternal immune response by impairment of decidual NK cell cytotoxicity, inhibition of CD4 + and CD8 + T-cell and B-cell proliferation, and induction of apoptosis of activated CD8 + T cells. Second, these HLA-G specific interactions contribute to stimulate placental development through secretion of angiogenic factors by decidual NK cells and macrophages, and to provide a protective effect for the outcome of pregnancy by the secretion of interleukin (IL-4 by decidual trophoblast antigen-specific CD4 + T cells.

  15. Studying early lethality of 45,XO (Turner's syndrome embryos using human embryonic stem cells.

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    Achia Urbach

    Full Text Available Turner's syndrome (caused by monosomy of chromosome X is one of the most common chromosomal abnormalities in females. Although 3% of all pregnancies start with XO embryos, 99% of these pregnancies terminate spontaneously during the first trimester. The common genetic explanation for the early lethality of monosomy X embryos, as well as the phenotype of surviving individuals is haploinsufficiency of pseudoautosomal genes on the X chromosome. Another possible mechanism is null expression of imprinted genes on the X chromosome due to the loss of the expressed allele. In contrast to humans, XO mice are viable, and fertile. Thus, neither cells from patients nor mouse models can be used in order to study the cause of early lethality in XO embryos. Human embryonic stem cells (HESCs can differentiate in culture into cells from the three embryonic germ layers as well as into extraembryonic cells. These cells have been shown to have great value in modeling human developmental genetic disorders. In order to study the reasons for the early lethality of 45,XO embryos we have isolated HESCs that have spontaneously lost one of their sex chromosomes. To examine the possibility that imprinted genes on the X chromosome play a role in the phenotype of XO embryos, we have identified genes that were no longer expressed in the mutant cells. None of these genes showed a monoallelic expression in XX cells, implying that imprinting is not playing a major role in the phenotype of XO embryos. To suggest an explanation for the embryonic lethality caused by monosomy X, we have differentiated the XO HESCs in vitro an in vivo. DNA microarray analysis of the differentiated cells enabled us to compare the expression of tissue specific genes in XO and XX cells. The tissue that showed the most significant differences between the clones was the placenta. Many placental genes are expressed at much higher levels in XX cells in compare to XO cells. Thus, we suggest that abnormal

  16. Early life allergen-induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion.

    Science.gov (United States)

    Barrios, Juliana; Patel, Kruti R; Aven, Linh; Achey, Rebecca; Minns, Martin S; Lee, Yoonjoo; Trinkaus-Randall, Vickery E; Ai, Xingbin

    2017-09-01

    Pulmonary neuroendocrine cells (PNECs) are the only innervated airway epithelial cells. To what extent neural innervation regulates PNEC secretion and function is unknown. Here, we discover that neurotrophin 4 (NT4) plays an essential role in mucus overproduction after early life allergen exposure by orchestrating PNEC innervation and secretion of GABA. We found that PNECs were the only cellular source of GABA in airways. In addition, PNECs expressed NT4 as a target-derived mechanism underlying PNEC innervation during development. Early life allergen exposure elevated the level of NT4 and caused PNEC hyperinnervation and nodose neuron hyperactivity. Associated with aberrant PNEC innervation, the authors discovered that GABA hypersecretion was required for the induction of mucin Muc5ac expression. In contrast, NT4-/- mice were protected from allergen-induced mucus overproduction and changes along the nerve-PNEC axis without any defects in inflammation. Last, GABA installation restored mucus overproduction in NT4-/- mice after early life allergen exposure. Together, our findings provide the first evidence for NT4-dependent neural regulation of PNEC secretion of GABA in a neonatal disease model. Targeting the nerve-PNEC axis may be a valid treatment strategy for mucus overproduction in airway diseases, such as childhood asthma.-Barrios, J., Patel, K. R., Aven, L., Achey, R., Minns, M. S., Lee, Y., Trinkaus-Randall, V. E., Ai, X. Early life allergen-induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion. © FASEB.

  17. CD6 attenuates early and late signaling events, setting thresholds for T-cell activation

    Science.gov (United States)

    Oliveira, Marta I; Gonçalves, Carine M; Pinto, Mafalda; Fabre, Stéphanie; Santos, Ana Mafalda; Lee, Simon F; Castro, Mónica A A; Nunes, Raquel J; Barbosa, Rita R; Parnes, Jane R; Yu, Chao; Davis, Simon J; Moreira, Alexandra; Bismuth, Georges; Carmo, Alexandre M

    2012-01-01

    The T lineage glycoprotein CD6 is generally considered to be a costimulator of T-cell activation. Here, we demonstrate that CD6 significantly reduces early and late T-cell responses upon superantigen stimulation or TCR triggering by Abs. Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react significantly less well compared with T cells not expressing the exogenous receptor. When the cytoplasmic domain of rat CD6 was removed, calcium responses were recovered, indicating that the inhibitory properties of CD6 are attributable to its cytoplasmic domain. Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deficient cells or cells expressing a cytoplasmic deletion mutant of human CD6. Similarly, calcium signals triggered by anti-CD3 were enhanced in human T lymphocytes following morpholino-mediated suppression of CD6 expression. Finally, the proliferation of T lymphocytes was increased when the CD6–CD166 interaction was blocked with anti-CD166 Abs, but inhibited when anti-CD6 Abs were used. Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufficient to restrain signaling in T cells. PMID:21956609

  18. In Situ Fixation Redefines Quiescence and Early Activation of Skeletal Muscle Stem Cells

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    Léo Machado

    2017-11-01

    Full Text Available State of the art techniques have been developed to isolate and analyze cells from various tissues, aiming to capture their in vivo state. However, the majority of cell isolation protocols involve lengthy mechanical and enzymatic dissociation steps followed by flow cytometry, exposing cells to stress and disrupting their physiological niche. Focusing on adult skeletal muscle stem cells, we have developed a protocol that circumvents the impact of isolation procedures and captures cells in their native quiescent state. We show that current isolation protocols induce major transcriptional changes accompanied by specific histone modifications while having negligible effects on DNA methylation. In addition to proposing a protocol to avoid isolation-induced artifacts, our study reveals previously undetected quiescence and early activation genes of potential biological interest.

  19. Defining the functional states of Th17 cells [v1; ref status: indexed, http://f1000r.es/51y

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    Youjin Lee

    2015-05-01

    Full Text Available The molecular mechanisms governing T helper (Th cell differentiation and function have revealed a complex network of transcriptional and protein regulators. Cytokines not only initiate the differentiation of CD4 Th cells into subsets but also influence the identity, plasticity and effector function of a T cell. Of the subsets, Th17 cells, named for producing interleukin 17 (IL-17 as their signature cytokine, secrete a cohort of other cytokines, including IL-22, IL-21, IL-10, IL-9, IFNγ, and GM-CSF.  In recent years, Th17 cells have emerged as key players in host defense against both extracellular pathogens and fungal infections, but they have also been implicated as one of the main drivers in the pathogenesis of autoimmunity, likely mediated in part by the cytokines that they produce. Advances in high throughput genomic sequencing have revealed unexpected heterogeneity in Th17 cells and, as a consequence, may have tremendous impact on our understanding of their functional diversity. The assortment in gene expression may also identify different functional states of Th17 cells. This review aims to understand the interplay between the cytokine regulators that drive Th17 cell differentiation and functional states in Th17 cells.

  20. y la perdedora es...

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    Alejandro Ramos Escobedo

    2007-01-01

    Full Text Available El presente artículo aborda la relación entre la aplicación de la ley en el caso del divorcio por mutuo consentimiento, las diferencias de género y la justicia para la mujer de un sector popular de Guadalajara, Jalisco, México. Se describen testimonios de mujeres, abogados y jueces que muestran que en términos económicos y materiales la aplicación de la ley no necesariamente significa justicia para la mujer. Los testimonios presentados corresponden a la experiencia de mujeres, así como a abogados y jueces encargados de llevar los casos de divorcio. El artículo es producto del proyecto general acerca del estudio de la toma de decisiones en el divorcio.

  1. Notch pathway regulates female germ cell meiosis progression and early oogenesis events in fetal mouse.

    Science.gov (United States)

    Feng, Yan-Min; Liang, Gui-Jin; Pan, Bo; Qin, Xun-Si; Zhang, Xi-Feng; Chen, Chun-Lei; Li, Lan; Cheng, Shun-Feng; De Felici, Massimo; Shen, Wei

    2014-01-01

    A critical process of early oogenesis is the entry of mitotic oogonia into meiosis, a cell cycle switch regulated by a complex gene regulatory network. Although Notch pathway is involved in numerous important aspects of oogenesis in invertebrate species, whether it plays roles in early oogenesis events in mammals is unknown. Therefore, the rationale of the present study was to investigate the roles of Notch signaling in crucial processes of early oogenesis, such as meiosis entry and early oocyte growth. Notch receptors and ligands were localized in mouse embryonic female gonads and 2 Notch inhibitors, namely DAPT and L-685,458, were used to attenuate its signaling in an in vitro culture system of ovarian tissues from 12.5 days post coitum (dpc) fetus. The results demonstrated that the expression of Stra8, a master gene for germ cell meiosis, and its stimulation by retinoic acid (RA) were reduced after suppression of Notch signaling, and the other meiotic genes, Dazl, Dmc1, and Rec8, were abolished or markedly decreased. Furthermore, RNAi of Notch1 also markedly inhibited the expression of Stra8 and SCP3 in cultured female germ cells. The increased methylation status of CpG islands within the Stra8 promoter of the oocytes was observed in the presence of DAPT, indicating that Notch signaling is probably necessary for maintaining the epigenetic state of this gene in a way suitable for RA stimulation. Furthermore, in the presence of Notch inhibitors, progression of oocytes through meiosis I was markedly delayed. At later culture periods, the rate of oocyte growth was decreased, which impaired subsequent primordial follicle assembly in cultured ovarian tissues. Taken together, these results suggested new roles of the Notch signaling pathway in female germ cell meiosis progression and early oogenesis events in mammals.

  2. Canonical Wnt signaling promotes early hematopoietic progenitor formation and erythroid specification during embryonic stem cell differentiation.

    Science.gov (United States)

    Tarafdar, Anuradha; Dobbin, Edwina; Corrigan, Pamela; Freeburn, Robin; Wheadon, Helen

    2013-01-01

    The generation of hematopoietic stem cells (HSCs) during development is a complex process linked to morphogenic signals. Understanding this process is important for regenerative medicine applications that require in vitro production of HSC. In this study we investigated the effects of canonical Wnt/β-catenin signaling during early embryonic differentiation and hematopoietic specification using an embryonic stem cell system. Our data clearly demonstrates that following early differentiation induction, canonical Wnt signaling induces a strong mesodermal program whilst maintaining a degree of stemness potential. This involved a complex interplay between β-catenin/TCF/LEF/Brachyury/Nanog. β-catenin mediated up-regulation of TCF/LEF resulted in enhanced brachyury levels, which in-turn lead to Nanog up-regulation. During differentiation, active canonical Wnt signaling also up-regulated key transcription factors and cell specific markers essential for hematopoietic specification, in particular genes involved in establishing primitive erythropoiesis. This led to a significant increase in primitive erythroid colony formation. β-catenin signaling also augmented early hematopoietic and multipotent progenitor (MPP) formation. Following culture in a MPP specific cytokine cocktail, activation of β-catenin suppressed differentiation of the early hematopoietic progenitor population, with cells displaying a higher replating capacity and a propensity to form megakaryocytic erythroid progenitors. This bias towards erythroid lineage commitment was also observed when hematopoietic progenitors were directed to undergo myeloid colony formation. Overall this study underscores the importance of canonical Wnt/β-catenin signaling in mesodermal specification, primitive erythropoiesis and early hematopietic progenitor formation during hematopoietic induction.

  3. Canonical Wnt signaling promotes early hematopoietic progenitor formation and erythroid specification during embryonic stem cell differentiation.

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    Anuradha Tarafdar

    Full Text Available The generation of hematopoietic stem cells (HSCs during development is a complex process linked to morphogenic signals. Understanding this process is important for regenerative medicine applications that require in vitro production of HSC. In this study we investigated the effects of canonical Wnt/β-catenin signaling during early embryonic differentiation and hematopoietic specification using an embryonic stem cell system. Our data clearly demonstrates that following early differentiation induction, canonical Wnt signaling induces a strong mesodermal program whilst maintaining a degree of stemness potential. This involved a complex interplay between β-catenin/TCF/LEF/Brachyury/Nanog. β-catenin mediated up-regulation of TCF/LEF resulted in enhanced brachyury levels, which in-turn lead to Nanog up-regulation. During differentiation, active canonical Wnt signaling also up-regulated key transcription factors and cell specific markers essential for hematopoietic specification, in particular genes involved in establishing primitive erythropoiesis. This led to a significant increase in primitive erythroid colony formation. β-catenin signaling also augmented early hematopoietic and multipotent progenitor (MPP formation. Following culture in a MPP specific cytokine cocktail, activation of β-catenin suppressed differentiation of the early hematopoietic progenitor population, with cells displaying a higher replating capacity and a propensity to form megakaryocytic erythroid progenitors. This bias towards erythroid lineage commitment was also observed when hematopoietic progenitors were directed to undergo myeloid colony formation. Overall this study underscores the importance of canonical Wnt/β-catenin signaling in mesodermal specification, primitive erythropoiesis and early hematopietic progenitor formation during hematopoietic induction.

  4. Fujinon intelligent color enhancement for the diagnosis of early esophageal squamous cell carcinoma and precancerous lesion.

    Science.gov (United States)

    Li, Yan Xia; Shen, Lei; Yu, Hong Gang; Luo, He Sheng; Yu, Jie Ping

    2014-08-01

    Esophageal squamous cell carcinoma is a common malignant tumor in recent years, and the key for improving the survival rate is early diagnosis and treatment. Computed virtual chromoendoscopy with the Fujinon intelligent color enhancement (FICE) system was reported to improve visualization of neoplastic and non-neoplastic lesions in gastroscopy and colonoscopy. The purpose of this study was to evaluate the value of FICE in the diagnosis of early esophageal squamous cell carcinoma and precancerous lesions. Two hundred fifty-seven patients with suspicious lesions of the esophagus were examined successively by FICE, magnifying FICE, Lugol chromoendoscopy, and magnifying Lugol chromoendoscopy in the hospital. The lesions and the intrapapillary capillary loop (IPCL, microvessels at the surface of esophageal carcinoma) were observed and compared with the pathologic diagnosis that was regarded as the golden standard. The positive rates of early esophageal squamous cell carcinoma were 92.6% and 88.9% as examined by FICE and Lugol chromoendoscopy (p>0.05), and 96.3% and 92.6% as examined by magnifying FICE and magnifying Lugol chromoendoscopy (p>0.05), respectively. The magnifying FICE could observe the IPCL of the esophagus clearly. Early esophageal squamous cell carcinoma and high-grade intraepithelial neoplasia were mainly type IV and type V. Low-grade intraepithelial neoplasia and esophagitis were type II and type III, and normal esophagus was type I; however, the observation of the IPCL by magnifying Lugol chromoendoscopy was not clear. Fujinon intelligent color enhancement and magnifying FICE are complements to Lugol chromoendoscopy and magnifying Lugol chromoendoscopy in the diagnosis of early esophageal lesions.

  5. Embryonic Stem Cell Proteins and MicroRNAs in the Etiology of Germ Cell Cancer

    NARCIS (Netherlands)

    R. Eini (Ronak)

    2013-01-01

    textabstractIn the early 1980s, a population of unique cells was isolated from the inner cell mass (ICM) of the mouse pre-implantation embryo named embryonic stem (ES) cells. These cells were generated by removing the ICM from pre-implantation blastocysts. The resulting cells were found to be

  6. Role of Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mice in vitro.

    Science.gov (United States)

    Bahena, I; Xu, E; Betancourt, M; Casas, E; Ducolomb, Y; González, C; Bonilla, E

    2014-11-01

    In a previous study, we have identified a set of conserved spermatogenic genes whose expression is restricted to testis and ovary and that are developmentally regulated. One of these genes, the transcription factor Mael, has been reported to play an essential role in mouse spermatogenesis. Nevertheless, the role of Mael in mouse oogenesis has not been defined. In order to analyse the role of Mael in mouse oogenesis, the expression of this gene was blocked during early oogenesis in mouse in vitro using RNAi technology. In addition, the role of Mael during differentiation of embryonic stem cells (ESC) into germ cells in vitro was analysed. Results show that downregulation of Mael by a specific short interfering RNA disrupted fetal oocyte growth and differentiation in fetal ovary explants in culture and the expression of several germ-cell markers in ESC during their differentiation. These results suggest that there is an important role for Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mouse in vitro.

  7. Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Dun [Population Council, 1230 York Avenue, New York, NY 10065 (United States); Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000 (China); Chen, Hai-Xiao, E-mail: Hxchen-1@163.net [Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000 (China); Yu, Hai-Qiang [Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Liang, Yong; Wang, Carrie [Population Council, 1230 York Avenue, New York, NY 10065 (United States); Lian, Qing-Quan [The 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000 (China); Deng, Hai-Teng, E-mail: dengh@mail.rockefeller.edu [Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ge, Ren-Shan, E-mail: rge@popcbr.rockefeller.edu [Population Council, 1230 York Avenue, New York, NY 10065 (United States); The 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000 (China)

    2010-08-15

    Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.

  8. Melanopsin ganglion cells extend dendrites into the outer retina during early postnatal development.

    Science.gov (United States)

    Renna, Jordan M; Chellappa, Deepa K; Ross, Christopher L; Stabio, Maureen E; Berson, David M

    2015-09-01

    Melanopsin ganglion cells express the photopigment melanopsin and are the first functional photoreceptors to develop in the mammalian retina. They have been shown to play a variety of important roles in visual development and behavior in the early postnatal period (Johnson et al., 2010; Kirkby and Feller, 2013; Rao et al., 2013; Renna et al., 2011). Here, we probed the maturation of the dendritic arbors of melanopsin ganglion cells during this developmental period in mice. We found that some melanopsin ganglion cells (mainly the M1-subtype) transiently extend their dendrites not only into the inner plexiform layer (where they receive synaptic inputs from bipolar and amacrine cells) but also into the outer plexiform layer, where in mature retina, rod and cone photoreceptors are thought to contact only bipolar and horizontal cells. Thus, some immature melanopsin ganglion cells are biplexiform. This feature is much less common although still present in the mature retina. It reaches peak incidence 8-12 days after birth, before the eyes open and bipolar cells are sufficiently mature to link rods and cones to ganglion cells. At this age, some outer dendrites of melanopsin ganglion cells lie in close apposition to the axon terminals of cone photoreceptors and express a postsynaptic marker of glutamatergic transmission, postsynaptic density-95 protein (PSD-95). These findings raise the possibility of direct, monosynaptic connections between cones and melanopsin ganglion cells in the early postnatal retina. We provide a detailed description of the developmental profile of these processes and consider their possible functional and evolutionary significance. © 2015 Wiley Periodicals, Inc.

  9. El Futuro es Hoy

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    Alfonso Latiff Conde

    2002-08-01

    Full Text Available

    En ninguna otra época de la historia como en la nuestra se ha producido una transición tan rápida hacia el futuro, en el campo de la medicina.

    Lo que había sido una centuria de evolución desde la era Industrial a la era de la Informática, en la pasada década se ha convertido en una revolución.

    La cirugía laparoscopica que ha constituído el despertar a la edad de la informática como la tecnología lider es considerada ahora un standard en la práctica médica. En la actualidad tecnologias más avanzadas prometen mayores progresos en la medicina.

    La mayor revolución médica ocurrió en la cirugía en las postrimerias del siglo diecinueve cuando algunos gigantes de la medicina todavía caminaban sobre la tierra. Estos visionarios comprendieron la magnitud del cambio y dieron nacimiento a la nueva disciplina de la cirugía. Entre ellos estaban Bilroth, Lister, Virchow y Morton.

    Nunca trabajaron juntos, pero la integración espontánea de sus investigaciones y sus habilidades clínicas hicieron posible la nueva cirugía. Fue la convergencia de sus visiones y sus tecnologias las que dieron un vuelco a la cirugía. Bilroth aportó nuevas técnicas y nuevos instrumentos Lister aportó la asepsia, Virchow la patología y Morton la anestesia.

    Los antiguos mitos y muchos hechos empiricos hicieron creer por miles de años en la inviolabilidad del cuerpo humano. Las herramientas científicas de la Era Industrial convirtieron en realidad lo imposible y la ciencia dio nacimiento a la moderna cirugía. En un corto periodo de tiempo se establecieron los fundamentos de una cirugía que permitiría a las siguientes generaciones liderar nuevos avances y tecnologias.

    Nada estremeció más los fundamentos de la medicina como esta explosión de la cirugía. Comprender el shock, la cirugía cardiaca y coronaria, los trasplantes, tuvieron un enorme impacto que conmocionó la medicina. Es obvio que los cambios producidos

  10. Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer

    Science.gov (United States)

    Kaur, Ekjot; Aich, Jyotirmoi; Dani, Prachi; Sethunath, Vidyalakshmi; Gardi, Nilesh; Chandrani, Pratik; Godbole, Mukul; Sonawane, Kavita; Prasad, Ratnam; Kannan, Sadhana; Agarwal, Beamon; Kane, Shubhada; Gupta, Sudeep; Dutt, Shilpee; Dutt, Amit

    2016-01-01

    Background Notch pathway plays a complex role depending on cellular contexts: promotes stem cell maintenance or induces terminal differentiation in potential cancer-initiating cells; acts as an oncogene in lymphocytes and mammary tissue or plays a growth-suppressive role in leukemia, liver, skin, and head and neck cancer. Here, we present a novel clinical and functional significance of NOTCH1 alterations in early stage tongue squamous cell carcinoma (TSCC). Patients and Methods We analyzed the Notch signaling pathway in 68 early stage TSCC primary tumor samples by whole exome and transcriptome sequencing, real-time PCR based copy number, expression, immuno-histochemical, followed by cell based biochemical and functional assays. Results We show, unlike TCGA HNSCC data set, NOTCH1 harbors significantly lower frequency of inactivating mutations (4%); is somatically amplified; and, overexpressed in 31% and 37% of early stage TSCC patients, respectively. HNSCC cell lines over expressing NOTCH1, when plated in the absence of attachment, are enriched in stem cell markers and form spheroids. Furthermore, we show that inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 inhibits spheroid forming capacity, transformation, survival and migration of the HNSCC cells suggesting an oncogenic role of NOTCH1 in TSCC. Clinically, Notch pathway activation is higher in tumors of non-smokers compared to smokers (50% Vs 18%, respectively, P=0.026) and is also associated with greater nodal positivity compared to its non-activation (93% Vs 64%, respectively, P=0.029). Conclusion We anticipate that these results could form the basis for therapeutic targeting of NOTCH1 in tongue cancer. PMID:27391340

  11. Autophagic flux is highly active in early mitosis and differentially regulated throughout the cell cycle

    Science.gov (United States)

    Li, Zhiyuan; Ji, Xinmiao; Wang, Dongmei; Liu, Juanjuan; Zhang, Xin

    2016-01-01

    Mitosis is a fast process that involves dramatic cellular remodeling and has a high energy demand. Whether autophagy is active or inactive during the early stages of mitosis in a naturally dividing cell is still debated. Here we aimed to use multiple assays to resolve this apparent discrepancy. Although the LC3 puncta number was reduced in mitosis, the four different cell lines we tested all have active autophagic flux in both interphase and mitosis. In addition, the autophagic flux was highly active in nocodazole-induced, double-thymidine synchronization released as well as naturally occurring mitosis in HeLa cells. Multiple autophagy proteins are upregulated in mitosis and the increased Beclin-1 level likely contributes to the active autophagic flux in early mitosis. It is interesting that although the autophagic flux is active throughout the cell cycle, early mitosis and S phase have relatively higher autophagic flux than G1 and late G2 phases, which might be helpful to degrade the damaged organelles and provide energy during S phase and mitosis. PMID:27213594

  12. T cell receptor sequencing of early-stage breast cancer tumors identifies altered clonal structure of the T cell repertoire.

    Science.gov (United States)

    Beausang, John F; Wheeler, Amanda J; Chan, Natalie H; Hanft, Violet R; Dirbas, Frederick M; Jeffrey, Stefanie S; Quake, Stephen R

    2017-11-28

    Tumor-infiltrating T cells play an important role in many cancers, and can improve prognosis and yield therapeutic targets. We characterized T cells infiltrating both breast cancer tumors and the surrounding normal breast tissue to identify T cells specific to each, as well as their abundance in peripheral blood. Using immune profiling of the T cell beta-chain repertoire in 16 patients with early-stage breast cancer, we show that the clonal structure of the tumor is significantly different from adjacent breast tissue, with the tumor containing ∼2.5-fold greater density of T cells and higher clonality compared with normal breast. The clonal structure of T cells in blood and normal breast is more similar than between blood and tumor, and could be used to distinguish tumor from normal breast tissue in 14 of 16 patients. Many T cell sequences overlap between tissue and blood from the same patient, including ∼50% of T cells between tumor and normal breast. Both tumor and normal breast contain high-abundance "enriched" sequences that are absent or of low abundance in the other tissue. Many of these T cells are either not detected or detected with very low frequency in the blood, suggesting the existence of separate compartments of T cells in both tumor and normal breast. Enriched T cell sequences are typically unique to each patient, but a subset is shared between many different patients. We show that many of these are commonly generated sequences, and thus unlikely to play an important role in the tumor microenvironment. Copyright © 2017 the Author(s). Published by PNAS.

  13. Early Molecular Events in Murine Gastric Epithelial Cells Mediated by Helicobacter pylori CagA.

    Science.gov (United States)

    Banerjee, Aditi; Basu, Malini; Blanchard, Thomas G; Chintalacharuvu, Subba R; Guang, Wei; Lillehoj, Erik P; Czinn, Steven J

    2016-10-01

    Murine models of Helicobacter pylori infection are used to study host-pathogen interactions, but lack of severe gastritis in this model has limited its usefulness in studying pathogenesis. We compared the murine gastric epithelial cell line GSM06 to the human gastric epithelial AGS cell line to determine whether similar events occur when cultured with H. pylori. The lysates of cells infected with H. pylori isolates or an isogenic cagA-deficient mutant were assessed for translocation and phosphorylation of CagA and for activation of stress pathway kinases by immunoblot. Phosphorylated CagA was detected in both cell lines within 60 minutes. Phospho-ERK 1/2 was present within several minutes and distinctly present in GSM06 cells at 60 minutes. Similar results were obtained for phospho-JNK, although the 54 kDa phosphoprotein signal was dominant in AGS, whereas the lower molecular weight band was dominant in GSM06 cells. These results demonstrate that early events in H. pylori pathogenesis occur within mouse epithelial cells similar to human cells and therefore support the use of the mouse model for the study of acute CagA-associated host cell responses. These results also indicate that reduced disease in H. pylori-infected mice may be due to lack of the Cag PAI, or by differences in the mouse response downstream of the initial activation events. © 2016 John Wiley & Sons Ltd.

  14. Evidences of early senescence in multiple myeloma bone marrow mesenchymal stromal cells.

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    Thibaud André

    Full Text Available BACKGROUND: In multiple myeloma, bone marrow mesenchymal stromal cells support myeloma cell growth. Previous studies have suggested that direct and indirect interactions between malignant cells and bone marrow mesenchymal stromal cells result in constitutive abnormalities in the bone marrow mesenchymal stromal cells. DESIGN AND METHODS: The aims of this study were to investigate the constitutive abnormalities in myeloma bone marrow mesenchymal stromal cells and to evaluate the impact of new treatments. RESULTS: We demonstrated that myeloma bone marrow mesenchymal stromal cells have an increased expression of senescence-associated β-galactosidase, increased cell size, reduced proliferation capacity and characteristic expression of senescence-associated secretory profile members. We also observed a reduction in osteoblastogenic capacity and immunomodulatory activity and an increase in hematopoietic support capacity. Finally, we determined that current treatments were able to partially reduce some abnormalities in secreted factors, proliferation and osteoblastogenesis. CONCLUSIONS: We showed that myeloma bone marrow mesenchymal stromal cells have an early senescent profile with profound alterations in their characteristics. This senescent state most likely participates in disease progression and relapse by altering the tumor microenvironment.

  15. Overlapping DNA methylation dynamics in mouse intestinal cell differentiation and early stages of malignant progression.

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    Marta Forn

    Full Text Available Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high, differentiated cells (EphB2negative, ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart.

  16. Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana.

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    Hirokazu Tanaka

    2013-05-01

    Full Text Available PIN-FORMED (PIN proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.

  17. Early events associated with infection of Epstein-Barr virus infection of primary B-cells.

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    Sabyasachi Halder

    2009-09-01

    Full Text Available Epstein Barr virus (EBV is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology was used to introduce an expression cassette of green fluorescent protein (GFP by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6-7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6-12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.

  18. Glycine-extended gastrin enhances somatostatin release from cultured rabbit fundic D-cells [v1; ref status: indexed, http://f1000r.es/8n

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    Ian LP Beales

    2013-02-01

    Full Text Available The role of the peptide hormone gastrin in stimulating gastric acid secretion is well established. Mature amidated gastrin is processed from larger peptide precursor forms. Increasingly these processing intermediates, such as glycine-extended gastrin (G-Gly and progastrin, have been shown to have biological activities of their own, often separate and complementary to gastrin. Although G-Gly is synthesized and secreted by gastric antral G-cells, the physiological functions of this putative mediator are unclear. Gastrin and cholecystokinin (CCK stimulate the secretion of somatostatin from gastric D-cells as part of the feedback control of gastric acid. In this study the effect of G-Gly and gastrin on the release of somatostatin from rabbit fundic D-cells was examined. D-cells were obtained by collagenase-EDTA digestion and elutriation and cultured for 48 hours. With a 2 hour exposure to the peptides, gastrin but not G-Gly stimulated somatostatin release. Treatment of D-cells for 24 hours with gastrin or G-Gly individually, significantly enhanced subsequent basal as well as CCK- and GLP-1-stimulated somatostatin release. Twenty four hours exposure to gastrin combined with G-Gly synergistically enhanced basal and agonist-stimulated somatostatin release and cellular somatostatin content. Gastrin and G-Gly may be important in the longer term regulation of D-cell function.

  19. Importance of early absolute lymphocyte count after allogeneic stem cell transplantation: a retrospective study.

    Science.gov (United States)

    Rigoni, L; Scroferneker, M L; Pitombeira, B S; Ottoni, E; Paz, A; Fischer, G; Michalowski, M; Pezzi, A; Amorin, B; Valim, V; Baggio, L; Laureano, Á; da Silva, M A; Silla, L; Daudt, L

    2015-03-01

    Early lymphocyte recovery after allogeneic hematopoietic stem cell transplantation (HSCT) is related to the prevention of serious infections and the clearing of residual tumor cells. We analyzed the absolute lymphocyte count at 20 (D+20) and 30 (D+30) days after HSCT in 100 patients with malignant hematologic diseases and correlated with the risk of transplant-related mortality, overall survival (OS), disease-free survival (DFS), nonrelapsed mortality (NRM), and risk of infection. Patients presenting with lymphocyte counts of <300 × 103/μL on D+30 have a 3.76 times greater risk of death in <100 days. Over a medium follow-up of 20 months OS, DFS, and NRM were similar between the groups. In our group of patients delayed lymphocyte recovery after HSCT was a predictor of early death post-HSCT. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Techniques for early diagnosis of oral squamous cell carcinoma: Systematic review.

    Science.gov (United States)

    Carreras-Torras, Clàudia; Gay-Escoda, Cosme

    2015-05-01

    The diagnosis of early oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC) is of paramount clinical importance given the mortality rate of late stage disease. The aim of this study is to review the literature to assess the current situation and progress in this area. A search in Cochrane and PubMed (January 2006 to December 2013) has been used with the key words "squamous cell carcinoma", "early diagnosis" "oral cavity", "Potentially Malignant Disorders" y "premalignant lesions". The inclusion criteria were the use of techniques for early diagnosis of OSCC and OPMD, 7 years aged articles and publications written in English, French or Spanish. The exclusion criteria were case reports and studies in other languages. Out of the 89 studies obtained initially from the search 60 articles were selected to be included in the systematic review: 1 metaanalysis, 17 systematic reviews, 35 prospective studies, 5 retrospective studies, 1 consensus and 1 semi-structured interviews. The best diagnostic technique is that which we have sufficient experience and training. Definitely tissue biopsy and histopathological examination should remain the gold standard for oral cancer diagnose. In this systematic review it has not been found sufficient scientific evidence on the majority of proposed techniques for early diagnosis of OSCC, therefore more extensive and exhaustive studies are needed.

  1. El sedentarismo es...

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    Alex Branco Fraga

    Full Text Available Resumen En este artículo se hizo un análisis crítico del sentido de sedentarismo/sedentario que circula en la literatura pertinente al campo de la actividad física y la salud. Destaca dos "familias" distintas de sedentarios surgidas en dos momentos históricos específicos: el sedentario "cooperiano", oriundo de los preceptos de la aptitud física difundidos en Brasil al principio de los años 1970 por Kenneth Cooper; el sedentario "agitoniano", proveniente de las recomendaciones de actividad física moderada de mediados de los años 1990, propagadas de modo más evidente en Brasil por el programa Agita São Paulo. Entre tantas acepciones posibles, prevalece la noción de que el sedentarismo es agente catalizador de muchas características "malas", una plaga silenciosa, que lentamente envenena el cuerpo y debilita el alma.

  2. Early Intervention Stem Cell-Based Therapy (EISCBT) for Corneal Burns and Trauma

    Science.gov (United States)

    2016-10-01

    corneal bandage (ReCoBand) that can be applied in the battlefield to prevent permanent scarring of the cornea after trauma , blast, or burn wounds . In the...AWARD NUMBER: W81XWH-14-1-0465 TITLE: Early Intervention Stem Cell-Based Therapy (EISCBT) for Corneal Burns and Trauma PRINCIPAL INVESTIGATOR...Intervention Stem Cell-Based Therapy (EISCBT) for Corneal Burns and Trauma 5a. CONTRACT NUMBER W81WH-14-1-0465 5b. GRANT NUMBER 5c. PROGRAM

  3. Simple mechanisms of early life - simulation model on the origin of semi-cells.

    Science.gov (United States)

    Klein, Adrian; Bock, Martin; Alt, Wolfgang

    2017-01-01

    The development of first cellular structures played an important role in the early evolution of life. Early evolution of life probably took place on a molecular level in a reactive environment. The iron-sulfur theory postulates the formation of cell-like structures on catalytic surfaces. Experiments show that H2S together with FeS and other metallic centers drive auto-catalytic surface reactions, in which organic molecules such as pyruvic and amino acids occur. It is questionable which mechanisms are needed to form cell-like structures under these conditions. To address this question, we implemented a model system featuring the fundamentals of molecular dynamics: heat, attraction, repulsion and formation of covalent bonds. Our basic model exhibits a series of essential processes: self-organization of lipid micelles and bilayers, formation of fluid filled cavities, flux of molecules along membranes, transport of energized groups towards sinks and whole colonies of cell-like structures on a larger scale. The results demonstrate that only a few features are sufficient for discovering hitherto non described phenomena of self-assembly and dynamics of cell-like structures as candidates for early evolving proto-cells. Significance statement The quest for a possible origin of life continues to be one of the most fascinating problems in biology. In one theoretical scenario, early life originated from a solution of reactive chemicals in the ancient deep sea, similar to conditions as to be found in thermal vents. Experiments have shown that a variety of organic molecules, the building blocks of life, form under these conditions. Based on such experiments, the iron-sulfur theory postulates the growth of cell-like structures at certain catalytic surfaces. For an explanation and proof of such a process we have developed a computer model simulating molecular assembly of lipid bilayers and formation of semi-cell cavities. The results demonstrate the possibility of cell-like self

  4. Autologous mesenchymal stem cells or meniscal cells: what is the best cell source for regenerative meniscus treatment in an early osteoarthritis situation?

    Science.gov (United States)

    Zellner, Johannes; Pattappa, Girish; Koch, Matthias; Lang, Siegmund; Weber, Johannes; Pfeifer, Christian G; Mueller, Michael B; Kujat, Richard; Nerlich, Michael; Angele, Peter

    2017-10-10

    Treatment of meniscus tears within the avascular region represents a significant challenge, particularly in a situation of early osteoarthritis. Cell-based tissue engineering approaches have shown promising results. However, studies have not found a consensus on the appropriate autologous cell source in a clinical situation, specifically in a challenging degenerative environment. The present study sought to evaluate the appropriate cell source for autologous meniscal repair in a demanding setting of early osteoarthritis. A rabbit model was used to test autologous meniscal repair. Bone marrow and medial menisci were harvested 4 weeks prior to surgery. Bone marrow-derived mesenchymal stem cells (MSCs) and meniscal cells were isolated, expanded, and seeded onto collagen-hyaluronan scaffolds before implantation. A punch defect model was performed on the lateral meniscus and then a cell-seeded scaffold was press-fit into the defect. Following 6 or 12 weeks, gross joint morphology and OARSI grade were assessed, and menisci were harvested for macroscopic, histological, and immunohistochemical evaluation using a validated meniscus scoring system. In conjunction, human meniscal cells isolated from non-repairable bucket handle tears and human MSCs were expanded and, using the pellet culture model, assessed for their meniscus-like potential in a translational setting through collagen type I and II immunostaining, collagen type II enzyme-linked immunosorbent assay (ELISA), and gene expression analysis. After resections of the medial menisci, all knees showed early osteoarthritic changes (average OARSI grade 3.1). However, successful repair of meniscus punch defects was performed using either meniscal cells or MSCs. Gross joint assessment demonstrated donor site morbidity for meniscal cell treatment. Furthermore, human MSCs had significantly increased collagen type II gene expression and production compared to meniscal cells (p cell-based tissue engineering approach was shown

  5. Relationship between red cell distribution width and early renal injury in patients with gestational diabetes mellitus.

    Science.gov (United States)

    Cheng, Dong; Zhao, Jiangtao; Jian, Liguo; Ding, Tongbin; Liu, Shichao

    2016-09-01

    Previous studies found that red cell distribution width was related to adverse cardiovascular events. However, few studies reported the relationship between red cell distribution width and early-stage renal injury in pregnant women with gestational diabetes mellitus. Using a cross-sectional design, 334 pregnant women with gestational diabetes mellitus were enrolled according to the criterion of inclusion and exclusion. Demographic and clinical examination data were collected. Depended on the urine albumin, study population were divided into case group (n = 118) and control group (n = 216). Compared with control group, the case group tend to be higher red cell distribution width level (13.6 ± 0.9 vs.12.5 ± 0.6, p gestational diabetes mellitus patients. The elevated red cell distribution width level might be a predictor of early-stage renal injury in pregnant women with gestational diabetes mellitus. As an easy and routine examination index, red cell distribution width may provide better clinical guidance when combined with other important indices.

  6. Comprehensive Evaluation of Streptococcus sanguinis Cell Wall-Anchored Proteins in Early Infective Endocarditis▿ †

    Science.gov (United States)

    Turner, Lauren Senty; Kanamoto, Taisei; Unoki, Takeshi; Munro, Cindy L.; Wu, Hui; Kitten, Todd

    2009-01-01

    Streptococcus sanguinis is a member of the viridans group of streptococci and a leading cause of the life-threatening endovascular disease infective endocarditis. Initial contact with the cardiac infection site is likely mediated by S. sanguinis surface proteins. In an attempt to identify the proteins required for this crucial step in pathogenesis, we searched for surface-exposed, cell wall-anchored proteins encoded by S. sanguinis and then used a targeted signature-tagged mutagenesis (STM) approach to evaluate their contributions to virulence. Thirty-three predicted cell wall-anchored proteins were identified—a number much larger than those found in related species. The requirement of each cell wall-anchored protein for infective endocarditis was assessed in the rabbit model. It was found that no single cell wall-anchored protein was essential for the development of early infective endocarditis. STM screening was also employed for the evaluation of three predicted sortase transpeptidase enzymes, which mediate the cell surface presentation of cell wall-anchored proteins. The sortase A mutant exhibited a modest (∼2-fold) reduction in competitiveness, while the other two sortase mutants were indistinguishable from the parental strain. The combined results suggest that while cell wall-anchored proteins may play a role in S. sanguinis infective endocarditis, strategies designed to interfere with individual cell wall-anchored proteins or sortases would not be effective for disease prevention. PMID:19703977

  7. The Red Blood Cell Membrane of Preterm Infants in the Early Neonatal Period

    Directory of Open Access Journals (Sweden)

    S. A. Perepelitsa

    2014-01-01

    Full Text Available Objective: to study the nanostructure of red blood cell membranes and erythrocyte index in preterm neonatal infants.Subjects and methods. The trial enrolled 47 neonatal infants, including 33 preterm infants who were included in a study group and 14 fullterm infants who formed a comparative group. The gestational age of the preterm infants was 33.3±1.9 weeks and the birth weight was 2065.4±304.8 g. Red blood cell counts, hemoglobin, and erythrocyte indices were estimat ed and the red blood cells were examined using an atomicforce microscope.Results. At birth, the preterm infants showed macrocytosis, intrauterine poikylocytosis, and the impaired nanostructure of red blood cell membranes. Intrauterine hypoxia affects the red blood cell membrane nanostructures: a phospholipid bilayer and a spectrin matrix, without damaging the membrane protein component. The detected changes are reversible and directed to maintaining the functional ability of red blood cells in a critical situation. At birth, gestational age, a baby's weight, hemoglobin, and blood cholesterol and standard bicarbonate levels influence the parameters of a red blood cell component. The early neonatal period was characterized by an active process on the red blood cell membranes and a change of morphological forms, suggesting the continuing postnatal rearrangement of erythropoiesis and a preterm infant's adaptation to new environmental conditions.

  8. Early intervention for spinal cord injury with human induced pluripotent stem cells oligodendrocyte progenitors.

    Directory of Open Access Journals (Sweden)

    Angelo H All

    Full Text Available Induced pluripotent stem (iPS cells are at the forefront of research in regenerative medicine and are envisaged as a source for personalized tissue repair and cell replacement therapy. Here, we demonstrate for the first time that oligodendrocyte progenitors (OPs can be derived from iPS cells generated using either an episomal, non-integrating plasmid approach or standard integrating retroviruses that survive and differentiate into mature oligodendrocytes after early transplantation into the injured spinal cord. The efficiency of OP differentiation in all 3 lines tested ranged from 40% to 60% of total cells, comparable to those derived from human embryonic stem cells. iPS cell lines derived using episomal vectors or retroviruses generated a similar number of early neural progenitors and glial progenitors while the episomal plasmid-derived iPS line generated more OPs expressing late markers O1 and RIP. Moreover, we discovered that iPS-derived OPs (iPS-OPs engrafted 24 hours following a moderate contusive spinal cord injury (SCI in rats survived for approximately two months and that more than 70% of the transplanted cells differentiated into mature oligodendrocytes that expressed myelin associated proteins. Transplanted OPs resulted in a significant increase in the number of myelinated axons in animals that received a transplantation 24 h after injury. In addition, nearly a 5-fold reduction in cavity size and reduced glial scarring was seen in iPS-treated groups compared to the control group, which was injected with heat-killed iPS-OPs. Although further investigation is needed to understand the mechanisms involved, these results provide evidence that patient-specific, iPS-derived OPs can survive for three months and improve behavioral assessment (BBB after acute transplantation into SCI. This is significant as determining the time in which stem cells are injected after SCI may influence their survival and differentiation capacity.

  9. Novel Polypyrrole-Coated Polylactide Scaffolds Enhance Adipose Stem Cell Proliferation and Early Osteogenic Differentiation

    Science.gov (United States)

    Pelto, Jani; Björninen, Miina; Pälli, Aliisa; Talvitie, Elina; Hyttinen, Jari; Mannerström, Bettina; Suuronen Seppanen, Riitta; Kellomäki, Minna; Miettinen, Susanna; Haimi, Suvi

    2013-01-01

    An electrically conductive polypyrrole (PPy) doped with a bioactive agent is an emerging functional biomaterial for tissue engineering. We therefore used chondroitin sulfate (CS)-doped PPy coating to modify initially electrically insulating polylactide resulting in novel osteogenic scaffolds. In situ chemical oxidative polymerization was used to obtain electrically conductive PPy coating on poly-96L/4D-lactide (PLA) nonwoven scaffolds. The coated scaffolds were characterized and their electrical conductivity was evaluated in hydrolysis. The ability of the coated and conductive scaffolds to enhance proliferation and osteogenic differentiation of human adipose stem cells (hASCs) under electrical stimulation (ES) in three-dimensional (3D) geometry was compared to the noncoated PLA scaffolds. Electrical conductivity of PPy-coated PLA scaffolds (PLA-PPy) was evident at the beginning of hydrolysis, but decreased during the first week of incubation due to de-doping. PLA-PPy scaffolds enhanced hASC proliferation significantly compared to the plain PLA scaffolds at 7 and 14 days. Furthermore, the alkaline phosphatase (ALP) activity of the hASCs was generally higher in PLA-PPy seeded scaffolds, but due to patient variation, no statistical significance could be determined. ES did not have a significant effect on hASCs. This study highlights the potential of novel PPy-coated PLA scaffolds in bone tissue engineering. PMID:23126228

  10. Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion.

    Science.gov (United States)

    Kaartinen, Tanja; Luostarinen, Annu; Maliniemi, Pilvi; Keto, Joni; Arvas, Mikko; Belt, Heini; Koponen, Jonna; Loskog, Angelica; Mustjoki, Satu; Porkka, Kimmo; Ylä-Herttuala, Seppo; Korhonen, Matti

    2017-06-01

    Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (TSCM, CD95+CD45RO-CD45RA+CD27+) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Urine cell-free DNA integrity as a marker for early bladder cancer diagnosis: preliminary data.

    Science.gov (United States)

    Casadio, Valentina; Calistri, Daniele; Tebaldi, Michela; Bravaccini, Sara; Gunelli, Roberta; Martorana, Giuseppe; Bertaccini, Alessandro; Serra, Luigi; Scarpi, Emanuela; Amadori, Dino; Silvestrini, Rosella; Zoli, Wainer

    2013-11-01

    Urine cell-free (UCF) DNA has recently been proposed as a potential marker for early bladder cancer diagnosis. It is known that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA. Therefore, we verified the potential role of UCF DNA integrity in early bladder cancer diagnosis. UCF DNA was isolated from 51 bladder cancer patients, 46 symptomatic patients, and 32 healthy volunteers. To verify UCF DNA integrity, sequences longer than 250 bp, c-Myc, BCAS1, and HER2, were quantified by real time PCR. At the best cutoff value of 0.1 ng/μl, UCF DNA integrity analysis showed a sensitivity of 0.73 (95% CI 0.61-0.85), and a specificity of 0.84 (95% CI 0.71-0.97) in healthy individuals and 0.83 (95% CI 0.72-0.94) in symptomatic patients. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of 0.834 (95% CI 0.739-0.930) for healthy individuals and 0.796 (95% CI 0.707-0.885) for symptomatic patients. These preliminary data suggest that UCF DNA integrity is a potentially good marker for early noninvasive diagnosis of bladder cancer. Its diagnostic performance does not seem to vary significantly, even in an "at risk" population of symptomatic individuals. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Trophoblast Cell Proliferation and Apoptosis in Placental Development During Early Gestation Period in Rats.

    Science.gov (United States)

    Erboga, Mustafa; Kanter, Mehmet

    2015-10-01

    To show apoptotic and mitotic activities in differentiation and proliferation of the trophoblast through the techniques of immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate-biotin nick end labeling (TUNEL), apart from achieving a morphological examination of placental development during early gestation in rats. Animals were sacrificed on days 7, 9, 11, and 13 of pregnancy. The samples removed from those rats were processed for purposes of microscopic analysis. The decidual structure resulting from the differentiation of the endometrial stromal cells in the uterus on days 7, 9, and 11 of pregnancy was determined. It was observed that the placenta, with an increasing trophoblast proliferation and differentiation, matures on day 13 of pregnancy, after which time the growth seems to be continuous. Density of PCNA-positive cells and PCNA immunostaining was observed to decrease in parallel with the age of pregnancy. The excessive number of apoptotic cells seen in the early periods of pregnancy decreased as the placenta matured. We speculate that the rat placenta grows to maturity by day 13 of pregnancy along with increased proliferation and apoptosis in the early days of pregnancy. In addition, a significant decrease of proliferation and apoptosis was observed in the placenta with increasing age of the pregnancy.

  13. Visualization of early influenza A virus trafficking in human dendritic cells using STED microscopy.

    Directory of Open Access Journals (Sweden)

    Faezzah Baharom

    Full Text Available Influenza A viruses (IAV primarily target respiratory epithelial cells, but can also replicate in immune cells, including human dendritic cells (DCs. Super-resolution microscopy provides a novel method of visualizing viral trafficking by overcoming the resolution limit imposed by conventional light microscopy, without the laborious sample preparation of electron microscopy. Using three-color Stimulated Emission Depletion (STED microscopy, we visualized input IAV nucleoprotein (NP, early and late endosomal compartments (EEA1 and LAMP1 respectively, and HLA-DR (DC membrane/cytosol by immunofluorescence in human DCs. Surface bound IAV were internalized within 5 min of infection. The association of virus particles with early endosomes peaked at 5 min when 50% of NP+ signals were also EEA1+. Peak association with late endosomes occurred at 15 min when 60% of NP+ signals were LAMP1+. At 30 min of infection, the majority of NP signals were in the nucleus. Our findings illustrate that early IAV trafficking in human DCs proceeds via the classical endocytic pathway.

  14. Impact of early lactation somatic cell count in heifers on somatic cell counts over the first lactation.

    Science.gov (United States)

    De Vliegher, S; Barkema, H W; Stryhn, H; Opsomer, G; de Kruif, A

    2004-11-01

    The objective of this study was to estimate the impact of somatic cell count in early lactation (SCCel) from Belgian dairy heifers on test-day somatic cell count (SCC) in first lactation. Geometric mean SCCel [5 to 14 d in milk (DIM)] of the 14,766 available samples was 104,000 cells/mL, and decreased from 178,000 at 5 DIM to 74,000 cells/mL at 14 DIM. Proportion of SCCel >200,000 cells/mL was 27.5. Heifers calving in the period April-June had highest SCCel. In total, 117,496 monthly SCC were measured. A multilevel regression analysis revealed that an increase of the natural log-transformed SCCel (LnSCCel) by one unit on average resulted in an increase of test-day natural log-transformed SCC (LnSCC) by 0.22 unit. The impact of LnSCCel on LnSCC depended on when LnSCCel was measured; an elevated LnSCCel at 14 DIM was more consequential than an equally elevated LnSCCel at 5 DIM. The probability of having a test-day SCC >200,000 cells/mL during the first lactation, also increased with an increasing LnSCCel. The negative effect of an elevated LnSCCel was still present, although to a lesser extent, in heifers with a second test-day SCC health problems in heifers in early lactation have a high prevalence and stresses that heifers should have a low SCCel, because an elevated SCCel will negatively influence test-day SCC during the whole first lactation.

  15. Stromal cell markers are differentially expressed in the synovial tissue of patients with early arthritis.

    Science.gov (United States)

    Choi, Ivy Y; Karpus, Olga N; Turner, Jason D; Hardie, Debbie; Marshall, Jennifer L; de Hair, Maria J H; Maijer, Karen I; Tak, Paul P; Raza, Karim; Hamann, Jörg; Buckley, Christopher D; Gerlag, Danielle M; Filer, Andrew

    2017-01-01

    Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers. We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables. Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

  16. Stromal cell markers are differentially expressed in the synovial tissue of patients with early arthritis.

    Directory of Open Access Journals (Sweden)

    Ivy Y Choi

    Full Text Available Previous studies have shown increased expression of stromal markers in synovial tissue (ST of patients with established rheumatoid arthritis (RA. Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied.ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA, parvovirus associated arthritis, reactive arthritis and RA, disease outcome (resolving vs persistent and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers.We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables.Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

  17. Changes in nucleosome repeat lengths precede replication in the early replicating metallothionein II gene region of cells synchronized in early S phase

    Energy Technology Data Exchange (ETDEWEB)

    D' Anna, J.A.; Tobey, R.A. (Los Alamos National Lab., NM (USA))

    1989-04-04

    Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. There the authors report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 {mu}g mL{sup {minus}1} aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression.

  18. Meta-Analysis of Cell-based CaRdiac stUdiEs (ACCRUE) in patients with acute myocardial infarction based on individual patient data

    DEFF Research Database (Denmark)

    Gyöngyösi, Mariann; Wojakowski, Wojciech; Lemarchand, Patricia

    2015-01-01

    therapy after acute myocardial infarction (AMI), including individual patient data from 12 randomized trials (ASTAMI, Aalst, BOOST, BONAMI, CADUCEUS, FINCELL, REGENT, REPAIR-AMI, SCAMI, SWISS-AMI, TIME, LATE-TIME; n=1252). METHODS AND RESULTS: The primary end point was freedom from combined major adverse......RATIONALE: The meta-Analysis of Cell-based CaRdiac study is the first prospectively declared collaborative multinational database, including individual data of patients with ischemic heart disease treated with cell therapy. OBJECTIVE: We analyzed the safety and efficacy of intracoronary cell...... cardiac and cerebrovascular events (including all-cause death, AMI recurrance, stroke, and target vessel revascularization). The secondary end point was freedom from hard clinical end points (death, AMI recurrence, or stroke), assessed with random-effects meta-analyses and Cox regressions for interactions...

  19. Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth.

    Science.gov (United States)

    Simmons, David G; Natale, David R C; Begay, Valerie; Hughes, Martha; Leutz, Achim; Cross, James C

    2008-06-01

    The labyrinth of the rodent placenta contains villi that are the site of nutrient exchange between mother and fetus. They are covered by three trophoblast cell types that separate the maternal blood sinusoids from fetal capillaries--a single mononuclear cell that is a subtype of trophoblast giant cell (sinusoidal or S-TGC) with endocrine function and two multinucleated syncytiotrophoblast layers, each resulting from cell-cell fusion, that function in nutrient transport. The developmental origins of these cell types have not previously been elucidated. We report here the discovery of cell-layer-restricted genes in the mid-gestation labyrinth (E12.5-14.5) including Ctsq in S-TGCs (also Hand1-positive), Syna in syncytiotrophoblast layer I (SynT-I), and Gcm1, Cebpa and Synb in syncytiotrophoblast layer II (SynT-II). These genes were also expressed in distinct layers in the chorion as early as E8.5, prior to villous formation. Specifically, Hand1 was expressed in apical cells lining maternal blood spaces (Ctsq is not expressed until E12.5), Syna in a layer immediately below, and Gcm1, Cebpa and Synb in basal cells in contact with the allantois. Cebpa and Synb were co-expressed with Gcm1 and were reduced in Gcm1 mutants. By contrast, Hand1 and Syna expression was unaltered in Gcm1 mutants, suggesting that Gcm1-positive cells are not required for the induction of the other chorion layers. These data indicate that the three differentiated trophoblast cell types in the labyrinth arise from distinct and autonomous precursors in the chorion that are patterned before morphogenesis begins.

  20. Effects of Herceptin on circulating tumor cells in HER2 positive early breast cancer.

    Science.gov (United States)

    Zhang, J-L; Yao, Q; Chen Y Wang, J-H; Wang, H; Fan, Q; Ling, R; Yi, J; Wang, L

    2015-03-20

    The objective of this study was to determine the changes in peripheral blood circulating tumor cells in HER2-positive early breast cancer before and after Herceptin therapy, and to explore the effects of the HER2 gene and Herceptin on circulating tumor cells. CK19 mRNA expression in peripheral blood was evaluated by qRT-PCR as an index of circulating tumor cells in 15 cases of HER-2-positive breast cancer and 18 cases of HER2-negative breast cancer before, and after chemotherapy as well. Ten cases of HER2-positive breast cancer continued on Herceptin therapy for 3 months after chemotherapy, and their peripheral blood was again drawn and assayed for CK-19 mRNA expression. Preoperatively, all cases of HER2-positive cancer were positive for CK19 mRNA in peripheral blood, but 6 cases of HER2-negative breast cancer were positive (33.3%), where there was a substantial difference between the two groups. After 6 cycles of adjuvant chemotherapy, CK19 positive rates in cases of HER2-positive and -negative breast cancer reduced by 93.3 and 11.1%, respectively, with a significant difference still existing. After 3 months of Herceptin therapy, expression of CK19 mRNA declined considerably in 10 cases of HER2 positive breast cancer (113.66 ± 88.65 vs 63.35 ± 49.27, P = 0.025). HER-2 gene expression closely correlated with circulating tumor cells in peripheral blood of early breast cancer patients. Moreover, Herceptin, a monoclonal antibody for HER2, can reduce the number of circulating tumor cells, which can be an early predictive factor for Herceptin therapy effectiveness against breast cancer.

  1. Flavopiridol causes early mitochondrial damage in chronic lymphocytic leukemia cells with impaired oxygen consumption and mobilization of intracellular calcium

    OpenAIRE

    Hussain, Syed-Rehan A.; Lucas, David M.; Johnson, Amy J.; Lin, Thomas S.; Bakaletz, Alan P.; Dang, Vinh X.; Viatchenko-Karpinski, Serge; Ruppert, Amy S.; Byrd, John C.; Kuppusamy, Periannan; Crouser, Elliott D.; Grever, Michael R.

    2008-01-01

    Effective administration of flavopiridol in advanced-stage chronic lymphocytic leukemia (CLL) is often associated with early biochemical evidence of tumor cell lysis. Previous work using other cell types showed that flavopiridol impacts mitochondria, and in CLL cells flavopiridol down-regulates the mitochondrial protein Mcl-1. We therefore investigated mitochondrial structure and function in flavopiridol-treated CLL patient cells and in the lymphoblastic cell line 697 using concentrations and...

  2. Localization of early germ cells in a stony coral, Euphyllia ancora: potential implications for a germline stem cell system in coral gametogenesis

    Science.gov (United States)

    Shikina, Shinya; Chung, Yi-Jou; Wang, Hsiang-Ming; Chiu, Yi-Ling; Shao, Zih-Fang; Lee, Yan-Horn; Chang, Ching-Fong

    2015-06-01

    Most corals exhibit annual or multiple gametogenic cycles. Thus far, coral gametogenesis has been studied in many species and locations during the past three decades; however, currently, only a few papers exist that describe the origin of germ cells, such as germline stem cells (GSCs), which support the continuous production of gametes in every reproductive cycle. To address this issue, in this study, we focused on and identified piwi gene, which has been used as a marker of germline cells, including GSCs, in various metazoans, in a scleractinian coral, Euphyllia ancora. Reverse-transcription PCR and Western blotting analyses revealed that E. ancora piwi-like ( Eapiwi) is expressed in mesentery tissues where the sites of gametogenesis are located for both sexes. Immunohistochemistry with a specific antibody against Eapiwi revealed strong immunoreactivity in the spermatogonia in males and in the oogonia and early oocytes in females, demonstrating that Eapiwi could be used as an early germ cell marker in E. ancora. Subsequent immunohistochemical analyses regarding the spatial and temporal distribution patterns of early germ cells in mesentery tissues revealed that early germ cells were present throughout the year in the mesentery tissue we examined, regardless of the sexual reproductive cycle. In particular, small numbers of early germ cells were observed in specific sites of mesentery tissues with fully matured gonads in both sexes. These early germ cells were not released together with mature gametes during the spawning period and remained in the mesentery tissues. These results suggested that these early germ cells most likely serve as a reservoir of germline cells and that some of these cells would produce differentiated germ cells for the upcoming sexual reproduction period; hence, these cells would function as GSCs. Our data provide new information for understanding continuous gamete production in corals.

  3. Improvement of early cell adhesion on Thai silk fibroin surface by low energy plasma.

    Science.gov (United States)

    Amornsudthiwat, Phakdee; Mongkolnavin, Rattachat; Kanokpanont, Sorada; Panpranot, Joongjai; Wong, Chiow San; Damrongsakkul, Siriporn

    2013-11-01

    Low energy plasma has been introduced to treat the surface of Thai silk fibroin which should be enhanced for cell adhesion due to its native hydrophobic surface. Plasma surface treatment could introduce desirable hydrophilic functionalities on the surface without using any chemicals. In this work, nitrogen glow discharge plasma was generated by a low energy AC50Hz power supply system. The plasma operating conditions were optimized to reach the highest nitrogen active species by using optical emission spectroscopy. X-ray photoelectron spectroscopy (XPS) revealed that amine, hydroxyl, ether, and carboxyl groups were induced on Thai silk fibroin surface after plasma treatment. The results on Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy confirmed that the plasma treated effects were only on the outermost layer since there was no change in the bulk chemistry. The surface topography was insignificantly changed from the detection with atomic force microscopy (AFM). The plasma-treated effects were the improved surface wettability and cell adhesion. After a 90-s treatment, the water contact angle was at 20°, while the untreated surface was at 70°. The early cell adhesion of L929 mouse fibroblast was accelerated. L929 cells only took 3h to reach 100% cell adhesion on 90 s N2 plasma-treated surface, while there was less than 50% cell adhesion on the untreated Thai silk fibroin surface after 6h of culture. The cell adhesion results were in agreement with the cytoskeleton development. L929 F-actin was more evident on 90 s N2 plasma-treated surface than others. It could be concluded that a lower energy AC50Hz plasma system enhanced early L929 mouse fibroblast adhesion on Thai silk fibroin surface without any significant change in surface topography and bulk chemistry. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Lactate dehydrogenase as a biomarker for early renal damage in patients with sickle cell disease

    Directory of Open Access Journals (Sweden)

    Mohammad S Alzahri

    2015-01-01

    Full Text Available Among many complications of sickle cell disease, renal failure is the main contributor to early mortality. It is present in up to 21% of patients with sickle cell disease. Although screening for microalbuminuria and proteinuria is the current acceptable practice to detect and follow renal damage in patients with sickle cell disease, there is a crucial need for other, more sensitive biomarkers. This becomes especially true knowing that those biomarkers start to appear only after more than 60% of the kidney function is lost. The primary purpose of this study is to determine whether lactate dehydrogenase (LDH correlates with other, direct and indirect bio-markers of renal insufficiency in patients with sickle cell disease and, therefore, could be used as a biomarker for early renal damage in patients with sickle cell disease. Fifty-five patients with an established diagnosis of sickle cell disease were recruited to in the study. Blood samples were taken and 24-h urine collection samples were collected. Using Statcrunch, a data analysis tool available on the web, we studied the correlation between LDH and other biomarkers of kidney function as well as the distribution and relationship between the variables. Regression analysis showed a significant negative correlation between serum LDH and creatinine clearance, R (correlation coefficient = -0.44, P = 0.0008. This correlation was more significant at younger age. This study shows that in sickle cell patients LDH correlates with creatinine clearance and, therefore, LDH could serve as a biomarker to predict renal insufficiency in those patients.

  5. Pluripotent and Metabolic Features of Two Types of Porcine iPSCs Derived from Defined Mouse and Human ES Cell Culture Conditions.

    Science.gov (United States)

    Zhang, Wei; Pei, Yangli; Zhong, Liang; Wen, Bingqiang; Cao, Suying; Han, Jianyong

    2015-01-01

    The domestic pig is an excellent animal model for stem cell research and clinical medicine. There is still no suitable culture condition to generate authentic porcine embryonic stem cells (pESCs) and high quality porcine induced pluripotent stem cells (piPSCs). In this study, we found that culture conditions affected pluripotent and metabolic features of piPSCs. Using defined human embryonic stem cell (hESC) and mouse ESC (mESC) culture conditions, we generated two types of piPSCs, one of which was morphologically similar to hESCs (here called hpiPSCs), the other resembled mESCs (here called mpiPSCs). Transcriptome analysis and signaling pathway inhibition results suggested that mpiPSCs shared more of mESC signaling pathways, such as the BMP pathway and JAK/STAT pathway and hpiPSCs shared more hESC signaling pathways, such as the FGF pathway. Importantly, the mpiPSCs performed embryonic chimera incorporation more efficiently than the hpiPSCs did. In addition, the mpiPSCs showed mitochondrial features of naive ESCs and lipid droplets accumulation. These evidences may facilitate understanding of the gene regulation network and metabolism in piPSCs and promote derivation of bona fide pESCs for translational medicine.

  6. Pluripotent and Metabolic Features of Two Types of Porcine iPSCs Derived from Defined Mouse and Human ES Cell Culture Conditions.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    Full Text Available The domestic pig is an excellent animal model for stem cell research and clinical medicine. There is still no suitable culture condition to generate authentic porcine embryonic stem cells (pESCs and high quality porcine induced pluripotent stem cells (piPSCs. In this study, we found that culture conditions affected pluripotent and metabolic features of piPSCs. Using defined human embryonic stem cell (hESC and mouse ESC (mESC culture conditions, we generated two types of piPSCs, one of which was morphologically similar to hESCs (here called hpiPSCs, the other resembled mESCs (here called mpiPSCs. Transcriptome analysis and signaling pathway inhibition results suggested that mpiPSCs shared more of mESC signaling pathways, such as the BMP pathway and JAK/STAT pathway and hpiPSCs shared more hESC signaling pathways, such as the FGF pathway. Importantly, the mpiPSCs performed embryonic chimera incorporation more efficiently than the hpiPSCs did. In addition, the mpiPSCs showed mitochondrial features of naive ESCs and lipid droplets accumulation. These evidences may facilitate understanding of the gene regulation network and metabolism in piPSCs and promote derivation of bona fide pESCs for translational medicine.

  7. Dynamics of cell wall assembly during early embryogenesis in the brown alga Fucus.

    Science.gov (United States)

    Torode, Thomas A; Siméon, Amandine; Marcus, Susan E; Jam, Murielle; Le Moigne, Marie-Anne; Duffieux, Delphine; Knox, J Paul; Hervé, Cécile

    2016-11-01

    Zygotes from Fucus species have been used extensively to study cell polarization and rhizoid outgrowth, and in this model system cell wall deposition aligns with the establishment of polarity. Monoclonal antibodies are essential tools for the in situ analysis of cell wall glycans, and here we report the characteristics of six monoclonal antibodies to alginates (BAM6-BAM11). The use of these, in conjunction with monoclonal antibodies to brown algal sulfated fucans, has enabled the study of the developmental dynamics of the Fucus zygote cell walls. Young zygotes are spherical and all alginate epitopes are deposited uniformly following cellulose deposition. At germination, sulfated fucans are secreted in the growing rhizoid wall. The redistribution of cell wall epitopes was investigated during treatments that cause reorientation of the growth axis (change in light direction) or disrupt rhizoid development (arabinogalactan-protein-reactive Yariv reagent). Alginate modeling was drastically impaired in the latter, and both treatments cause a redistribution of highly sulfated fucan epitopes. The dynamics of cell wall glycans in this system have been visualized in situ for the first time, leading to an enhanced understanding of the early developmental mechanisms of Fucus species. These sets of monoclonal antibodies significantly extend the available molecular tools for brown algal cell wall studies. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  8. On the fate of primordial germ cells injected into early mouse embryos.

    Science.gov (United States)

    Leitch, Harry G; Okamura, Daiji; Durcova-Hills, Gabriela; Stewart, Colin L; Gardner, Richard L; Matsui, Yasuhisa; Papaioannou, Virginia E

    2014-01-15

    Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a new embryo in the next generation. PGCs are also the cell of origin of multilineage teratocarcinomas. In vitro, mouse PGCs can give rise to embryonic germ (EG) cells - pluripotent stem cells that can contribute to primary chimaeras when introduced into pre-implantation embryos. Thus, PGCs can give rise to pluripotent cells in the course of the developmental cycle, during teratocarcinogenesis and by in vitro culture. However, there is no evidence that PGCs can differentiate directly into somatic cell types. Furthermore, it is generally assumed that PGCs do not contribute to chimaeras following injection into the early mouse embryo. However, these data have never been formally published. Here, we present the primary data from the original PGC-injection experiments performed 40 years ago, alongside results from more recent studies in three separate laboratories. These results have informed and influenced current models of the relationship between pluripotency and the germline cycle. Current technologies allow further experiments to confirm and expand upon these findings and allow definitive conclusions as to the developmental potency of PGCs. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Transcription in pronuclei and one- to four-cell embryos drives early development in a nematode

    Science.gov (United States)

    Wang, Jianbin; Garrey, Julianne; Davis, Richard E.

    2014-01-01

    Summary Background A long-standing view of development is that transcription is silenced in the oocyte until early divisions in the embryo. The point at which major transcription is reactivated varies between organisms, but is usually after the 2-cell stage. However, this model may not be universal. Results We used RNA-seq and exploited the protracted development of the parasitic nematode Ascaris suum, to provide a comprehensive time course of mRNA expression, degradation, and translation during early development. Surprisingly, we find that ~4,000 genes are transcribed prior to pronuclear fusion and in the 1–4 cell embryos. Intriguingly, we do not detect maternal contribution of many orthologs of maternal C. elegans mRNAs, but instead find these are newly transcribed in the A. suum zygote prior to pronuclear fusion. Ribosome profiling demonstrates that, in general, early embryonic mRNAs are not stored for subsequent translation, but are directly translated following their synthesis. The role of maternally contributed and zygotically transcribed genes differs between the nematodes A. suum and C. elegans despite the fact that the two nematodes appear to exhibit highly similar morphological patterns during early development. Conclusions Our study indicates that major transcription can occur immediately after fertilization and prior to pronuclear fusion in metazoa, suggesting that newly transcribed genes appear to drive A. suum early development. Furthermore, the mechanisms used for controlling the timing of the expression of key conserved genes has been altered between the two nematodes, illustrating significant plasticity in the regulatory networks that play important roles in developmental outcomes in nematodes. PMID:24374308

  10. Early stage hot spot analysis through standard cell base random pattern generation

    Science.gov (United States)

    Jeon, Joong-Won; Song, Jaewan; Kim, Jeong-Lim; Park, Seongyul; Yang, Seung-Hune; Lee, Sooryong; Kang, Hokyu; Madkour, Kareem; ElManhawy, Wael; Lee, SeungJo; Kwan, Joe

    2017-04-01

    Due to limited availability of DRC clean patterns during the process and RET recipe development, OPC recipes are not tested with high pattern coverage. Various kinds of pattern can help OPC engineer to detect sensitive patterns to lithographic effects. Random pattern generation is needed to secure robust OPC recipe. However, simple random patterns without considering real product layout style can't cover patterning hotspot in production levels. It is not effective to use them for OPC optimization thus it is important to generate random patterns similar to real product patterns. This paper presents a strategy for generating random patterns based on design architecture information and preventing hotspot in early process development stage through a tool called Layout Schema Generator (LSG). Using LSG, we generate standard cell based on random patterns reflecting real design cell structure - fin pitch, gate pitch and cell height. The output standard cells from LSG are applied to an analysis methodology to assess their hotspot severity by assigning a score according to their optical image parameters - NILS, MEEF, %PV band and thus potential hotspots can be defined by determining their ranking. This flow is demonstrated on Samsung 7nm technology optimizing OPC recipe and early enough in the process avoiding using problematic patterns.

  11. Gene expression profiling of early hepatic stellate cell activation reveals a role for Igfbp3 in cell migration.

    Directory of Open Access Journals (Sweden)

    Inge Mannaerts

    Full Text Available BACKGROUND: Scarring of the liver is the result of prolonged exposure to exogenous or endogenous stimuli. At the onset of fibrosis, quiescent hepatic stellate cells (HSCs activate and transdifferentiate into matrix producing, myofibroblast-like cells. AIM AND METHODS: To identify key players during early HSC activation, gene expression profiling was performed on primary mouse HSCs cultured for 4, 16 and 64 hours. Since valproic acid (VPA can partly inhibit HSC activation, we included VPA-treated cells in the profiling experiments to facilitate this search. RESULTS: Gene expression profiling confirmed early changes for known genes related to HSC activation such as alpha smooth muscle actin (Acta2, lysyl oxidase (Lox and collagen, type I, alpha 1 (Col1a1. In addition we noticed that, although genes which are related to fibrosis change between 4 and 16 hours in culture, most gene expression changes occur between 16 and 64 hours. Insulin-like growth factor binding protein 3 (Igfbp3 was identified as a gene strongly affected by VPA treatment. During normal HSC activation Igfbp3 is up regulated and this can thus be prevented by VPA treatment in vitro and in vivo. siRNA-mediated silencing of Igfbp3 in primary mouse HSCs induced matrix metalloproteinase (Mmp 9 mRNA expression and strongly reduced cell migration. The reduced cell migration after Igfbp3 knock-down could be overcome by tissue inhibitor of metalloproteinase (TIMP 1 treatment. CONCLUSION: Igfbp3 is a marker for culture-activated HSCs and plays a role in HSC migration. VPA treatment prevents Igfbp3 transcription during activation of HSCs in vitro and in vivo.

  12. Early activation of teleost B cells in response to rhabdovirus infection.

    Science.gov (United States)

    Abós, Beatriz; Castro, Rosario; González Granja, Aitor; Havixbeck, Jeffrey J; Barreda, Daniel R; Tafalla, Carolina

    2015-02-01

    demonstrated that VHSV infection provoked immediate transcriptional effects on B cells, at least partially mediated by intracellular PRR signaling. VHSV also activated NF-κB and increased IgM(+) cell survival. Interestingly, VHSV activated B lymphocytes toward an antigen-presenting profile, suggesting an important role of IgM(+) cells in VHSV presentation. Our results provide a first description of the effects provoked by fish rhabdoviruses through their early interaction with teleost B cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Early Development of Squamous Cell Carsinoma in Two Sister Cases with pidermodysplasia Verruciformis

    Directory of Open Access Journals (Sweden)

    Ömer Çalka

    2010-06-01

    Full Text Available Epidermodysplasia verruciformis (Lewandowsky-Lutz syndrome is an uncommon disease characterized by multiple plane warts, pityriasis versicolor-like lesions, defects of cell-mediated immunity, and tendency to develop skin malignancies, primarily on sun-exposed areas. Most commonly it is inherited as an autosomal recessive trait. Squamous cell carcinoma is the most common type of skin cancer found in patient with epidermodysplasia verruciformis. Human papilloma virus 5, 8, and 47 are found in more than 90% of epidermodysplasia verruciformis skin cancers. Treatment for epidermodysplasia verruciformis consists largely of preventive measures. Photoprotection remains essential for management. In this report, two sister case of epidermodisplasia verruciformis with plane warts, pityriasis versicolor-like lesions, and squamous cell carcinomas on sun-exposed areas of skin was presented for it is a rarely encountered disease and associated with early development of malignancy.

  14. Early Depletion of Primordial Germ Cells in Zebrafish Promotes Testis Formation

    Directory of Open Access Journals (Sweden)

    Keh-Weei Tzung

    2015-01-01

    Full Text Available As complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome.

  15. T helper cell 2 immune skewing in pregnancy/early life

    DEFF Research Database (Denmark)

    McFadden, J P; Thyssen, J P; Basketter, D A

    2015-01-01

    During the last 50 years there has been a significant increase in Western societies of atopic disease and associated allergy. The balance between functional subpopulations of T helper cells (Th) determines the quality of the immune response provoked by antigen. One such subpopulation - Th2 cells...... - is associated with the production of IgE antibody and atopic allergy, whereas, Th1 cells antagonize IgE responses and the development of allergic disease. In seeking to provide a mechanistic basis for this increased prevalence of allergic disease, one proposal has been the 'hygiene hypothesis', which argues...... that in Westernized societies reduced exposure during early childhood to pathogenic microorganisms favours the development of atopic allergy. Pregnancy is normally associated with Th2 skewing, which persists for some months in the neonate before Th1/Th2 realignment occurs. In this review, we consider...

  16. Molecular biology of the stress response in the early embryo and its stem cells.

    Science.gov (United States)

    Puscheck, Elizabeth E; Awonuga, Awoniyi O; Yang, Yu; Jiang, Zhongliang; Rappolee, Daniel A

    2015-01-01

    Stress is normal during early embryogenesis and transient, elevated stress is commonplace. Stress in the milieu of the peri-implantation embryo is a summation of maternal hormones, and other elements of the maternal milieu, that signal preparedness for development and implantation. Examples discussed here are leptin, adrenaline, cortisol, and progesterone. These hormones signal maternal nutritional status and provide energy, but also signal stress that diverts maternal and embryonic energy from an optimal embryonic developmental trajectory. These hormones communicate endocrine maternal effects and local embryonic effects although signaling mechanisms are not well understood. Other in vivo stresses affect the embryo such as local infection and inflammation, hypoxia, environmental toxins such as benzopyrene, dioxin, or metals, heat shock, and hyperosmotic stress due to dehydration or diabetes. In vitro, stresses include shear during handling, improper culture media and oxygen levels, cryopreservation, and manipulations of the embryo to introduce sperm or mitochondria. We define stress as any stimulus that slows stem cell accumulation or diminishes the ability of cells to produce normal and sufficient parenchymal products upon differentiation. Thus stress deflects downwards the normal trajectories of development, growth and differentiation. Typically stress is inversely proportional to embryonic developmental and proliferative rates, but can be proportional to induction of differentiation of stem cells in the peri-implantation embryo. When modeling stress it is most interesting to produce a 'runting model' where stress exposures slow accumulation but do not create excessive apoptosis or morbidity. Windows of stress sensitivity may occur when major new embryonic developmental programs require large amounts of energy and are exacerbated if nutritional flow decreases and removes energy from the normal developmental programs and stress responses. These windows correspond

  17. Symbiosis in cell evolution: Life and its environment on the early earth

    Science.gov (United States)

    Margulis, L.

    1981-01-01

    The book treats cell evolution from the viewpoint of the serial endosymbiosis theory of the origin of organelles. Following a brief outline of the symbiotic theory, which holds that eukaryotes evolved by the association of free-living bacteria with a host prokaryote, the diversity of life is considered, and five kingdoms of organisms are distinguished: the prokaryotic Monera and the eukaryotic Protoctista, Animalia, Fungi and Plantae. Symbiotic and traditional direct filiation theories of cell evolution are compared. Recent observations of cell structure and biochemistry are reviewed in relation to early cell evolution, with attention given to the geological context for the origin of eukaryotic cells, the origin of major bacterial anaerobic pathways, the relationship between aerobic metabolism and atmospheric oxygen, criteria for distinguishing symbiotic organelles from those that originated by differentiation, and the major classes of eukaryotic organelles: mitochondria, cilia, microtubules, the mitotic and meiotic apparatuses, and pastids. Cell evolution during the Phanerozoic is also discussed with emphasis on the effects of life on the biosphere

  18. Analytical and computational modeling of early penetration of non-enveloped icosahedral viruses into cells.

    Science.gov (United States)

    Katzengold, Rona; Zaharov, Evgeniya; Gefen, Amit

    2016-07-27

    As obligate intracellular parasites, all viruses penetrate target cells to initiate replication and infection. This study introduces two approaches for evaluating the contact loads applied to a cell during early penetration of non-enveloped icosahedral viruses. The first approach is analytical modeling which is based on Hertz's theory for the contact of two elastic bodies; here we model the virus capsid as a triangle and the cell as an order-of-magnitude larger sphere. The second approach is finite element modeling, where we simulate three types of viruses: adeno-, papilloma- and polio- viruses, each interacting with a cell section. We find that the peak contact pressures and forces generated at the initial virus-cell contact depend on the virus geometry - that is both size and shape. With respect to shape, we show that the icosahedral virus shape induces greater peak pressures compared to a spherical virus shape. With respect to size, it is shown that the larger the virus is the greater are the contact loads in the attacked cell. Utilization of our modeling can be substantially useful not only for basic science studies, but also in other, more applied fields, such as in the field of gene therapy, or in `phage' virus studies.

  19. Development of tissue-engineered models of oral dysplasia and early invasive oral squamous cell carcinoma.

    Science.gov (United States)

    Colley, H E; Hearnden, V; Jones, A V; Weinreb, P H; Violette, S M; Macneil, S; Thornhill, M H; Murdoch, C

    2011-11-08

    Current organotypic models of dysplasia and oral squamous cell carcinoma (OSCC) lack the complexity that mimics in vivo tissue. Here we describe a three-dimensional in vitro model of the oral epithelium that replicates tumour progression from dysplasia to an invasive phenotype. The OSCC cell lines were seeded as a cell suspension (D20, Cal27) or as multicellular tumour spheroids (FaDu) with oral fibroblasts on to a de-epidermised acellular dermis to generate tissue-engineered models and compared with patient biopsies. The D20 and Cal27 cells generated a model of epithelial dysplasia. Overtime Cal27 cells traversed the basement membrane and invaded the connective tissue to reproduce features of early invasive OSCC. When seeded onto a model of the normal oral mucosa, FaDu spheroids produced a histological picture mimicking carcinoma in situ with severe cellular atypia juxtaposed to normal epithelium. It is possible to culture in vitro models with the morphological appearance and histological characteristics of dysplasia and tumour cell invasion seen in vivo using native dermis. Such models could facilitate study of the molecular processes involved in malignant transformation, invasion and tumour growth as well as in vitro testing of new treatments, diagnostic tests and drug delivery systems for OSCC. 2011 Cancer Research UK

  20. Prognostic Factors and Treatment of Early-stage Small-cell Lung Cancer.

    Science.gov (United States)

    Käsmann, Lukas; Bolm, Louisa; Janssen, Stefan; Rades, Dirk

    2017-03-01

    Only 0.1-0.17% of all lung cancer patients are diagnosed with stage I or II small cell lung cancer (SCLC). Radiochemotherapy remains the standard treatment for limited stage disease. This study focused on prognostic factors in early stage SCLC treated with radiochemotherapy. Seven factors in eight patients with early stage SCLC were analyzed concerning the impact on overall survival, namely gender, age, Karnofsky performance score, N-category, UICC-stage, concurrent chemotherapy and prophylactic cranial irradiation (PCI). Median overall survival was 46 months. On univariate analysis, UICC stage I (48 vs. 24 months, p=0.022) and PCI (48 vs. 20 months, p=0.004) were significantly associated with improved overall survival. On multivariate analysis, PCI was an independent positive prognostic factor (pstage and PCI were identified as significant predictors of survival in early stage SCLC. PCI qualified as an independent positive prognostic factor and should be administered in early-stage SCLC. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  1. Early body weight loss during concurrent chemo-radiotherapy for non-small cell lung cancer.

    Science.gov (United States)

    Op den Kamp, Céline M H; De Ruysscher, Dirk K M; van den Heuvel, Marieke; Elferink, Meike; Houben, Ruud M A; Oberije, Cary J G; Bootsma, Gerben P; Geraedts, Wiel H; Pitz, Cordula C M; Langen, Ramon C; Wouters, Emiel F M; Schols, Annemie M W J; Dingemans, Anne-Marie C

    2014-06-01

    Radiation-esophagitis and weight loss are frequently observed toxicities in patients treated with concurrent chemo-radiotherapy (CT-RT) for non-small cell lung cancer (NSCLC) and might be related. The purpose was to investigate whether weight loss already starts early after initiation of CT-RT and precedes radiation-esophagitis. In a retrospective cohort, weight and esophagitis grade ≥2 were assessed during the first weeks of (CT-)RT in patients treated with concurrent (n = 102) or sequential (n = 92) therapy. In a prospective validation study, data on body weight, esophagitis grade ≥2, nutritional intake and muscle strength were obtained before, during and following CT-RT. In the retrospective cohort, early weight loss was observed in concurrently treated patients (p = 0.002), independent of esophagitis ≥ grade 2. Early weight loss was also observed in the prospective cohort (p = 0.003) and was not accompanied by decreases in nutritional intake. In addition lower limb muscle strength rapidly declined (p = 0.042). In the later weeks of treatment, further body weight loss occurred (p nutritional supplementation and body weight was only partly recovered after 4 weeks post CT-RT (p = 0.003). Weight loss during concurrent CT-RT for NSCLC starts early and prior to onset of esophagitis, requiring timely and intense nutritional rehabilitation.

  2. Fish Stem Cell Cultures

    Science.gov (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer. PMID:21547056

  3. Fish stem cell cultures.

    Science.gov (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-04-13

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  4. Capsaicin from chili (Capsicum spp. inhibits vascular smooth muscle cell proliferation [v1; ref status: indexed, http://f1000r.es/4yk

    Directory of Open Access Journals (Sweden)

    Rongxia Liu

    2015-01-01

    Full Text Available Accelerated vascular smooth muscle cell (VSMC proliferation is implied in cardiovascular disease and significantly contributes to vessel lumen reduction following surgical interventions such as percutaneous transluminal coronary angioplasty or bypass surgery. Therefore, identification and characterization of compounds and mechanisms able to counteract VSMC proliferation is of potential therapeutic relevance. This work reveals the anti-proliferative effect of the natural product capsaicin from Capsicum spp. by quantification of metabolic activity and DNA synthesis in activated VSMC. The observed in vitro activity profile of capsaicin warrants further research on its mechanism of action and potential for therapeutic application.

  5. Outcomes from a prospective trial of endoscopic radiofrequency ablation of early squamous cell neoplasia of the esophagus

    NARCIS (Netherlands)

    Bergman, Jacques J. G. H. M.; Zhang, Yue-Ming; He, Shun; Weusten, Bas; Xue, Liyan; Fleischer, David E.; Lu, Ning; Dawsey, Sanford M.; Wang, Gui-Qi

    2011-01-01

    Radiofrequency ablation (RFA) is safe and effective for eradicating neoplasia in Barrett's esophagus. To evaluate RFA for eradicating early esophageal squamous cell neoplasia (ESCN) defined as moderate-grade squamous intraepithelial neoplasia (MGIN) and high-grade squamous intraepithelial neoplasia

  6. X Chromosome Inactivation and Differentiation Occur Readily in ES Cells Doubly-Deficient for MacroH2A1 and MacroH2A2

    Science.gov (United States)

    Tanasijevic, Borko; Rasmussen, Theodore P.

    2011-01-01

    Macrohistones (mH2As) are unusual histone variants found exclusively in vertebrate chromatin. In mice, the H2afy gene encodes two splice variants, mH2A1.1 and mH2A1.2 and a second gene, H2afy2, encodes an additional mH2A2 protein. Both mH2A isoforms have been found enriched on the inactive X chromosome (Xi) in differentiated mammalian female cells, and are incorporated into the chromatin of developmentally-regulated genes. To investigate the functional significance of mH2A isoforms for X chromosome inactivation (XCI), we produced male and female embryonic stem cell (ESC) lines with stably-integrated shRNA constructs that simultaneously target both mH2A1 and mH2A2. Surprisingly, we find that female ESCs deficient for both mH2A1 and mH2A2 readily execute and maintain XCI upon differentiation. Furthermore, male and female mH2A-deficient ESCs proliferate normally under pluripotency culture conditions, and respond to several standard differentiation procedures efficiently. Our results show that XCI can readily proceed with substantially reduced total mH2A content. PMID:21738686

  7. Treatment of Early Stage Non-Small Cell Lung Cancer: Surgery or Stereotactic Ablative Radiotherapy?

    Science.gov (United States)

    Uzel, Esengül Koçak; Abacıoğlu, Ufuk

    2015-01-01

    The management of early-stage Non-small Cell Lung Cancer (NSCLC) has improved recently due to advances in surgical and radiation modalities. Minimally-invasive procedures like Video-assisted thoracoscopic surgery (VATS) lobectomy decreases the morbidity of surgery, while the numerous methods of staging the mediastinum such as endobronchial and endoscopic ultrasound-guided biopsies are helping to achieve the objectives much more effectively. Stereotactic Ablative Radiotherapy (SABR) has become the frontrunner as the standard of care in medically inoperable early stage NSCLC patients, and has also been branded as tolerable and highly effective. Ongoing researches using SABR are continuously validating the optimal dosing and fractionation schemes, while at the same time instituting its role for both inoperable and operable patients. PMID:25759766

  8. Variation in treatment and outcome in the early stage oral cavity squamous cell carcinoma.

    Science.gov (United States)

    Gogarty, Darragh S; Lennon, Paul; Deady, Sandra; Barry O'Sullivan, J; McArdle, Orla; Leader, Mary; Sheahan, Patrick; O'Neill, James Paul

    2017-02-01

    This study aims to determine the survival impact of patient characteristics and treatment options associated with the early stage oral cavity squamous cell carcinoma, OCSCC. The methods are analysis of Irish cancer database examining T1/2, N0, and M0 cases of OCSCC from 1997 to 2007 inclusive. In total, 397 cases were identified. Anterolateral tongue accounted for 52.9 % of cases. Increased age at diagnosis and smoking are independent prognostic survival indicators associated with poorer outcomes. Surgery as the initial intervention was associated with significantly better survival outcomes, while surgery and adjuvant radiotherapy significantly worse outcomes. Surgical intervention is recommended as the first-line treatment in the early stage OCSCC in combination with elective neck dissection.

  9. Reduced Expression of FOXP3 and Regulatory T-Cell Function in Severe Forms of Early-onset Autoimmune Enteropathy

    NARCIS (Netherlands)

    Moes, Nicolette; Rieux-Laucat, Frederic; Begue, Bernadette; Verdier, Julien; Neven, Benedicte; Patey, Natacha; Torgerson, Troy T.; Picard, Capucine; Stolzenberg, Marie-Claude; Ruemmele, Corinne; Rings, Edmond Hhm; Casanova, Jean-Laurent; Piloquet, Hugues; Biver, Armand; Breton, Anne; Ochs, Hans D.; Hermine, Olivier; Fischer, Alain; Goulet, Olivier; Cerf-Bensussan, Nadine; Ruemmele, Frank M.

    BACKGROUND & AIMS: Little is known about the pathophysiology of early onset forms of autoimmune enteropathy (AIE). AIE has been associated with mutations in FOXP3-a transcription factor that controls regulatory T-cell development and function. We analyzed the molecular basis of neonatal or early

  10. Changes in oscillatory dynamics in the cell cycle of early Xenopus laevis embryos.

    Directory of Open Access Journals (Sweden)

    Tony Y-C Tsai

    2014-02-01

    Full Text Available During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min and the subsequent 11 cycles are short (∼30 min and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.

  11. Increased prevalence of lymphoid tissue inducer cells in the cerebrospinal fluid of patients with early multiple sclerosis

    DEFF Research Database (Denmark)

    Degn, Matilda; Modvig, Signe; Dyring-Andersen, Beatrice

    2016-01-01

    BACKGROUND: Inflammatory cytokines produced by cells of the immune system are believed to play a central role in the pathogenesis of multiple sclerosis (MS). Innate lymphoid cells (ILCs) have been shown to produce and secrete a wide range of the cytokines involved in MS pathogenesis; however...... of LTi cells in the CSF, suggesting a favoured recruitment of blood derived LTi cells. CONCLUSION: Our data suggests a role for ILCs, and in particular the LTi subset, in the early stages of MS. This finding represents an important contribution to the understanding of early inflammation in MS, and adds...

  12. Genetic mutation screen in early non--small-cell lung cancer (NSCLC) specimens.

    Science.gov (United States)

    Bar, Jair; Damianovich, Maya; Hout Siloni, Goni; Dar, Erel; Cohen, Yoram; Perelman, Marina; Ben Nun, Alon; Simansky, David; Yellin, Alon; Urban, Damien; Onn, Amir

    2014-03-01

    Testing for genetic abnormalities in epithelial growth factor receptor (EGFR), anaplastic lymphoma receptor tyrosine kinase (ALK), and potentially additional genes is a critical tool in the care of advanced NSCLC. There is conflicting evidence for the role of such tests in early NSCLC. We report a single-institute Sequenom testing for a wide range of mutations and their clinical correlations in early-resected NSCLC specimens. Early NSCLC paraffin-embedded, formalin-fixed (FFPE) specimens were collected, DNA extracted, and using Sequenom-based matrix-assisted laser desorption/ionization-time of flight analysis, mutations in 22 oncogenes and tumor suppressor genes were evaluated. Clinical data was collected retrospectively. The technique was found to be feasible. Thirty-six of 96 patients (37.5%) had any genetic abnormality identified, and 8 (8.3%) had 2 or more mutations. Kirsten rat sarcoma viral oncogene homolog (KRAS) and EGFR were the most common genes to appear mutated (15.6%); phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) was the gene to be found most commonly in tumors with co-mutations. Transversions were found mostly in KRAS gene mutations and to be nonprognostic. No difference in the spectrum of mutations was found between squamous-cell and non-squamous-cell lung cancers. Ever-smokers showed a trend for worse prognosis, with a similar spectrum of mutations. Sequenom-based mutation screen is feasible using FFPE samples. More than a third of the patients were found to harbor some genetic abnormality, and 8% were found to have more than a single mutated gene. Wide-range gene screens using large sample depositories are required for further insight into the important genes at play in early NSCLC. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Abnormal neural precursor cell regulation in the early postnatal Fragile X mouse hippocampus.

    Science.gov (United States)

    Sourial, Mary; Doering, Laurie C

    2017-07-01

    The regulation of neural precursor cells (NPCs) is indispensable for a properly functioning brain. Abnormalities in NPC proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome. Yet, no studies have examined NPCs from the early postnatal Fragile X mouse hippocampus despite the importance of this developmental time point, which marks the highest expression level of FMRP, the protein missing in Fragile X, in the rodent hippocampus and is when hippocampal NPCs have migrated to the dentate gyrus (DG) to give rise to lifelong neurogenesis. In this study, we examined NPCs from the early postnatal hippocampus and DG of Fragile X mice (Fmr1-KO). Immunocytochemistry on neurospheres showed increased Nestin expression and decreased Ki67 expression, which collectively indicated aberrant NPC biology. Intriguingly, flow cytometric analysis of the expression of the antigens CD15, CD24, CD133, GLAST, and PSA-NCAM showed a decreased proportion of neural stem cells (GLAST + CD15 + CD133 + ) and an increased proportion of neuroblasts (PSA-NCAM + CD15 + ) in the DG of P7 Fmr1-KO mice. This was mirrored by lower expression levels of Nestin and the mitotic marker phospho-histone H3 in vivo in the P9 hippocampus, as well as a decreased proportion of cells in the G 2 /M phases of the P7 DG. Thus, the absence of FMRP leads to fewer actively cycling NPCs, coinciding with a decrease in neural stem cells and an increase in neuroblasts. Together, these results show the importance of FMRP in the developing hippocampal formation and suggest abnormalities in cell cycle regulation in Fragile X. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  14. Ex vivo analysis of the contribution of FGF10(+) cells to airway smooth muscle cell formation during early lung development.

    Science.gov (United States)

    El Agha, Elie; Kheirollahi, Vahid; Moiseenko, Alena; Seeger, Werner; Bellusci, Saverio

    2017-07-01

    Airway smooth muscle cells (ASMCs) have been widely studied during embryonic lung development. These cells have been shown to control epithelial bifurcation during branching morphogenesis. Fibroblast growth factor 10-positive (FGF10(+) ) cells, originally residing in the submesothelial mesenchyme, contribute to ASMC formation in the distal lung. The reported work aims at monitoring the response of FGF10(+) progenitors and differentiated ASMCs to growth factor treatment in real time using lineage tracing in the background of an air-liquid interface (ALI) culture system. FGF ligands impose divergent effects on iterative lung branching in vitro. Moreover, time-lapse imaging and endpoint analysis show that FGF9 treatment leads to amplification of the FGF10(+) lineage and represses its differentiation to ASMCs. Sonic hedgehog (SHH) treatment reduces the amplification of this lineage and leads to decreased lung branching. Finally, differentiated ASMCs in proximal regions fail to expand upon FGF9 treatment. Our data demonstrate, in real time, that FGF9 is an important regulator of amplification, migration, and subsequent differentiation of ASMC progenitors during early lung development. The attained results agree with previous findings regarding ASMC formation and highlight the complexity of growth factor signaling networks in controlling mesenchymal cell-fate decisions in the developing mouse lung. Developmental Dynamics 246:531-538, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. On the intrinsic disorder status of the major players in programmed cell death pathways [v1; ref status: indexed, http://f1000r.es/1me

    Directory of Open Access Journals (Sweden)

    Alexey V Uversky

    2013-09-01

    Full Text Available Earlier computational and bioinformatics analysis of several large protein datasets across 28 species showed that proteins involved in regulation and execution of programmed cell death (PCD possess substantial amounts of intrinsic disorder. Based on the comprehensive analysis of these datasets by a wide array of modern bioinformatics tools it was concluded that disordered regions of PCD-related proteins are involved in a multitude of biological functions and interactions with various partners, possess numerous posttranslational modification sites, and have specific evolutionary patterns (Peng et al. 2013. This study extends our previous work by providing information on the intrinsic disorder status of some of the major players of the three major PCD pathways: apoptosis, autophagy, and necroptosis. We also present a detailed description of the disorder status and interactomes of selected proteins that are involved in the p53-mediated apoptotic signaling pathways.

  16. All trans-retinoic acid (ATRA) induces re-differentiation of early transformed breast epithelial cells.

    Science.gov (United States)

    Arisi, Maria F; Starker, Rebecca A; Addya, Sankar; Huang, Yong; Fernandez, Sandra V

    2014-06-01

    Retinoids have been used as potential chemotherapeutic or chemopreventive agents because of their differentiative, anti-proliferative, pro-apoptotic and antioxidant properties. We investigated the effect of all trans-retinoic acid (ATRA) at different stages of the neoplastic transformation using an in vitro model of breast cancer progression. This model was previously developed by treating the MCF-10F human normal breast epithelial cells with high dose of estradiol and consists of four cell lines which show a progressive neoplastic transformation: MCF-10F, normal stage; trMCF, transformed MCF-10F; bsMCF, invasive stage; and caMCF, tumorigenic stage. In 3D cultures, MCF-10F cells form tubules resembling the structures in the normal mammary gland. After treatment with estradiol, these cells formed tubules and spherical masses which are indicative of transformation. Cells that only formed spherical masses in collagen were isolated (trMCF clone 11) and treated with ATRA. After treatment with 10 or 1 µM ATRA, the trMCF clone 11 cells showed tubules in collagen; 10 and 43% of the structures were tubules in cells treated with 10 and 1 µM ATRA, respectively. Gene expression studies showed that 207 genes upregulated in transformed trMCF clone 11 cells were downregulated after 1 µM ATRA treatment to levels comparable to those found in the normal breast epithelial cells MCF-10F. Furthermore, 236 genes that were downregulated in trMCF clone 11 were upregulated after 1 µM ATRA treatment to similar levels shown in normal epithelial cells. These 443 genes defined a signature of the ATRA re-programming effect. Our results showed that 1 µM ATRA was able to re-differentiate transformed cells at early stages of the neoplastic process and antagonistically regulate breast cancer associated genes. The invasive and tumorigenic cells did not show any changes in morphology after ATRA treatment. These results suggest that ATRA could be used as a chemopreventive agent to

  17. "The Only 13-Year-Old on Planet Earth without a Cell Phone": Meanings of Cell Phones in Early Adolescents' Everyday Lives

    Science.gov (United States)

    Blair, Bethany L.; Fletcher, Anne C.

    2011-01-01

    Cellular telephones have become an increasingly prevalent feature of contemporary American life, with usage often beginning during early adolescence. With this in mind, twenty 7th graders and their mothers participated in separate qualitative interviews regarding early adolescents' use of cell phones as well as perceived risks and benefits of such…

  18. The importance of drug transporters in human pluripotent stem cells and in early tissue differentiation.

    Science.gov (United States)

    Apáti, Ágota; Szebényi, Kornélia; Erdei, Zsuzsa; Várady, György; Orbán, Tamás I; Sarkadi, Balázs

    2016-01-01

    Drug transporters are large transmembrane proteins which catalyse the movement of a wide variety of chemicals, including drugs as well as xeno- and endobiotics through cellular membranes. The major groups of these proteins include the ATP-binding cassette transporters which in eukaryotes work as ATP-fuelled drug 'exporters' and the Solute Carrier transporters, with various transport directions and mechanisms. In this review, we discuss the key ATP-binding cassette and Solute Carrier drug transporters which have been reported to contribute to the function and/or protection of undifferentiated human stem cells and during tissue differentiation. We review the various techniques for studying transporter expression and function in stem cells, and the role of drug transporters in foetal and placental tissues is also discussed. We especially focus on the regulation of transporter expression by factors modulating cell differentiation properties and on the function of the transporters in adjustment to environmental challenges. The relatively new and as yet unexplored territory of transporters in stem cell biology may rapidly expand and bring important new information regarding the metabolic and epigenetic regulation of 'stemness' and the early differentiation properties. Drug transporters are clearly important protective and regulatory components in stem cells and differentiation.

  19. Cell wall structures leading to cultivar differences in softening rates develop early during apple (Malus x domestica) fruit growth.

    Science.gov (United States)

    Ng, Jovyn K T; Schröder, Roswitha; Sutherland, Paul W; Hallett, Ian C; Hall, Miriam I; Prakash, Roneel; Smith, Bronwen G; Melton, Laurence D; Johnston, Jason W

    2013-11-19

    There is a paucity of information regarding development of fruit tissue microstructure and changes in the cell walls during fruit growth, and how these developmental processes differ between cultivars with contrasting softening behaviour. In this study we compare two apple cultivars that show different softening rates during fruit development and ripening. We investigate whether these different softening behaviours manifest themselves late during ethylene-induced softening in the ripening phase, or early during fruit expansion and maturation. 'Scifresh' (slow softening) and 'Royal Gala' (rapid softening) apples show differences in cortical microstructure and cell adhesion as early as the cell expansion phase. 'Scifresh' apples showed reduced loss of firmness and greater dry matter accumulation compared with 'Royal Gala' during early fruit development, suggesting differences in resource allocation that influence tissue structural properties. Tricellular junctions in 'Scifresh' were rich in highly-esterified pectin, contributing to stronger cell adhesion and an increased resistance to the development of large airspaces during cell expansion. Consequently, mature fruit of 'Scifresh' showed larger, more angular shaped cells than 'Royal Gala', with less airspaces and denser tissue. Stronger cell adhesion in ripe 'Scifresh' resulted in tissue fracture by cell rupture rather than by cell-to-cell-separation as seen in 'Royal Gala'. CDTA-soluble pectin differed in both cultivars during development, implicating its involvement in cell adhesion. Low pectin methylesterase activity during early stages of fruit development coupled with the lack of immuno-detectable PG was associated with increased cell adhesion in 'Scifresh'. Our results indicate that cell wall structures leading to differences in softening rates of apple fruit develop early during fruit growth and well before the induction of the ripening process.

  20. Early body weight loss during concurrent chemo-radiotherapy for non-small cell lung cancer

    OpenAIRE

    Op Den Kamp, Celine M. H; de Ruysscher, Dirk; van den Heuvel, Marieke; Elferink, Meike; Houben, Ruud M. A.; Oberije, Cary J. G.; Bootsma, Gerben P.; Geraedts, Wiel H; Pitz, Cordula C. M; Langen, Ramon C.; Wouters, Emiel F.M.; Schols, Annemie M.W.J.; Dingemans, Anne-Marie C.

    2014-01-01

    Background and purpose Radiation-esophagitis and weight loss are frequently observed toxicities in patients treated with concurrent chemo-radiotherapy (CT-RT) for non-small cell lung cancer (NSCLC) and might be related. The purpose was to investigate whether weight loss already starts early after initiation of CT-RT and precedes radiation-esophagitis. Materials and methods In a retrospective cohort, weight and esophagitis grade ≥2 were assessed during the first weeks of (CT-)RT in patients tr...

  1. SSX2-4 expression in early-stage non-small cell lung cancer

    DEFF Research Database (Denmark)

    Greve, K B V; Pøhl, M; Olsen, K E

    2014-01-01

    The expression of cancer/testis antigens SSX2, SSX3, and SSX4 in non-small cell lung cancers (NSCLC) was examined, since they are considered promising targets for cancer immunotherapy due to their immunogenicity and testis-restricted normal tissue expression. We characterized three SSX antibodies...... was only detected in 5 of 143 early-stage NSCLCs, which is rare compared to other cancer/testis antigens (e.g. MAGE-A and GAGE). However, further studies are needed to determine whether SSX can be used as a prognostic or predictive biomarker in NSCLC....

  2. Early detection of tumor cells by innate immune cells leads to T(reg) recruitment through CCL22 production by tumor cells.

    Science.gov (United States)

    Faget, Julien; Biota, Cathy; Bachelot, Thomas; Gobert, Michael; Treilleux, Isabelle; Goutagny, Nadège; Durand, Isabelle; Léon-Goddard, Sophie; Blay, Jean Yves; Caux, Christophe; Ménétrier-Caux, Christine

    2011-10-01

    In breast carcinomas, patient survival seems to be negatively affected by the recruitment of regulatory T cells (T(reg)) within lymphoid aggregates by CCL22. However, the mechanisms underpinning this process, which may be of broader significance in solid tumors, have yet to be described. In this study, we determined how CCL22 production is controlled in tumor cells. In human breast carcinoma cell lines, CCL22 was secreted at low basal levels that were strongly increased in response to inflammatory signals [TNF-α, IFN-γ, and interleukin (IL)-1β], contrasting with CCL17. Primary breast tumors and CD45(+) infiltrating immune cells appeared to cooperate in driving CCL22 secretion, as shown clearly in cocultures of breast tumor cell lines and peripheral blood mononuclear cells (PBMC) or their supernatants. We determined that monocyte-derived IL-1β and TNF-α are key players as monocyte depletion or neutralization of these cytokines attenuated secretion of CCL22. However, when purified monocytes were used, exogenous human IFN-γ was also required to generate this response suggesting a role for IFN-γ-producing cells within PBMCs. In this setting, we found that human IFN-γ could be replaced by the addition of (i) IL-2 or K562-activated natural killer (NK) cells or (ii) resting NK cells in the presence of anti-MHC class I antibody. Taken together, our results show a dialogue between NK and tumor cells leading to IFN-γ secretion, which in turn associates with monocyte-derived IL-1β and TNF-α to drive production of CCL22 by tumor cells and subsequent recruitment of T(reg). As one validation of this conclusion in primary breast tumors, we showed that NK cells and macrophages tend to colocalize within tumors. In summary, our findings suggest that at early times during tumorigenesis, the detection of tumor cells by innate effectors (monocytes and NK cells) imposes a selection for CCL22 secretion that recruits T(reg) to evade this early antitumor immune response.

  3. Squamous cell carcinoma of the esophagus and multiple primary tumors of the upper aerodigestive tract Carcinoma epidermoide do esôfago e múltiplos tumores primários do trato aerodigestivo alto

    Directory of Open Access Journals (Sweden)

    Ulysses RIBEIRO Jr.

    1999-12-01

    Full Text Available Squamous cell carcinoma of the esophagus is frequently associated with other, synchronous or metachronous tumors, in the upper aerodigestive tract. All 264 patients with squamous cell carcinoma of the esophagus, treated in the Gastrointestinal Surgery, Esophagus section, of the "Hospital das Clínicas" (São Paulo University Medical School, Brazil, between 1979 and 1989 were analyzed retrospectively with regards to the occurrence of multiple primary tumors in the upper aerodigestive tract. Multiple primary tumors were encountered in 10 (3.8% patients. All patients were male and the mean age at the time of the first primary was 52.2 years. Tobacco smoke and alcohol were the principal carcinogens in these patients (n = 10. The sites of the tumors were: larynx (n = 4, tongue (n = 4, lung (n = 2, and oral cavity (n = 1. Two simultaneous, three synchronous and five metachronous multiple primary carcinomas were detected. The esophagus was the second primary tumor in nine patients. The mean overall survival after the diagnosis of the second primary was 2.8 months (SD = 0.89. Inquiry regarding other malignancies, associated with panendoscopy should be carry out prior to the treatment of the first primary to diagnose simultaneous or synchronous primary tumors, and careful follow-up should be performed after treatment of the first primary to detect new tumors in these high-risk patients.Carcinoma epidermóide do esôfago está freqüentemente associado a outros, sincrônicos ou metacrônicos tumores do trato aerodigestivo alto. Foram analisados, retrospectivamente, 264 pacientes com carcinoma de esôfago tratados na Disciplina de Cirurgia do Aparelho Digestivo, Divisão de Cirurgia do Esôfago, do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, entre 1979 e 1989, com o intuito de se observar a ocorrência de múltiplos tumores primários do trato aerodigestivo alto. Observaram-se 10 (3.8% pacientes com múltiplos tumores

  4. Reconstructing a B-cell clonal lineage. I. Statistical inference of unobserved ancestors [v1; ref status: indexed, http://f1000r.es/z6

    Directory of Open Access Journals (Sweden)

    Thomas B Kepler

    2013-04-01

    Full Text Available One of the key phenomena in the adaptive immune response to infection and immunization is affinity maturation, during which antibody genes are mutated and selected, typically resulting in a substantial increase in binding affinity to the eliciting antigen. Advances in technology on several fronts have made it possible to clone large numbers of heavy-chain light-chain pairs from individual B cells and thereby identify whole sets of clonally related antibodies. These collections could provide the information necessary to reconstruct their own history - the sequence of changes introduced into the lineage during the development of the clone - and to study affinity maturation in detail. But the success of such a program depends entirely on accurately inferring the founding ancestor and the other unobserved intermediates. Given a set of clonally related immunoglobulin V-region genes, the method described here allows one to compute the posterior distribution over their possible ancestors, thereby giving a thorough accounting of the uncertainty inherent in the reconstruction. I demonstrate the application of this method on heavy-chain and light-chain clones, assess the reliability of the inference, and discuss the sources of uncertainty.

  5. Dental Pulp Stem Cells Model Early Life and Imprinted DNA Methylation Patterns.

    Science.gov (United States)

    Dunaway, Keith; Goorha, Sarita; Matelski, Lauren; Urraca, Nora; Lein, Pamela J; Korf, Ian; Reiter, Lawrence T; LaSalle, Janine M

    2017-04-01

    Early embryonic stages of pluripotency are modeled for epigenomic studies primarily with human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSCs). For analysis of DNA methylation however, ESCs and iPSCs do not accurately reflect the DNA methylation levels found in preimplantation embryos. Whole genome bisulfite sequencing (WGBS) approaches have revealed the presence of large partially methylated domains (PMDs) covering 30%-40% of the genome in oocytes, preimplantation embryos, and placenta. In contrast, ESCs and iPSCs show abnormally high levels of DNA methylation compared to inner cell mass (ICM) or placenta. Here we show that dental pulp stem cells (DPSCs), derived from baby teeth and cultured in serum-containing media, have PMDs and mimic the ICM and placental methylome more closely than iPSCs and ESCs. By principal component analysis, DPSC methylation patterns were more similar to two other neural stem cell types of human derivation (EPI-NCSC and LUHMES) and placenta than were iPSCs, ESCs or other human cell lines (SH-SY5Y, B lymphoblast, IMR90). To test the suitability of DPSCs in modeling epigenetic differences associated with disease, we compared methylation patterns of DPSCs derived from children with chromosome 15q11.2-q13.3 maternal duplication (Dup15q) to controls. Differential methylation region (DMR) analyses revealed the expected Dup15q hypermethylation at the imprinting control region, as well as hypomethylation over SNORD116, and novel DMRs over 147 genes, including several autism candidate genes. Together these data suggest that DPSCs are a useful model for epigenomic and functional studies of human neurodevelopmental disorders. Stem Cells 2017;35:981-988. © 2016 AlphaMed Press.

  6. Molecular features of renal cell carcinoma: early diagnostics and perspectives for therapy

    Directory of Open Access Journals (Sweden)

    O. V. Kovaleva

    2014-01-01

    Full Text Available Kidney cancer (renal cell carcinoma is one of the major problems of modern urological oncology. In Russia renal cell carcinoma accountsfor 4.3 % of all cancers. The global incidence of renal cell carcinoma has increased over the past two decades. Worldwide renal cell carcinoma accounts for 3.6 % of all cancers and is 10th frequent malignancy. For some malignancies, for instance tumours of prostate, there are markers known that allowed improved early diagnostics. Kidney cancer, however, remains to be hard to diagnose and to treat, since the symptoms can be detected on advanced stages of the disease. In Russia 75.4 % of renal cell carcinoma cases detected at the stage of local and locally advanced disease. Though there are various target drugs on the market aimed to treat this disease, the results of renal cell carcinoma treatment did not reach any substantial success. Most of existing target drugs for kidney cancer treatment include inhibitors of a single signalingpathway regulated by VHL1, which expression is lost in the vast majority of renal-cell carcinomas. Till now existing drugs did not reach sufficient efficacy. Therefore, it is highly important to search for new signaling pathways, regulating such cellular processes as proliferation, migration and apoptosis. Further, prognostic markers and therapy targets identified so far are not sufficient and poorly specific. Therefore identification and validation of new markers, and especially new specific targets for the treatment of kindey oncopathologies is highly important and timely task.

  7. Analysis of early mesothelial cell responses to Staphylococcus epidermidis isolated from patients with peritoneal dialysis-associated peritonitis.

    Science.gov (United States)

    McGuire, Amanda L; Mulroney, Kieran T; Carson, Christine F; Ram, Ramesh; Morahan, Grant; Chakera, Aron

    2017-01-01

    The major complication of peritoneal dialysis (PD) is the development of peritonitis, an infection within the abdominal cavity, primarily caused by bacteria. PD peritonitis is associated with significant morbidity, mortality and health care costs. Staphylococcus epidermidis is the most frequently isolated cause of PD-associated peritonitis. Mesothelial cells are integral to the host response to peritonitis, and subsequent clinical outcomes, yet the effects of infection on mesothelial cells are not well characterised. We systematically investigated the early mesothelial cell response to clinical and reference isolates of S. epidermidis using primary mesothelial cells and the mesothelial cell line Met-5A. Using an unbiased whole genome microarray, followed by a targeted panel of genes known to be involved in the human antibacterial response, we identified 38 differentially regulated genes (adj. p-value peritonitis. This study provides new insights into early mesothelial cell responses to infection with S. epidermidis, and confirms the importance of validating findings in primary mesothelial cells.

  8. The NK Cell Response to Mouse Cytomegalovirus Infection Affects the Level and Kinetics of the Early CD8+ T-Cell Response

    Science.gov (United States)

    Mitrović, Maja; Arapović, Jurica; Jordan, Stefan; Fodil-Cornu, Nassima; Ebert, Stefan; Vidal, Silvia M.; Krmpotić, Astrid; Reddehase, Matthias J.

    2012-01-01

    Natural killer (NK) cells and CD8+ T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating the CD8+ T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting of a CD8+ T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157. Our results indicate that the requirement for CD8+ T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8+ T cells has only a minor effect on the early control of wild-type MCMV, CD8+ T cells are essential in the control of Δm157 virus. The frequencies of virus epitope-specific CD8+ T cells and their activation status were higher in mice infected with Δm157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection, particularly in Δm157-infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced CD8+ T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H–m157 interaction. PMID:22156533

  9. Role of chemotherapy and targeted therapy in early-stage non-small cell lung cancer.

    Science.gov (United States)

    Nagasaka, Misako; Gadgeel, Shirish M

    2018-01-01

    Adjuvant platinum based chemotherapy is accepted as standard of care in stage II and III non-small cell lung cancer (NSCLC) patients and is often considered in patients with stage IB disease who have tumors ≥ 4 cm. The survival advantage is modest with approximately 5% at 5 years. Areas covered: This review article presents relevant data regarding chemotherapy use in the perioperative setting for early stage NSCLC. A literature search was performed utilizing PubMed as well as clinical trial.gov. Randomized phase III studies in this setting including adjuvant and neoadjuvant use of chemotherapy as well as ongoing trials on targeted therapy and immunotherapy are also discussed. Expert commentary: With increasing utilization of screening computed tomography scans, it is possible that the percentage of early stage NSCLC patients will increase in the coming years. Benefits of adjuvant chemotherapy in early stage NSCLC patients remain modest. There is a need to better define patients most likely to derive survival benefit from adjuvant therapy and spare patients who do not need adjuvant chemotherapy due to the toxicity of such therapy. Trials for adjuvant targeted therapy, including adjuvant EGFR-TKI trials and trials of immunotherapy drugs are ongoing and will define the role of these agents as adjuvant therapy.

  10. Punção aspirativa por agulha fina para diagnóstico de mastocitoma em cães Fine needle aspiration for diagnosis of mast cell tumors in dogs

    Directory of Open Access Journals (Sweden)

    G.E. Lavalle

    2003-08-01

    Full Text Available Fine needle aspiration (FNA associated with the cytological diagnosis mast cell tumor is a widely employed technique in human medicine, but it is still underused in veterinary medicine. The aim of this study was to demonstrate the efficacy of FNA technique for the diagnosis of mast cell tumors in dogs. Over one year period all dogs referred to the Veterinary Hospital of the Universidade Federal de Minas Gerais with tumor-like formations of the skin were submitted to FNA. In order to detect metastasis, both skin lesions and the regional lymph nodes were subjected to FNA. After surgical removal of the lesions, histological examination indicated a complete agreement with the cytological diagnosis. In conclusion, FNA technique is a good choice for diagnosis of mast cell tumors in dogs. In addition, FNA allows an adequate and early therapeutic planning.

  11. The polycomb group protein Suz12 is required for embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Hansen, Jacob Bo Højberg

    2007-01-01

    results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation...

  12. Obstructive apneas induce early activation of mesenchymal stem cells and enhancement of endothelial wound healing

    Directory of Open Access Journals (Sweden)

    Montserrat Josep M

    2010-07-01

    Full Text Available Abstract Background The aim was to test the hypothesis that the blood serum of rats subjected to recurrent airway obstructions mimicking obstructive sleep apnea (OSA induces early activation of bone marrow-derived mesenchymal stem cells (MSC and enhancement of endothelial wound healing. Methods We studied 30 control rats and 30 rats subjected to recurrent obstructive apneas (60 per hour, lasting 15 s each, for 5 h. The migration induced in MSC by apneic serum was measured by transwell assays. MSC-endothelial adhesion induced by apneic serum was assessed by incubating fluorescent-labelled MSC on monolayers of cultured endothelial cells from rat aorta. A wound healing assay was used to investigate the effect of apneic serum on endothelial repair. Results Apneic serum showed significant increase in chemotaxis in MSC when compared with control serum: the normalized chemotaxis indices were 2.20 ± 0.58 (m ± SE and 1.00 ± 0.26, respectively (p Conclusions The early increases induced by recurrent obstructive apneas in MSC migration, adhesion and endothelial repair suggest that these mechanisms play a role in the physiological response to the challenges associated to OSA.

  13. Early alternating chemotherapy and radiotherapy schedule in limited disease stage small cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Purohit, A.; Charloux, A.; Dietemann, A.; Quoix, E. [Strasbourg-1 Univ., 67 (France); Jung, G.-M.; Schumacher, C. [Centre Paul-Strauss, 67 - Strasbourg (France); Fraisse, P. [Hopital de Hautepierre, Strasbourg (France). Service de Pneumologie

    1995-09-01

    44 patients with limited small cell lung cancer were treated with six cycles of chemotherapy (cisplatinum 60 mg/m{sup 2}day 1, doxorubicin 40 mg/m{sup 2}day 1, etoposide 100 mg/m{sup 2}days 1-3) alternating with three courses of mediastinal irradiation, the first one starting 7 days after the first day of chemotherapy. A total dose of 55 Gy was delivered. Prophylactic cranial irradiation (30 Gy after the third cycle of chemotherapy) was left to the physician`s discretion. 4 patients had radical surgery before combined modality treatment. 29 patients finished the scheduled program. The complete response rate (bronchoscopically confirmed) was 25.6% after two cycles of chemotherapy and 41% at the end of treatment. Median survival time was 17.2 months, with an estimated survival of 32% at 2 years. Main toxicity was haematological with one early toxic death and six premature interruptions of treatment. We conclude that this treatment modality is feasible and efficacious. Prospective studies comparing chemotherapy with alternating or concurrent early radiotherapy schedules in limited disease small cell lung cancer are needed to determine the best treatment modality. (author).

  14. Sufficient numbers of early germ cells are essential for female sex development in zebrafish.

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    Xiangyan Dai

    Full Text Available The sex determination for zebrafish is controlled by a combination of genetic and environmental factors. The determination of sex in zebrafish has been suggested to rely on a mechanism that is affected by germ cell-derived signals. To begin our current study, a simplified and efficient germ cell-specific promoter of the dead end (dnd gene was identified. Utilizing the metrodinazole (MTZ/ bacterial nitroreductase (NTR system for inducible germ cell ablation, several stable Tg (dnd:NTR-EGFP(-3'UTR and Tg (dnd:NTR-EGFP(+3'UTR zebrafish lines were then generated with the identified promoter. A thorough comparison of the expression patterns and tissue distributions of endogenous dnd and ntr-egfp transcripts in vivo revealed that the identified 2032-bp zebrafish dnd promoter can recapitulate dnd expression faithfully in stable transgenic zebrafish. The correlation between the levels of the germ cell-derived signals and requirement for maintaining the female fate has been also explored with different durations of the MTZ treatments. Our results revealed the decreasing ratios of female presented in the treated transgenic group are fairly associated with the reducing levels of the early germ cell-derived signals. After the juvenile transgenic fish treated with 5 mM MTZ for 20 days, all MTZ-treated transgenic fish exclusively developed into males with subfertilities. Taken together, our results identified here a simplified and efficient dnd promoter, and provide clear evidence indicating that it was not the presence but the sufficiency of signals derived from germ cells that is essential for female sex development in zebrafish. Our model also provides a unique system for sex control in zebrafish studies.

  15. Mesenchymal stromal cells improve early lymphocyte recovery and T cell reconstitution after autologous hematopoietic stem cell transplantation in patients with malignant lymphomas.

    Science.gov (United States)

    Batorov, Egor V; Shevela, Ekaterina Ya; Tikhonova, Marina A; Batorova, Dariya S; Ushakova, Galina Yu; Sizikova, Svetlana A; Sergeevicheva, Vera V; Gilevich, Andrey V; Kryuchkova, Irina V; Ostanin, Alexandr A; Chernykh, Elena R

    2015-10-01

    Mesenchymal stromal cells (MSCs) possess a multi-lineage potential and immunoregulatory activities and provide a great potential in cell-based technologies. However, MSC suppressive activity raises concerns regarding the possible adverse effect of MSCs on the immune recovery. The influence of autologous MSC co-transplantation on recovery of T cell subsets in patients receiving autologous hematopoietic stem cell transplantation (AHSCT) for malignant lymphomas and multiple myeloma were characterized. Co-transplantation of MSCs improved lymphocyte recovery most effectively in patients with low input of hematopoietic stem cells or low absolute lymphocyte count in apheresis product. MSC co-transplantation improved early recovery of both memory and naive T cells with more prominent effect on naive CD4(+) T cells. Patients with MSC co-transplantation showed more effective reconstitution of recent thymic emigrants. These data indicate the positive impact of MSCs on immune reconstitution and note MSC co-transplantation is feasible to optimize the outcomes of AHSCT in malignant lymphoma patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Carcinoma de pequenas células do esôfago: estudo clínico patológico de dois casos Small cell carcinoma of the esophagus: clinical pathologic study of two cases

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    Maria Aparecida Coelho de Arruda Henry

    2008-03-01

    Full Text Available RACIONAL: O carcinoma de pequenas células primário do esôfago é tumor raro, agressivo, morfologicamente indistinguível de seu correspondente no pulmão. OBJETIVO: Apresentar os aspectos clínico-patológicos de dois pacientes com carcinoma de pequenas células do esôfago. RELATO DE CASOS: Paciente 1: masculino, 56 anos com disfagia progressiva há seis meses e emagrecimento, com antecedentes de tabagismo e etilismo. A endoscopia mostrou lesão vegetante dos 30 aos 40 cm da arcada dentária superior e o exame anatomopatológico, diagnosticou neoplasia maligna indiferenciada de pequenas células com marcadores imunoistoquímicos positivos para cromogranina e sinaptofisina, caracterizando a linhagem neuroendócrina da neoplasia. Após dois ciclos de quimioterapia (cisplatina e etoposide associada à radioterapia ele apresentou remissão da disfagia. Paciente 2: masculino, 55 anos, com queixas de pirose, disfagia, rouquidão há seis meses, com emagrecimento de 10 kg no período. A endoscopia mostrou lesão vegetante à 30 cm da arcada dentária superior, obstrutiva. O exame anatomopatológico revelou carcinoma de pequenas células, com os mesmos marcadores imunoistoquímicos positivos para linhagem neuroendócrina. Tomografia computadorizada mostrou metástases hepáticas. Frente ao estadio avançado da doença optou-se pela indicação de gastrostomia. O paciente desenvolveu pneumonia e faleceu dois meses após o diagnóstico. CONCLUSÃO: A evolução dos portadores de carcinoma de pequenas células do esôfago depende do estadiamento da doença e apesar da alta agressividade biológica, este tumor apresenta boa resposta à quimioterapia associada à radioterapia.BACKGOUND: Small-cell Carcinoma of the Esophagus is a rare tumor, aggressive, and morphologically indistinguishable from its correspondent well-known tumor in the lung. AIM: To present the clinical-pathological aspects of two patients presenting small-cell carcinoma of the esophagus

  17. B1 SOX coordinate cell specification with patterning and morphogenesis in the early zebrafish embryo.

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    Yuichi Okuda

    2010-05-01

    Full Text Available The B1 SOX transcription factors SOX1/2/3/19 have been implicated in various processes of early embryogenesis. However, their regulatory functions in stages from the blastula to early neurula remain largely unknown, primarily because loss-of-function studies have not been informative to date. In our present study, we systematically knocked down the B1 sox genes in zebrafish. Only the quadruple knockdown of the four B1 sox genes sox2/3/19a/19b resulted in very severe developmental abnormalities, confirming that the B1 sox genes are functionally redundant. We characterized the sox2/3/19a/19b quadruple knockdown embryos in detail by examining the changes in gene expression through in situ hybridization, RT-PCR, and microarray analyses. Importantly, these phenotypic analyses revealed that the B1 SOX proteins regulate the following distinct processes: (1 early dorsoventral patterning by controlling bmp2b/7; (2 gastrulation movements via the regulation of pcdh18a/18b and wnt11, a non-canonical Wnt ligand gene; (3 neural differentiation by regulating the Hes-class bHLH gene her3 and the proneural-class bHLH genes neurog1 (positively and ascl1a (negatively, and regional transcription factor genes, e.g., hesx1, zic1, and rx3; and (4 neural patterning by regulating signaling pathway genes, cyp26a1 in RA signaling, oep in Nodal signaling, shh, and mdkb. Chromatin immunoprecipitation analysis of the her3, hesx1, neurog1, pcdh18a, and cyp26a1 genes further suggests a direct regulation of these genes by B1 SOX. We also found an interesting overlap between the early phenotypes of the B1 sox quadruple knockdown embryos and the maternal-zygotic spg embryos that are devoid of pou5f1 activity. These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo through partnering in part with Pou5f1 and possibly with other factors, and suggest that the B1 sox functions are central to coordinating cell fate

  18. Wnt5a regulates the cell proliferation and adipogenesis via MAPK-independent pathway in early stage of obesity.

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    Tang, Qi; Chen, Chang; Zhang, Yan; Dai, Minjia; Jiang, Yichen; Wang, Hang; Yu, Mei; Jing, Wei; Tian, Weidong

    2017-08-29

    The early stage of obesity is an important stage in the development of obesity. However, there are few studies which explored the property or changes in obesity at early stage especially involving Wnt5a. The associated gene expression of Wnt5a on cell regeneration and the effect of Wnt5a on rat adipose-derived stem cell (rASC) proliferation and adipogenesis need additional study. Here, we investigated the changes in obesity at early stage and how Wnt5a regulates rASC regeneration, proliferation, and adipogenesis. Our data revealed that obesity at early stage measured by Lee index presented a state with impaired adipogenesis and more infiltrated inflammatory cells but without significant changes in adipocyte sizes and inflammatory factors. The process might be associated with anti-canonical Wnt pathway and a reciprocal Wnt5a/JNK pathway. Besides the gene expression of Wnt5a decreased from cell passage 1 to passage 3. The cell proliferation was regulated by increasing dose of Wnt5a with the maximal effect at 50 ng/mL and 50 ng/mL Wnt5a suppressed adipogenic differentiation at middle-late stage of adipogenesis via anti-β-catenin and a mitogen-activated protein kinase (MAPK) signaling-independent manner. Accordingly, the research helps to gain further insights into the early stage of obesity and its associated changes on a cellular and molecular level. © 2017 International Federation for Cell Biology.

  19. Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells.

    Science.gov (United States)

    Lin, Shu; Yuan, Zuo-Fei; Han, Yumiao; Marchione, Dylan M; Garcia, Benjamin A

    2016-07-15

    How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Fetal Nerve Cell Transplantation in Early Post-Resuscitation Period in Rats

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    Damira Tazhibayeva

    2015-02-01

    was less than in the control group. Moreover, cell therapy improved the reflexes in the experimental animals. Conclusions. The study revealed the positive neuroprotective effect of the fetal nerve cells on the recovery in the early post-resuscitation period. This was confirmed by the normalization of enzymatic reactions, improvement reflective activity, and increase in the survival rate of the resuscitated animals in the group treated with fetal nerve cell transplantation. These findings warrant future research on the mechanisms associated with reflex improvement. 

  1. Maternal Serum B-Cell Activating Factor Levels: Candidate Early Biomarker for Hypertensive Disorders of Pregnancy.

    Science.gov (United States)

    Stohl, Hindi E; Lee, Richard H; Manetta, Joseph; Kikly, Kristine; Korst, Lisa M; Stohl, William

    2017-11-01

    Hypertensive disorders of pregnancy are a leading cause of maternal and perinatal morbidity and mortality. Early suppression of B-cell lymphopoiesis is necessary for a normal pregnancy. Dysregulation of factors critical to B-cell survival may result in pregnancy complications, including hypertension. In this prospective observational study at a single medical center, serum levels of BAFF (B-cell activating factor) were measured in pregnant participants at each trimester, at delivery, and postpartum and in nonpregnant controls at a single time point. Comparisons were made between nonpregnant and pregnant subjects and between time periods of pregnancy. First-trimester serum BAFF levels were further tested for association with hypertensive disorders of pregnancy. The study included 149 healthy pregnant women, 25 pregnant women with chronic hypertension, and 48 nonpregnant controls. Median first-trimester serum BAFF level (ng/mL) for healthy women (0.90) was lower than median serum BAFF levels for women with chronic hypertension (0.96; P=0.013) and controls (1.00; P=0.002). Serum BAFF levels steadily declined throughout pregnancy, with the median second-trimester level lower than the corresponding first-trimester level (0.77; P=0.003) and the median third-trimester level lower than the corresponding second-trimester level (0.72; P=0.025). The median first-trimester serum BAFF level was elevated in women who subsequently developed hypertension compared with women who remained normotensive (1.02 versus 0.85; P=0.012), with the area under the receiver operating characteristic curve being 0.709. First-trimester serum BAFF level may be an early and clinically useful predictor of hypertensive disorders of pregnancy. © 2017 American Heart Association, Inc.

  2. VHL and HIF-1α: gene variations and prognosis in early-stage clear cell renal cell carcinoma.

    Science.gov (United States)

    Lessi, Francesca; Mazzanti, Chiara Maria; Tomei, Sara; Di Cristofano, Claudio; Minervini, Andrea; Menicagli, Michele; Apollo, Alessandro; Masieri, Lorenzo; Collecchi, Paola; Minervini, Riccardo; Carini, Marco; Bevilacqua, Generoso

    2014-03-01

    Von Hipple-Lindau gene (VHL) inactivation represents the most frequent abnormality in clear cell renal cell carcinoma (ccRCC). Hypoxia-inducible factor-1α (HIF-1α) expression is regulated by O2 level. In normal O2 conditions, VHL binds HIF-1α and allows HIF-1α proteasomal degradation. A single-nucleotide polymorphism (SNP) has been found located in the oxygen-dependent degradation domain at codon 582 (C1772T, rs11549465, Pro582Ser). In hypoxia, VHL/HIF-1α interaction is abolished and HIF-1α activates target genes in the nucleus. This study analyzes the impact of genetic alterations and protein expression of VHL and the C1772T SNP of HIF-1α gene (HIF-1α) on prognosis in early-stage ccRCC (pT1a, pT1b, and pT2). Mutational analysis of the entire VHL sequence and the genotyping of HIF-1α C1772T SNP were performed together with VHL promoter methylation analysis and loss of heterozygosis (LOH) analysis at (3p25) locus. Data obtained were correlated with VHL and HIF-1α protein expression and with tumor-specific survival (TSS). VHL mutations, methylation status, and LOH were detected in 51, 11, and 12% of cases, respectively. Our results support the association between biallelic alterations and/or VHL silencing with a worse TSS. Moreover, we found a significant association between the HIF-1α C1772C genotype and a worse TSS. The same association was found when testing the presence of HIF-1α protein in the nucleus. Our results highlight the role of VHL/HIF-1α pathway in RCC and support the molecular heterogeneity of early-stage ccRCC. More important, we show the involvement of HIF-1α C1772T SNP in ccRCC progression.

  3. Validation of a Pediatric Early Warning Score in Hospitalized Pediatric Oncology and Hematopoietic Stem Cell Transplant Patients.

    Science.gov (United States)

    Agulnik, Asya; Forbes, Peter W; Stenquist, Nicole; Rodriguez-Galindo, Carlos; Kleinman, Monica

    2016-04-01

    To evaluate the correlation of a Pediatric Early Warning Score with unplanned transfer to the PICU in hospitalized oncology and hematopoietic stem cell transplant patients. We performed a retrospective matched case-control study, comparing the highest documented Pediatric Early Warning Score within 24 hours prior to unplanned PICU transfers in hospitalized pediatric oncology and hematopoietic stem cell transplant patients between September 2011 and December 2013. Controls were patients who remained on the inpatient unit and were matched 2:1 using age, condition (oncology vs hematopoietic stem cell transplant), and length of hospital stay. Pediatric Early Warning Scores were documented by nursing staff at least every 4 hours as part of routine care. Need for transfer was determined by a PICU physician called to evaluate the patient. A large tertiary/quaternary free-standing academic children's hospital. One hundred ten hospitalized pediatric oncology patients (42 oncology, 68 hematopoietic stem cell transplant) requiring unplanned PICU transfer and 220 matched controls. None. Using the highest score in the 24 hours prior to transfer for cases and a matched time period for controls, the Pediatric Early Warning Score was highly correlated with the need for PICU transfer overall (area under the receiver operating characteristic = 0.96), and in the oncology and hematopoietic stem cell transplant groups individually (area under the receiver operating characteristic = 0.95 and 0.96, respectively). The difference in Pediatric Early Warning Score results between the cases and controls was noted as early as 24 hours prior to PICU admission. Seventeen patients died (15.4%). Patients with higher Pediatric Early Warning Scores prior to transfer had increased PICU mortality (p = 0.028) and length of stay (p = 0.004). We demonstrate that our institution's Pediatric Early Warning Score is highly correlated with the need for unplanned PICU transfer in hospitalized oncology and

  4. Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas.

    Science.gov (United States)

    Won, Minho; Ro, Hyunju; Dawid, Igor B

    2015-10-06

    The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.

  5. Tumor-specific suppressor T-cells which inhibit the in vitro generation of cytolytic T-cells from immune and early tumor-bearing host spleens.

    Science.gov (United States)

    Bear, H D

    1986-04-01

    Spleen cells from DBA/2 mice, after immunization with syngeneic P815 mastocytoma cells and Corynebacterium parvum, respond to P815 in vitro with a brisk, secondary-type generation of cytotoxic cells. This cytotoxicity is mediated by antigen-specific T-lymphocytes and correlates with resistance to in vivo challenge. This model confirms the observations of previous investigators made in semisyngeneic hosts using an in vivo transfer model. Spleen cells from "early" tumor-bearing hosts (TBHs), 7-12 days after intradermal (i.d.) inoculation of 10(6) P815 cells alone, made a similar, but generally higher, cytotoxic T-lymphocyte (CTL) response in vitro. Spleen cells from "late" TBHs (18-28 days) completely suppressed the in vitro CTL response of immune cells (e.g., from 71% specific release in controls down to 8% at an effector: target ratio of 40:1). Early i.d. TBH spleen cells, because of their higher level response, appeared to be resistant to this suppression (85% release for controls and 84% when suppressor cells were added at 40:1). By testing early TBH CTL at lower effector: target ratios, however, suppression by late TBH spleen cells could be readily demonstrated. When TBHs were inoculated s.c. instead of i.d. or with lower doses of tumor cells, responses were lower and susceptibility of splenic CTLs to suppression was increased. At intermediate times after tumor inoculation (14-20 days), spleen cells from TBHs still can respond in vitro, but they are completely suppressed by spleen cells from late TBHs. The suppressor cells are antigen-specific, radiation-sensitive, Thy1+ cells.

  6. Clinical role of early dynamic FDG-PET/CT for the evaluation of renal cell carcinoma

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    Nakajima, Reiko; Abe, Koichiro; Sakai, Shuji [Tokyo Women' s Medical University, Department of Diagnostic Imaging and Nuclear Medicine, Tokyo (Japan); Kondo, Tsunenori; Tanabe, Kazunari [Tokyo Women' s Medical University, Department of Urology, Tokyo (Japan)

    2016-06-15

    We studied the usefulness of early dynamic (ED) and whole-body (WB) FDG-PET/CT for the evaluation of renal cell carcinoma (RCC). One hundred patients with 107 tumours underwent kidney ED and WB FDG-PET/CT. We visually and semiquantitatively evaluated the FDG accumulation in RCCs in the ED and WB phases, and compared the accumulation values with regard to histological type (clear cell carcinoma [CCC] vs. non-clear cell carcinoma [N-CCC]), the TNM stage (high stage [3-4] vs. low stage [1-2]), the Fuhrman grade (high grade [3-4] vs. low grade [1-2]) and presence versus absence of venous (V) and lymphatic (Ly) invasion. In the ED phase, visual evaluation revealed no significant differences in FDG accumulation in terms of each item. However, the maximum standardized uptake value and tumour-to-normal tissue ratios were significantly higher in the CCCs compared to the N-CCCs (p < 0.001). In the WB phase, in contrast, significantly higher FDG accumulation (p < 0.001) was found in RCCs with a higher TNM stage, higher Furman grade, and the presence of V and Ly invasion in both the visual and the semiquantitative evaluations. ED and WB FDG-PET/CT is a useful tool for the evaluation of RCCs. (orig.)

  7. Clinical role of early dynamic FDG-PET/CT for the evaluation of renal cell carcinoma.

    Science.gov (United States)

    Nakajima, Reiko; Abe, Koichiro; Kondo, Tsunenori; Tanabe, Kazunari; Sakai, Shuji

    2016-06-01

    We studied the usefulness of early dynamic (ED) and whole-body (WB) FDG-PET/CT for the evaluation of renal cell carcinoma (RCC). One hundred patients with 107 tumours underwent kidney ED and WB FDG-PET/CT. We visually and semiquantitatively evaluated the FDG accumulation in RCCs in the ED and WB phases, and compared the accumulation values with regard to histological type (clear cell carcinoma [CCC] vs. non-clear cell carcinoma [N-CCC]), the TNM stage (high stage [3-4] vs. low stage [1-2]), the Fuhrman grade (high grade [3-4] vs. low grade [1-2]) and presence versus absence of venous (V) and lymphatic (Ly) invasion. In the ED phase, visual evaluation revealed no significant differences in FDG accumulation in terms of each item. However, the maximum standardized uptake value and tumour-to-normal tissue ratios were significantly higher in the CCCs compared to the N-CCCs (p evaluations. ED and WB FDG-PET/CT is a useful tool for the evaluation of RCCs. • ED and WB FDG-PET/ CT helps to assess patients with RCC • ED FDG-PET/CT enabled differentiation between CCC and N-CCC • FDG accumulation in the WB phase reflects tumour aggressiveness • Management of RCC is improved by ED and WB FDG-PET/CT.

  8. The majority of early primordial germ cells acquire pluripotency by AKT activation.

    Science.gov (United States)

    Matsui, Yasuhisa; Takehara, Asuka; Tokitake, Yuko; Ikeda, Makiko; Obara, Yuka; Morita-Fujimura, Yuiko; Kimura, Tohru; Nakano, Toru

    2014-12-01

    Primordial germ cells (PGCs) are undifferentiated germ cells in embryos, the fate of which is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotent embryonic germ cells (EGCs) in culture in the presence of particular extracellular factors, such as combinations of Steel factor (KITL), LIF and bFGF (FGF2). Early PGCs form EGCs more readily than do later PGCs, and PGCs lose the ability to form EGCs by embryonic day (E) 15.5. Here, we examined the effects of activation of the serine/threonine kinase AKT in PGCs during EGC formation; notably, AKT activation, in combination with LIF and bFGF, enhanced EGC formation and caused ∼60% of E10.5 PGCs to become EGCs. The results indicate that the majority of PGCs at E10.5 could acquire pluripotency with an activated AKT signaling pathway. Importantly, AKT activation did not fully substitute for bFGF and LIF, and AKT activation without both LIF and bFGF did not result in EGC formation. These findings indicate that AKT signal enhances and/or collaborates with signaling pathways of bFGF and of LIF in PGCs for the acquisition of pluripotency. © 2014. Published by The Company of Biologists Ltd.

  9. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia

    Science.gov (United States)

    Zhang, Jinghui; Ding, Li; Holmfeldt, Linda; Wu, Gang; Heatley, Sue L.; Payne-Turner, Debbie; Easton, John; Chen, Xiang; Wang, Jianmin; Rusch, Michael; Lu, Charles; Chen, Shann-Ching; Wei, Lei; Collins-Underwood, J. Racquel; Ma, Jing; Roberts, Kathryn G.; Pounds, Stanley B.; Ulyanov, Anatoly; Becksfort, Jared; Gupta, Pankaj; Huether, Robert; Kriwacki, Richard W.; Parker, Matthew; McGoldrick, Daniel J.; Zhao, David; Alford, Daniel; Espy, Stephen; Bobba, Kiran Chand; Song, Guangchun; Pei, Deqing; Cheng, Cheng; Roberts, Stefan; Barbato, Michael I.; Campana, Dario; Coustan-Smith, Elaine; Shurtleff, Sheila A.; Raimondi, Susana C.; Kleppe, Maria; Cools, Jan; Shimano, Kristin A.; Hermiston, Michelle L.; Doulatov, Sergei; Eppert, Kolja; Laurenti, Elisa; Notta, Faiyaz; Dick, John E.; Basso, Giuseppe; Hunger, Stephen P.; Loh, Mignon L.; Devidas, Meenakshi; Wood, Brent; Winter, Stuart; Dunsmore, Kimberley P.; Fulton, Robert S.; Fulton, Lucinda L.; Hong, Xin; Harris, Christopher C.; Dooling, David J.; Ochoa, Kerri; Johnson, Kimberly J.; Obenauer, John C.; Evans, William E.; Pui, Ching-Hon; Naeve, Clayton W.; Ley, Timothy J.; Mardis, Elaine R.; Wilson, Richard K.; Downing, James R.; Mullighan, Charles G.

    2012-01-01

    Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL. PMID:22237106

  10. The kinetics of early T and B cell immune recovery after bone marrow transplantation in RAG-2-deficient SCID patients.

    Directory of Open Access Journals (Sweden)

    Atar Lev

    Full Text Available The kinetics of T and B cell immune recovery after bone marrow transplantation (BMT is affected by many pre- and post-transplant factors. Because of the profoundly depleted baseline T and B cell immunity in recombination activating gene 2 (RAG-2-deficient severe combined immunodeficiency (SCID patients, some of these factors are eliminated, and the immune recovery after BMT can then be clearly assessed. This process was followed in ten SCID patients in parallel to their associated transplant-related complications. Early peripheral presence of T and B cells was observed in 8 and 4 patients, respectively. The latter correlated with pre-transplant conditioning therapy. Cells from these patients carried mainly signal joint DNA episomes, indicative of newly derived B and T cells. They were present before the normalization of the T cell receptor (TCR and the B cell receptor (BCR repertoire. Early presentation of the ordered TCR gene rearrangements after BMT occurred simultaneously, but this pattern was heterogeneous over time, suggesting different and individual thymic recovery processes. Our findings early after transplant could suggest the long-term patients' clinical outcome. Early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites after BMT suggests donor stem cell origin rather than peripheral expansion, and is indicative of successful outcome. Peripheral detection of TCR excision circles and kappa-deleting recombination excision circles in RAG-2-deficient SCID post-BMT are early markers of T and B cell reconstitution, and can be used to monitor outcome and tailor specific therapy for patients undergoing BMT.

  11. es

    Directory of Open Access Journals (Sweden)

    Esther Bernal Valls

    Full Text Available El uso de ejercicios en el tratamiento de pacientes con déficit vestibular crónico está incrementándose de forma notable, lo que evidencia que se trata de un procedimiento que resulta beneficioso para este tipo de pacientes. Los buenos resultados que se obtienen sugieren que los ejercicios vestibulares dan lugar a una estabilidad postural y a una disminución de la sensación de desequilibrio.

  12. Effect of Somatic Cell Types and Culture Medium on in vitro Maturation, Fertilization and Early Development Capability of Buffalo Oocytes

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    H. Jamil*, H. A. Samad, N. Rehman, Z. I. Qureshi and L. A. Lodhi

    2011-04-01

    Full Text Available This study was designed to evaluate the efficacy of different somatic cell types and media in supporting in vitro maturation (IVM, in vitro fertilization (IVF and early embryonic development competence of buffalo follicular oocytes. Cumulus oocyte complexes were collected for maturation from follicles (>6mm of buffalo ovaries collected at the local abattoir. Oocytes were co-cultured in tissue culture medium (TCM-199 with either granulosa cells, cumulus cells, or buffalo oviductal epithelial cells (BOEC @ 3x106 cells/ml or in TCM-199 without helper cells (control at 39°C and 5%CO2 in humidified air. Fresh semen was prepared in modified Ca++ free Tyrode medium. Fertilization was carried out in four types of media: i Tyrode lactate albumin pyruvate (TALP, ii TALP+BOEC, iii modified Ca++ free Tyrode and iv modified Ca++ free Tyrode+BOEC. Fertilized oocytes were cultured for early embryonic development in TCM-199 with and without BOEC. Higher maturation rates were observed in the granulosa (84.24% and cumulus cells (83.44% than BOEC co culture system (73.37%. Highest fertilization rate was obtained in modified Ca++ free Tyrode with BOEC co culture (70.42%, followed by modified Ca++ free Tyrode alone (63.77%, TALP with BOEC (36.92% and TALP alone (10.94%. Development of early embryos (8-cell stage improved in TCM-199 with BOEC co culture than TCM-199 alone. From the results of this study, it can be concluded that addition of somatic cells (granulosa cells, cumulus cells results in higher maturation rates of buffalo follicular oocytes than BOEC co culture system, while fertilization rate improved in modified Ca++ free Tyrode with and without BOEC. Addition of BOEC to TCM-199 improved the developmental capacity of early embryo.

  13. Peroxisome-proliferator activator receptor-gamma activation decreases attachment of endometrial cells to peritoneal mesothelial cells in an in vitro model of the early endometriotic lesion.

    Science.gov (United States)

    Kavoussi, S K; Witz, C A; Binkley, P A; Nair, A S; Lebovic, D I

    2009-10-01

    The aim of this study was to investigate whether peroxisome proliferator-activated receptor (PPAR)-gamma activation has an effect on the attachment of endometrial cells to peritoneal mesothelial cells in a well-established in vitro model of the early endometriotic lesion. The endometrial epithelial cell line EM42 and mesothelial cell line LP9 were used for this study. EM42 cells, LP9 cells or both were treated with the PPAR-gamma agonist ciglitazone (CTZ) at varying concentrations (10, 20 and 40 microM) x 48 h with subsequent co-culture of EM42 and LP9 cells. The rate of EM42 attachment and invasion through LP9 cells was then assessed and compared with control (EM42 and LP9 cells co-cultured without prior treatment with CTZ). Next, attachment of CTZ-treated and untreated EM42 cells to hyaluronic acid (HA), a cell adhesion molecule (CAM) on peritoneal mesothelial cells, were assessed. Although there was no difference in EM42 attachment when LP9 cells alone were treated with CTZ, treatment of EM42 cells with 40 microM CTZ decreased EM42 attachment to LP9 cells by 27% (P CTZ decreased EM42 attachment to LP9 by 37% (P CTZ decreased attachment to HA by 66% (P = 0.056). CTZ did not decrease invasion of EM42 cells through the LP9 monolayer. CTZ may inhibit EM42 cell proliferation. In conclusion, CTZ significantly decreased EM42 attachment to LP9 cells and HA in an in vitro model of the early endometriotic lesion.

  14. SPOC1-mediated antiviral host cell response is antagonized early in human adenovirus type 5 infection.

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    Sabrina Schreiner

    Full Text Available Little is known about immediate phases after viral infection and how an incoming viral genome complex counteracts host cell defenses, before the start of viral gene expression. Adenovirus (Ad serves as an ideal model, since entry and onset of gene expression are rapid and highly efficient, and mechanisms used 24-48 hours post infection to counteract host antiviral and DNA repair factors (e.g. p53, Mre11, Daxx are well studied. Here, we identify an even earlier host cell target for Ad, the chromatin-associated factor and epigenetic reader, SPOC1, recently found recruited to double strand breaks, and playing a role in DNA damage response. SPOC1 co-localized with viral replication centers in the host cell nucleus, interacted with Ad DNA, and repressed viral gene expression at the transcriptional level. We discovered that this SPOC1-mediated restriction imposed upon Ad growth is relieved by its functional association with the Ad major core protein pVII that enters with the viral genome, followed by E1B-55K/E4orf6-dependent proteasomal degradation of SPOC1. Mimicking removal of SPOC1 in the cell, knock down of this cellular restriction factor using RNAi techniques resulted in significantly increased Ad replication, including enhanced viral gene expression. However, depletion of SPOC1 also reduced the efficiency of E1B-55K transcriptional repression of cellular promoters, with possible implications for viral transformation. Intriguingly, not exclusive to Ad infection, other human pathogenic viruses (HSV-1, HSV-2, HIV-1, and HCV also depleted SPOC1 in infected cells. Our findings provide a general model for how pathogenic human viruses antagonize intrinsic SPOC1-mediated antiviral responses in their host cells. A better understanding of viral entry and early restrictive functions in host cells should provide new perspectives for developing antiviral agents and therapies. Conversely, for Ad vectors used in gene therapy, counteracting mechanisms

  15. Role of Chemotherapy and Targeted Therapy in Early-Stage Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Gadgeel, Shirish M

    2017-01-01

    On the basis of several randomized trials and meta-analyses, adjuvant chemotherapy is the accepted standard of care for certain patients with early-stage non-small cell lung cancer (NSCLC). Patients with stage II, IIIA, or large (≥ 4 cm) IB tumors are candidates for adjuvant chemotherapy. The survival improvement with adjuvant chemotherapy is approximately 5% at 5 years, though certain trials have suggested that it can be 8% to 10%. Neoadjuvant chemotherapy also has shown a survival advantage, though the volume of data with this approach is far less than that of adjuvant chemotherapy. The combination of cisplatin and vinorelbine is the most well-studied regimen, but current consensus is to use four cycles of any of the platinum-based chemotherapy regimens commonly used as front-line therapy for patients with advanced-stage NSCLC. Trials to define biomarkers that can predict benefit from adjuvant chemotherapy have not been successful, but results of other such trials are still awaited. On the basis of the benefit observed with targeted agents in patients with advanced-stage disease and driver genetic alterations in their tumors, ongoing trials are evaluating the utility of these targeted agents as adjuvant therapy. Similarly, clinical benefit observed with checkpoint inhibitors has prompted assessment of these drugs in patients with early-stage NSCLC. It is very likely, in the future, that factors other than the anatomy of the tumor will be used to select patients with early-stage NSCLC for systemic therapy and that the choice of systemic therapy will extend beyond platinum-based chemotherapy.

  16. Targeting regulatory T cells to improve vaccine immunogenicity in early life

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    Jorjoh eNdure

    2014-09-01

    Full Text Available Human newborns and infants are bombarded with multiple pathogens on leaving the sterile intra-uterine environment, and yet have suboptimal innate immunity and limited immunological memory, thus leading to increased susceptibility to infections in early life. They are thus the target age group for a host of vaccines against common bacterial and viral pathogens. They are also the target group for many vaccines in development, including those against tuberculosis (TB, malaria and HIV infection. However, neonatal and infant responses to many vaccines are suboptimal, and in the case of the polysaccharide vaccines, it has been necessary to develop the alternative conjugated formulations in order to induce immunity in early life. Immunoregulatory factors are an intrinsic component of natural immunity necessary to dampen or control immune responses, with the caveat that they may also decrease immunity to infections or lead to chronic infection. This review explores the key immunoregulatory factors at play in early life, with a particular emphasis on regulatory T cells (Tregs. It goes on to explore the role that Tregs play in limting vaccine immunogenicity, and describes animal and human studies in which Tregs have been depleted in order to enhance vaccine responses. A deeper understanding of the role that Tregs play in limiting or controlling vaccine induced immunity would provide strategies to improve vaccine immunogenicity in this critical age group. New adjuvants and drugs are being developed that can transiently suppress Treg function, and their use as part of human vaccination strategies against infections is becoming a real prospect for the future.

  17. Early host cell targets of Yersinia pestis during primary pneumonic plague.

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    Roger D Pechous

    Full Text Available Inhalation of Yersinia pestis causes primary pneumonic plague, a highly lethal syndrome with mortality rates approaching 100%. Pneumonic plague progression is biphasic, with an initial pre-inflammatory phase facilitating bacterial growth in the absence of host inflammation, followed by a pro-inflammatory phase marked by extensive neutrophil influx, an inflammatory cytokine storm, and severe tissue destruction. Using a FRET-based probe to quantitate injection of effector proteins by the Y. pestis type III secretion system, we show that these bacteria target alveolar macrophages early during infection of mice, followed by a switch in host cell preference to neutrophils. We also demonstrate that neutrophil influx is unable to limit bacterial growth in the lung and is ultimately responsible for the severe inflammation during the lethal pro-inflammatory phase.

  18. Basal cell carcinoma with halo phenomenon in a young female: Significance of dermatoscopy in early diagnosis

    Directory of Open Access Journals (Sweden)

    Pinar Yuksel Basak

    2015-01-01

    Full Text Available Halo phenomenon of nevus may be observed as a circular reaction, although it is unusual around tumors. A 29-year-old woman presented with a pigmented lesion on the cheek since three years. She noted whitening of the skin around the lesion almost after a year following its appearance. Dermatologic examination revealed a pigmented nodular lesion with a hypopigmented halo on the left infraorbital region. The clinical impression was halo nevus, whereas basal cell carcinoma (BCC was considered in dermatoscopic differential diagnosis. The diagnosis was infiltrative-type BCC histopathologically. The persistence of a perilesional halo around an enlarging pigmented lesion should be carefully examined with accompanying dermatoscopic findings even in young patients for early diagnosis of tumoral lesions.

  19. Proton-Based Stereotactic Ablative Radiotherapy in Early-Stage Non-Small-Cell Lung Cancer

    Science.gov (United States)

    Grant, Jonathan D.; Chang, Joe Y.

    2014-01-01

    Stereotactic ablative radiotherapy (SABR), a recent implementation in the practice of radiation oncology, has been shown to confer high rates of local control in the treatment of early stage non-small-cell lung cancer (NSCLC). This technique, which involves limited invasive procedures and reduced treatment intervals, offers definitive treatment for patients unable or unwilling to undergo an operation. The use of protons in SABR delivery confers the added physical advantage of normal tissue sparing due to the absence of collateral radiation dose delivered to regions distal to the target. This may translate into clinical benefit and a decreased risk of clinical toxicity in patients with nearby critical structures or limited pulmonary reserve. In this review, we present the rationale for proton-based SABR, principles relating to the delivery and planning of this modality, and a summary of published clinical studies. PMID:25136582

  20. Detection of early malignant changes in tissue cultured cells using a novel tumorigenicity assay in nude mice

    Energy Technology Data Exchange (ETDEWEB)

    Wells, R.S.; Campbell, E.W.; Holland, L.M.; Schwartzendruber, D.E.; Kraemer, P.M.

    1979-01-01

    Cultured cells were tested for tumorigenicity in nude mice using a new test involving implantation of cells grown on small gelatin sponges. The test was applied to mouse, hamster, and human cells, and the results were compared to a conventional tumorigenicity assay (injection of cell suspensions). The sponge assay was at least as sensitive as the conventional assay in all cases tested so far. In several instances the sponge assay could detect events not seen in the standard assay. Use of the sponge assay has led to interesting possibilities for studying in vitro and early in vivo cellular changes that may be associated with the ability to form tumors. In studies with two human squamous cell carcinoma cell lines, the two assays yielded comparable results. Such cell lines might provide a system in which the lack of terminal differentiation within a cell population could be examined as a parameter of neoplastic change.

  1. Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients

    Science.gov (United States)

    Gurioli, Giorgia; Martignano, Filippo; Foca, Flavia; Gunelli, Roberta; Cicchetti, Giacomo; De Giorgi, Ugo; Zoli, Wainer; Calistri, Daniele

    2015-01-01

    Introduction. The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract. Patients and Methods. A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group). Prostate-specific antigen (PSA) levels were determined. Urine cell-free (UCF) DNA was isolated and sequences longer than 250 bp corresponding to 3 genes (c-MYC, HER2, and AR) were quantified by Real-Time PCR to assess UCF-DNA integrity. Results. UCF-DNA was quantifiable in all samples, while UCF-DNA integrity was evaluable in all but 16 samples. Receiver operating characteristic analysis showed an area under the curve of 0.5048 for UCF-DNA integrity and 0.8423 for PSA. Sensitivity was 0.58 and 0.95 for UCF-DNA integrity and PSA, respectively. Specificity was 0.44 and 0.69, respectively. Conclusions. UCF-DNA integrity showed lower accuracy than PSA and would not seem to be a reliable marker for early prostate cancer diagnosis. Despite this, we believe that UCF-DNA could represent a source of other biomarkers and could detect gene alterations. PMID:26412928

  2. Drug Repositioning Discovery for Early- and Late-Stage Non-Small-Cell Lung Cancer

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    Chien-Hung Huang

    2014-01-01

    Full Text Available Drug repositioning is a popular approach in the pharmaceutical industry for identifying potential new uses for existing drugs and accelerating the development time. Non-small-cell lung cancer (NSCLC is one of the leading causes of death worldwide. To reduce the biological heterogeneity effects among different individuals, both normal and cancer tissues were taken from the same patient, hence allowing pairwise testing. By comparing early- and late-stage cancer patients, we can identify stage-specific NSCLC genes. Differentially expressed genes are clustered separately to form up- and downregulated communities that are used as queries to perform enrichment analysis. The results suggest that pathways for early- and late-stage cancers are different. Sets of up- and downregulated genes were submitted to the cMap web resource to identify potential drugs. To achieve high confidence drug prediction, multiple microarray experimental results were merged by performing meta-analysis. The results of a few drug findings are supported by MTT assay or clonogenic assay data. In conclusion, we have been able to assess the potential existing drugs to identify novel anticancer drugs, which may be helpful in drug repositioning discovery for NSCLC.

  3. Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

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    Joseph Landry

    2008-10-01

    Full Text Available We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor, the largest subunit of NURF (Nucleosome Remodeling Factor in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/- embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/- embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

  4. Early diagnosis and treatment of renal cell carcinoma of native kidney in kidney transplantation.

    Science.gov (United States)

    García Álvarez, T; Mazuecos Blanca, A; Navas García, N; Calle García, L; Vallejos Roca, E; Moreno Salazar, A; Soto Villalba, J; Collantes Mateos, R; Rivero Sánchez, M

    2011-01-01

    The frecuency of malignancies in renal transplant (RT) patients is increasing. Renal cell carcinoma (RCC) of native kidneys is one of the most frequent and its outcome can be more aggressive than in general population. To evaluate the incidence and prognosis of RCC in renal transplant patients followed in our transplantation unit. Between January 1997 and December 2009, 683 patients underwent kidney transplant at our hospital. Ultrasonography of the native kidneys was annually performed in all renal transplant patients. When suspect solid masses were found at ultrasonography, patients underwent computed tomography. If the suspicion was confirmed, nephrectomy was performed. 14 radical nephrectomies were performed in 12 patients due to suspect CCR. In 11 nephrectomies (corresponding to 9 patients), anatomopathologic diagnosis was CCR (incidence 1.5%). Histologic stage was T1N0M0 in all cases. In the other 3 RT, the diagnosis was complicated renal cyst. Those patients without carcinoma had polycystic kidney disease. The time on dialysis before CCR diagnosis was 36.7 ± 24.3 months and the interval between RT and diagnosis was 39 ± 25.8 months. After a mean follow-up of 58.6 ± 38.6 months, the outcome of all cases has been excellent, without tumor recurrence. Annual renal ultrasonography plays a key role in the early diagnosis of CRR. The early treatment of this pathology is associated with an excellent prognosis in RT patients.

  5. Rapid and sensitive detection of early esophageal squamous cell carcinoma with fluorescence probe targeting dipeptidylpeptidase IV

    Science.gov (United States)

    Onoyama, Haruna; Kamiya, Mako; Kuriki, Yugo; Komatsu, Toru; Abe, Hiroyuki; Tsuji, Yosuke; Yagi, Koichi; Yamagata, Yukinori; Aikou, Susumu; Nishida, Masato; Mori, Kazuhiko; Yamashita, Hiroharu; Fujishiro, Mitsuhiro; Nomura, Sachiyo; Shimizu, Nobuyuki; Fukayama, Masashi; Koike, Kazuhiko; Urano, Yasuteru; Seto, Yasuyuki

    2016-01-01

    Early detection of esophageal squamous cell carcinoma (ESCC) is an important prognosticator, but is difficult to achieve by conventional endoscopy. Conventional lugol chromoendoscopy and equipment-based image-enhanced endoscopy, such as narrow-band imaging (NBI), have various practical limitations. Since fluorescence-based visualization is considered a promising approach, we aimed to develop an activatable fluorescence probe to visualize ESCCs. First, based on the fact that various aminopeptidase activities are elevated in cancer, we screened freshly resected specimens from patients with a series of aminopeptidase-activatable fluorescence probes. The results indicated that dipeptidylpeptidase IV (DPP-IV) is specifically activated in ESCCs, and would be a suitable molecular target for detection of esophageal cancer. Therefore, we designed, synthesized and characterized a series of DPP-IV-activatable fluorescence probes. When the selected probe was topically sprayed onto endoscopic submucosal dissection (ESD) or surgical specimens, tumors were visualized within 5 min, and when the probe was sprayed on biopsy samples, the sensitivity, specificity and accuracy reached 96.9%, 85.7% and 90.5%. We believe that DPP-IV-targeted activatable fluorescence probes are practically translatable as convenient tools for clinical application to enable rapid and accurate diagnosis of early esophageal cancer during endoscopic or surgical procedures. PMID:27245876

  6. Feasibility of using fluorescence in situ hybridization (FISH) to detect early gene changes in sputum cells from uranium miners

    Energy Technology Data Exchange (ETDEWEB)

    Neft, R.E.; Rogers, J.L.; Belinsky, S.A. [and others

    1995-12-01

    Epidemiological studies have shown that combined exposure to radon progeny and tobacco smoke produce a greater than additive or synergistic increase in lung cancer risk. Lung cancer results from multiple genetic changes over a long period of time. An early change that occurs in lung cancer is trisomy 7 which is found in 50% of non-small cell lung cancer and in the far margins of resected lung tumors. The 80% mortality associated with lung cancer is in part related to the high proportion of patients who present with an advanced, unresectable tumor. Therefore, early detection of patients at risk for tumor development is critical to improve treatment of this disease. Currently, it is difficult to detect lung cancer early while it is still amendable by surgery. Saccomanno, G. has shown that premalignant cytologic changes in sputum cells collected from uranium miners can be detected by a skilled, highly trained cytopathologist. A more objective alternative for identifying premalignant cells in sputum may be to determine whether an early genetic change such as trisomy 7 is present in these cells. Fluorescence in situ hybridization (FISH) can be used to identify cells with trisomy 7. The results of this investigation indicate that FISH may prove to be an accurate, efficient method to test at-risk individuals for genetic alterations in bronchial epithelial cells from sputum.

  7. Drosophila caspases involved in developmentally regulated programmed cell death of peptidergic neurons during early metamorphosis.

    Science.gov (United States)

    Lee, Gyunghee; Wang, Zixing; Sehgal, Ritika; Chen, Chun-Hong; Kikuno, Keiko; Hay, Bruce; Park, Jae H

    2011-01-01

    A great number of obsolete larval neurons in the Drosophila central nervous system are eliminated by developmentally programmed cell death (PCD) during early metamorphosis. To elucidate the mechanisms of neuronal PCD occurring during this period, we undertook genetic dissection of seven currently known Drosophila caspases in the PCD of a group of interneurons (vCrz) that produce corazonin (Crz) neuropeptide in the ventral nerve cord. The molecular death program in the vCrz neurons initiates within 1 hour after pupariation, as demonstrated by the cytological signs of cell death and caspase activation. PCD was significantly suppressed in dronc-null mutants, but not in null mutants of either dredd or strica. A double mutation lacking both dronc and strica impaired PCD phenotype more severely than did a dronc mutation alone, but comparably to a triple dredd/strica/dronc mutation, indicating that dronc is a main initiator caspase, while strica plays a minor role that overlaps with dronc's. As for effector caspases, vCrz PCD requires both ice and dcp-1 functions, as they work cooperatively for a timely removal of the vCrz neurons. Interestingly, the activation of the Ice and Dcp-1 is not solely dependent on Dronc and Strica, implying an alternative pathway to activate the effectors. Two remaining effector caspase genes, decay and damm, found no apparent functions in the neuronal PCD, at least during early metamorphosis. Overall, our work revealed that vCrz PCD utilizes dronc, strica, dcp-1, and ice wherein the activation of Ice and Dcp-1 requires a novel pathway in addition to the initiator caspases.

  8. The keys to conservative treatment of early-stage squamous cell carcinoma of the tonsillar region.

    Science.gov (United States)

    Laccourreye, O; Castelnau-Marchand, P; Rubin, F; Badoual, C; Halimi, P; Giraud, P

    2017-09-01

    To analyze the medical literature devoted to work-up, epidemiology, local control, survival, complications and sequelae after conservative treatment for early-stage squamous cell carcinoma of the tonsillar region. An analysis of the PubMed (1975-2016) database was performed using the following keywords and associations: "tonsil/tonsillar region/oropharynx" AND "squamous cell carcinoma" AND "early-stage (I-II; T1-2N0M0)" AND "radiation therapy/radiotherapy" OR "conservative surgery/oropharyngectomy/transoral surgery/radical tonsillectomy". The search retrieved 10 retrospective series documenting local control and/or survival in series with more than 50 cases and a minimum 2 years' follow-up after conservative treatment; no prospective studies, meta-analyses and/or Cochrane analyses were found. Magnetic resonance imaging is the key radiological exam for local extension assessment. Human papilloma virus infection (HPV) is a risk factor that must be screened for systematically, since it induces tumoral radio-sensitivity and increases the risk of specific synchronous and metachronous second primaries. Whatever conservative treatment used, local control and survival rates higher than 85% were achieved. Implementing intensity-modulated radiation therapy reduced the incidence and severity of radiation-related complications and sequelae. Transoral surgery yielded very low morbidity/mortality rates, enabled association to ipsilateral neck dissection, and allowed radiation therapy to be reserved for the management of metachronous second primaries. Transoral surgery appeared to be the first-line option in the majority of cases. Lifetime follow-up adapted to HPV status is mandatory. The development of HPV vaccination does not mean that campaigns against smoking and alcohol abuse are of diminished importance. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  9. Expansion in microcarrier-spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells.

    Science.gov (United States)

    Lin, Youshan Melissa; Lim, Jessica Fang Yan; Lee, Jialing; Choolani, Mahesh; Chan, Jerry Kok Yen; Reuveny, Shaul; Oh, Steve Kah Weng

    2016-06-01

    Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. heMSC expanded on either collagen-coated Cytodex 3 microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100β, MMP13 and ALPL, were investigated and compared in both conditions. BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100β) but not of hypertrophic genes (e.g., MMP13 and ALPL). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights

  10. Early diagnosis of diabetic vascular complications: impairment of red blood cell deformability

    Science.gov (United States)

    Shin, Sehyun; Ku, Yunhee; Park, Cheol-Woo; Suh, Jang-Soo

    2006-02-01

    Reduced deformability of red blood cells (RBCs) may play an important role on the pathogenesis of chronic vascular complications of diabetes mellitus. However, available techniques for measuring RBC deformability often require washing process after each measurement, which is not optimal for day-to-day clinical use at point of care. The objectives of the present study are to develop a device and to delineate the correlation of impaired RBC deformability with diabetic nephropathy. We developed a disposable ektacytometry to measure RBC deformability, which adopted a laser diffraction technique and slit rheometry. The essential features of this design are its simplicity (ease of operation and no moving parts) and a disposable element which is in contact with the blood sample. We studied adult diabetic patients divided into three groups according to diabetic complications. Group I comprised 57 diabetic patients with normal renal function. Group II comprised 26 diabetic patients with chronic renal failure (CRF). Group III consisted of 30 diabetic subjects with end-stage renal disease (ESRD) on hemodialysis. According to the renal function for the diabetic groups, matched non-diabetic groups were served as control. We found substantially impaired red blood cell deformability in those with normal renal function (group I) compared to non-diabetic control (P = 0.0005). As renal function decreases, an increased impairment in RBC deformability was found. Diabetic patients with chronic renal failure (group II) when compared to non-diabetic controls (CRF) had an apparently greater impairment in RBC deformability (P = 0.07). The non-diabetic cohort (CRF), on the other hand, manifested significant impairment in red blood cell deformability compared to healthy control (P = 0.0001). The newly developed slit ektacytometer can measure the RBC deformability with ease and accuracy. In addition, progressive impairment in cell deformability is associated with renal function loss in all

  11. Modeling abnormal early development with induced pluripotent stem cells from aneuploid syndromes.

    Science.gov (United States)

    Li, Wen; Wang, Xianming; Fan, Wenxia; Zhao, Ping; Chan, Yau-Chi; Chen, Shen; Zhang, Shiqiang; Guo, Xiangpeng; Zhang, Ya; Li, Yanhua; Cai, Jinglei; Qin, Dajiang; Li, Xingyan; Yang, Jiayin; Peng, Tianran; Zychlinski, Daniela; Hoffmann, Dirk; Zhang, Ruosi; Deng, Kang; Ng, Kwong-Man; Menten, Bjorn; Zhong, Mei; Wu, Jiayan; Li, Zhiyuan; Chen, Yonglong; Schambach, Axel; Tse, Hung-Fat; Pei, Duanqing; Esteban, Miguel A

    2012-01-01

    Many human diseases share a developmental origin that manifests during childhood or maturity. Aneuploid syndromes are caused by supernumerary or reduced number of chromosomes and represent an extreme example of developmental disease, as they have devastating consequences before and after birth. Investigating how alterations in gene dosage drive these conditions is relevant because it might help treat some clinical aspects. It may also provide explanations as to how quantitative differences in gene expression determine phenotypic diversity and disease susceptibility among natural populations. Here, we aimed to produce induced pluripotent stem cell (iPSC) lines that can be used to improve our understanding of aneuploid syndromes. We have generated iPSCs from monosomy X [Turner syndrome (TS)], trisomy 8 (Warkany syndrome 2), trisomy 13 (Patau syndrome) and partial trisomy 11;22 (Emanuel syndrome), using either skin fibroblasts from affected individuals or amniocytes from antenatal diagnostic tests. These cell lines stably maintain the karyotype of the donors and behave like embryonic stem cells in all tested assays. TS iPSCs were used for further studies including global gene expression analysis and tissue-specific directed differentiation. Multiple clones displayed lower levels of the pseudoautosomal genes ASMTL and PPP2R3B than the controls. Moreover, they could be transformed into neural-like, hepatocyte-like and heart-like cells, but displayed insufficient up-regulation of the pseudoautosomal placental gene CSF2RA during embryoid body formation. These data support that abnormal organogenesis and early lethality in TS are not caused by a tissue-specific differentiation blockade, but rather involves other abnormalities including impaired placentation.

  12. Leukotrienes inhibit early stages of HIV-1 infection in monocyte-derived microglia-like cells

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    Bertin Jonathan

    2012-03-01

    Full Text Available Abstract Background Microglia are one of the main cell types to be productively infected by HIV-1 in the central nervous system (CNS. Leukotriene B4 (LTB4 and cysteinyl-leukotrienes such as LTC4 are some of the proinflammatory molecules produced in infected individuals that contribute to neuroinflammation. We therefore sought to investigate the role of leukotrienes (LTs in HIV-1 infection of microglial cells. Methods To evaluate the role of LTs on HIV-1 infection in the CNS, monocyte-derived microglial-like cells (MDMis were utilized in this study. Leukotriene-treated MDMis were infected with either fully replicative brain-derived HIV-1 isolates (YU2 or R5-tropic luciferase-encoding particles in order to assess viral production and expression. The efficacy of various steps of the replication cycle was evaluated by means of p24 quantification by ELISA, luciferase activity determination and quantitative real-time polymerase chain reaction (RT-PCR. Results We report in this study that virus replication is reduced upon treatment of MDMis with LTB4 and LTC4. Additional experiments indicate that these proinflammatory molecules alter the pH-independent entry and early post-fusion events of the viral life cycle. Indeed, LT treatment induced a diminution in integrated proviral DNA while reverse-transcribed viral products remained unaffected. Furthermore, decreased C-C chemokine receptor type 5 (CCR5 surface expression was observed in LT-treated MDMis. Finally, the effect of LTs on HIV-1 infection in MDMis appears to be mediated partly via a signal transduction pathway involving protein kinase C. Conclusions These data show for the first time that LTs influence microglial cell infection by HIV-1, and may be a factor in the control of viral load in the CNS.

  13. Pesquisaje y dilema del asesoramiento genético en parejas de riesgo de anemia a hematíes falciformes Screening and dilemma of the genetic counselling in couples at risk for sickle cell anemia

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    Mabel Domínguez Mena

    2005-04-01

    Full Text Available Se realizó un estudio descriptivo en la Consulta de Desarrollo de la Ginecología del municipio La Lisa, en el período comprendido de enero de 1999 hasta diciembre de 2003. La muestra la conformaron 338 embarazadas portadoras de anemia a hematíes falciformes, a las que se les brindó asesoramiento genético que incluyó la repetición del estudio de electroforesis de hemoglobina y la realización al esposo. Los datos fueron procesados en el cálculo porcentual. Se detectaron 28parejas de riesgo (7,4 %, de las cuales 21 (75, 0 % optaron por el diagnóstico prenatal de hemoglobina fetal. Se encontraron 4 fetos con anemia a hematíes falciformes (19 % y 9 portadores (42,8 %. No se realizaron diagnóstico prenatal 7 pacientes (25 %, de ellas 5 (71,4 % por edad gestacional avanzada y 2 (28, 5 % por negarse a la realización del proceder médico. El asesoramiento genético fue no directivo, respetando las decisiones personales, confiabilidad, explicando la relación riesgo/beneficio, y obteniendo en todos los casos el consentimiento informado para el diagnóstico prenatal.A descriptive study was undertaken at the Office of Genetics Development of La Lisa municipality from January 1999 to December 2003. The sample was composed of 338 pregnant women carriers of sickle cell anemia that received genetical counselling, which included the conduction of hemoglobin electrophoresis in the expectants and their husbands. The data were processed by percentage calculation. 28 risk couples were detected (7.2 %, of which 21 (75 % chose the prenatal diagnosis of fetal hemoglobin. 4 fetoes with sickle cell anemia (19 % and 9 carriers (42.8 % were found. 7 patients did not have prenatal diganosis (25 %, 5 of them (71.4 % due to advanced gestational age and 2 (28.5 % for rejecting to do so. The genetical counselling was not directive. The personal decisions were respected and reliability was guaranteed. The risk/benefit relation was explained and in all cases the

  14. ¿Cuándo es demasiado pronto y cuándo demasiado tarde para la cirugía en la enfermedad de Crohn? When is it too early or too late for surgery in Crohn's disease?

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    J. I. Fernández-Blanco Hernáiz

    2008-01-01

    Full Text Available El abordaje quirúrgico de la enfermedad de Crohn (EC, entendido como último esfuerzo de tratamiento frente a modalidades en las que la terapia médica ha fracasado, supone una pérdida de perspectiva, que puede posponer el retraso en la recuperación del paciente y le retrae de una mejor calidad de su vida, cuando se considera que un 50% de los pacientes mantienen inactiva su enfermedad durante años tras procedimientos quirúrgicos seleccionados; unas tasas no alcanzadas por los tratamientos más eficaces. El riesgo de precisar algún procedimiento quirúrgico en el curso de la EC alcanza al 75% de sus portadores, más del 50% en el primer año desde el diagnóstico, y prácticamente el 100% de los pacientes en la evolución de su proceso cuando se contemplan lesiones de localización perianal. Es por eso que el gastroenterólogo debe estar entrenado en la selección de quién, cuándo y por qué debe ser operado. Retrasar la cirugía a fases de enfermedad avanzada incrementa la morbilidad y, si es cierto que las actuales terapias médicas permiten una mayor tasa de remisiones, también lo es que incrementar la duración del proceso y la edad de los pacientes contribuye a mayor riesgo quirúrgico y peor perspectiva en el tratamiento de complicaciones agudas y de manifestaciones crónicas de la EC, a menudo clínicamente desconsideradas, como la mesenteritis retráctil, los estados de hipercoagulabilidad y la aparición de fenómenos de malignización. Salvando las indicaciones absolutas de actuación quirúrgica inicial en el manejo de pacientes con EC, como sangrado intestinal masivo, desarrollo de megacolon tóxico o perforación libre; otros condicionantes para el tratamiento quirúrgico deben ser reevaluados a la luz de nuestros conocimientos actuales. La genotipación del paciente constituye un elemento clínico que contribuye a identificar riesgos específicos y facilita la selección terapéutica. Desgraciadamente, hasta que estos an

  15. Identification of long noncoding RNAs for the detection of early stage lung squamous cell carcinoma by microarray analysis.

    Science.gov (United States)

    Cheng, Zule; Bai, Yanan; Wang, Ping; Wu, Zhenhua; Zhou, Lin; Zhong, Ming; Jin, Qinghui; Zhao, Jianlong; Mao, Hailei; Mao, Hongju

    2017-02-21

    The aberrant expressions of long noncoding RNAs have been reported in numerous cancers, which have facilitated the cancer diagnosis. However, the expression profile of lncRNAs in early stage lung squamous cell carcinoma has not been well discussed. The present study aimed to examine the expression profile of lncRNAs in early stage lung squamous cell carcinoma and identify lncRNA biomarkers for diagnosis. Through high-throughput lncRNA microarray, we screened thousands of aberrantly expressed lncRNAs and mRNAs in early stage lung squamous cell carcinoma tissues compared to their corresponding adjacent nontumorous tissues. Bioinformatics analyses were used to investigate the functions of aberrantly expressed mRNAs and their associated lncRNAs. After that, in order to identify lncRNA biomarkers for early detection, candidate lncRNA biomarkers were selected based on our established filtering pipeline and validated by real-time quantitative polymerase chain reaction on a total of 63 pairs of tumor samples. Five lncRNAs were finally identified which were able to distinguish early stage tumor and normal samples with high sensitivity (92%) and specificity (83%). These results imply that lncRNAs may be powerful biomarker for early diagnosis.

  16. Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by Laser Capture Microdissection

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    Bonnet Agnes

    2011-08-01

    Full Text Available Abstract Background Successful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult. The recently developed laser capture microdissection (LCM technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA. Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis. Results We developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15 and three granulosa cell-specific genes (KL, GATA4, AMH. A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte. Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA. Conclusions

  17. Immature MEF2C-dysregulated T-cell leukemia patients have an early T-cell precursor acute lymphoblastic leukemia gene signature and typically have non-rearranged T-cell receptors

    Science.gov (United States)

    Zuurbier, Linda; Gutierrez, Alejandro; Mullighan, Charles G.; Canté-Barrett, Kirsten; Gevaert, A. Olivier; de Rooi, Johan; Li, Yunlei; Smits, Willem K.; Buijs-Gladdines, Jessica G.C.A.M.; Sonneveld, Edwin; Look, A. Thomas; Horstmann, Martin; Pieters, Rob; Meijerink, Jules P.P.

    2014-01-01

    Three distinct immature T-cell acute lymphoblastic leukemia entities have been described including cases that express an early T-cell precursor immunophenotype or expression profile, immature MEF2C-dysregulated T-cell acute lymphoblastic leukemia cluster cases based on gene expression analysis (immature cluster) and cases that retain non-rearranged TRG@ loci. Early T-cell precursor acute lymphoblastic leukemia cases exclusively overlap with immature cluster samples based on the expression of early T-cell precursor acute lymphoblastic leukemia signature genes, indicating that both are featuring a single disease entity. Patients lacking TRG@ rearrangements represent only 40% of immature cluster cases, but no further evidence was found to suggest that cases with absence of bi-allelic TRG@ deletions reflect a distinct and even more immature disease entity. Immature cluster/early T-cell precursor acute lymphoblastic leukemia cases are strongly enriched for genes expressed in hematopoietic stem cells as well as genes expressed in normal early thymocyte progenitor or double negative-2A T-cell subsets. Identification of early T-cell precursor acute lymphoblastic leukemia cases solely by defined immunophenotypic criteria strongly underestimates the number of cases that have a corresponding gene signature. However, early T-cell precursor acute lymphoblastic leukemia samples correlate best with a CD1 negative, CD4 and CD8 double negative immunophenotype with expression of CD34 and/or myeloid markers CD13 or CD33. Unlike various other studies, immature cluster/early T-cell precursor acute lymphoblastic leukemia patients treated on the COALL-97 protocol did not have an overall inferior outcome, and demonstrated equal sensitivity levels to most conventional therapeutic drugs compared to other pediatric T-cell acute lymphoblastic leukemia patients. PMID:23975177

  18. Sustained human hematopoiesis in sheep transplanted in utero during early gestation with fractionated adult human bone marrow cells.

    Science.gov (United States)

    Srour, E F; Zanjani, E D; Brandt, J E; Leemhuis, T; Briddell, R A; Heerema, N A; Hoffman, R

    1992-03-15

    Sheep were transplanted in utero during early gestation with subpopulations of adult human bone marrow (BM) cells enriched for human progenitor and hematopoietic stem cells (HSC). Chimerism was documented in three of seven transplanted fetuses using monoclonal antibodies against human-specific hematopoietic cell lineages and/or cytogenetic analysis of BM and peripheral blood cells of recipients. Only chimeric sheep BM cells expressing CD45 (6.0% of total BM cells) formed human hematopoietic colonies in response to human recombinant cytokines as determined by cytogenetic analysis. Sorted CD45+ BM cells developed human T-cell colonies containing CD3+, CD4+, and CD8+ cells. DNA from chimeric BM cells obtained 3 months after birth displayed a finger printing pattern identical to that of DNA from the human donor of the HSC graft. These studies indicate that first trimester sheep fetuses are tolerant of adult human HSC grafts, thus permitting the creation of xenogeneic chimera expressing human myeloid and lymphoid lineages. The present findings also suggest that HSC grafts from immunologically competent, HLA-mismatched adult donors may be useful for correcting human genetic diseases in utero during early gestation.

  19. Transcriptome analysis of the hormone-sensing cells in mammary epithelial reveals dynamic changes in early pregnancy.

    Science.gov (United States)

    De Silva, Duvini; Kunasegaran, Kamini; Ghosh, Sujoy; Pietersen, Alexandra M

    2015-01-27

    Alveoli, the milk-producing units of the mammary gland, are generated during pregnancy by collaboration of different epithelial cell types. We present the first analysis of transcriptional changes within the hormone sensing population during pregnancy. Hormone-receptor positive (HR+) cells play a key role in the initiation of alveologenesis as they sense systemic hormonal changes and translate these into local instructions for neighboring HR- cells. We recently showed that IGF2 is produced specifically by HR+ cells in early pregnancy, but is undetectable in the virgin state. Here, we define the transcriptome of HR+ cells in early pregnancy with the aim to elucidate additional changes that are unique for this dynamic developmental time window. We harvested mammary glands from virgin, 3-day and 7-day pregnant mice and isolated a few hundred hormone-sensing cells per animal by FACS for microarray analysis. There was a high concordance between animals with a clear induction of cell cycle progression genes at day 3 of pregnancy and molecules involved in paracrine signalling at day 7. These findings underscore the proliferative capacity of HR+ cells upon specific stimuli and elucidate developmentally-restricted changes in cellular communication. Since the majority of breast cancers are HR+, with a variable proportion of HR+ cells per tumor, we anticipate that this data set will aid further studies into the regulation of HR+ cell proliferation and the role of heterotypic signalling within tumors.

  20. Sepúlveda es arte

    OpenAIRE

    García Poza, Almudena

    2017-01-01

    Uno de los principales contenidos en la formación y educación de los niños, es el conocimiento de su pasado más cercano. Partiendo de esta idea fundamental es casi una exigencia como docentes, ofrecer a los alumnos los contenidos y las herramientas necesarias para que conozcan su pasado histórico y las manifestaciones artísticas que expresaron su cultura. Conocer, comprender y valorar nuestro pasado va a convertir la práctica educativa en un aprendizaje empírico donde los conocimientos tengan...

  1. GABA agonist promoted formation of low affinity GABA receptors on cerebellar granule cells is restricted to early development

    DEFF Research Database (Denmark)

    Belhage, B; Hansen, G H; Schousboe, A

    1988-01-01

    The ability of the GABA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) to promote formation of low affinity GABA receptors on cerebellar granule cells was tested using primary cultures of these neurons. Granule cells were exposed to THIP (150 microM) for 6 hr after, respec...... cells expressed a high density of high affinity GABA receptors. It is concluded that the ability of THIP to promote formation of low affinity GABA receptors on cerebellar granule cells is restricted to an early developmental period....

  2. Imidacloprid Exposure Suppresses Neural Crest Cells Generation during Early Chick Embryo Development.

    Science.gov (United States)

    Wang, Chao-Jie; Wang, Guang; Wang, Xiao-Yu; Liu, Meng; Chuai, Manli; Lee, Kenneth Ka Ho; He, Xiao-Song; Lu, Da-Xiang; Yang, Xuesong

    2016-06-15

    Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

  3. DNA Methylation Profiles of Blood Cells Are Distinct between Early-Onset Obese and Control Individuals

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    Je-Keun Rhee

    2017-03-01

    Full Text Available Obesity is a highly prevalent, chronic disorder that has been increasing in incidence in young patients. Both epigenetic and genetic aberrations may play a role in the pathogenesis of obesity. Therefore, in-depth epigenomic and genomic analyses will advance our understanding of the detailed molecular mechanisms underlying obesity and aid in the selection of potential biomarkers for obesity in youth. Here, we performed microarray-based DNA methylation and gene expression profiling of peripheral white blood cells obtained from six young, obese individuals and six healthy controls. We observed that the hierarchical clustering of DNA methylation, but not gene expression, clearly segregates the obese individuals from the controls, suggesting that the metabolic disturbance that occurs as a result of obesity at a young age may affect the DNA methylation of peripheral blood cells without accompanying transcriptional changes. To examine the genome-wide differences in the DNA methylation profiles of young obese and control individuals, we identified differentially methylated CpG sites and investigated their genomic and epigenomic contexts. The aberrant DNA methylation patterns in obese individuals can be summarized as relative gains and losses of DNA methylation in gene promoters and gene bodies, respectively. We also observed that the CpG islands of obese individuals are more susceptible to DNA methylation compared to controls. Our pilot study suggests that the genome-wide aberrant DNA methylation patterns of obese individuals may advance not only our understanding of the epigenomic pathogenesis but also early screening of obesity in youth.

  4. Data fusion for detection of early-stage lung cancer cells using evidential reasoning

    Science.gov (United States)

    Liu, Lei-Jian; Yang, Jingyu; Lu, Jian

    1993-08-01

    Data fusion has already been widely used in various applications in which multiple sources of information are presented. One of the most widely used applications of data fusion is in the field of object recognition and classification, since it can efficiently improve the accuracy and the ability of fault tolerance. This presentation describes a cytological color image processing system, using data fusion method, developed for the detection of early stage lung cancers used in the health inspection. As most of the existing microscopic diagnostic systems use morphological, textual and gray or color features respectively, which results in the instability of the diagnosis, this system makes use of all these features by fusing multiple classification results obtained using morphological, textual and chromatic features respectively. Data fusion is achieved using Dempster-Shafer's Evidential Reasoning (DSER). In the current system, all the nuclei are first segmented by thresholding in a special color space which is a non-linear transformation of the (R,G,B) color space. Then, using morphological, textual and chromatic features respectively, the segmented cells are classified as normal or abnormal cells. Using DSER, the classification results obtained above are fused into a final result. Finally, a decision strategy based on the fused data is presented to get the final classification results. And experiment results are given to show the feasibility of the data fusion approach proposed here.

  5. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development.

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    Jeongwoo Kwon

    Full Text Available ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10 is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ, and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR, supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development.

  6. Early detection of apoptosis by staining of acid-treated apoptotic cells with FITC-labeled lectin from Narcissus pseudonarcissus.

    Science.gov (United States)

    Heyder, Petra; Gaipl, Udo S; Beyer, Thomas D; Voll, Reinhard E; Kern, Peter M; Stach, Christian; Kalden, Joachim R; Herrmann, Martin

    2003-10-01

    Exposure of anionic phospholipids and modified carbohydrates are main parts of the apoptotic death program. Cells undergoing apoptosis can be identified by various methods, detecting surface changes or modifications of their organelles, respectively. We describe a method for the detection of early apoptosis by staining of cells with fluorescein isothiocyanate (FITC)-labeled lectin from Narcissus pseudonarcissus (NPn). Apoptosis in cells or in cell lines was induced by various stimuli. To detect apoptosis the cells were stained with FITC-labeled lectin of NPn. After a short-term acid treatment they were analyzed by flow cytometry. The instability of the cytoplasmic membrane against acid and the binding of NPn were very early features of apoptotic cell death. The NPn lectin staining procedure detected apoptosis with high sensitivity. The staining was stable for at least 12 h. The method described in this study is suitable for the detection of the very early phases of apoptosis. The NPn lectin staining after short-term acid treatment can, therefore, be added to the list of reliable tools for the research of cell death. Copyright 2003 Wiley-Liss, Inc.

  7. Expressão citofotométrica do marcador CD34 no carcinoma epidermóide de esôfago Citophotometric expression of CD34 in squamous cell carcinoma of the esophagus

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    Olímpia Alves Teixeira Lima

    2007-12-01

    Full Text Available RACIONAL: O câncer de esôfago está entre as seis neoplasias malignas mais comuns do mundo. Devido à sua grande agressividade clínica, o subtipo carcinoma epidermóide constitui um dos tumores de pior prognóstico, com alto índice de morbi-mortalidade. Marcadores de biologia molecular tem sido apontados como forte coadjuvante no diagnóstico e graduação de tumores. A angiogênese, evento essencial para a progressão tumoral, pode ser estudada pelo marcador CD34. OBJETIVO: Determinar por citofotometria, usando o sistema SAMBA 4000, a expressão do marcador CD34 no carcinoma epidermóide de esôfago e, correlacioná-los com dados clínico-patológicos (idade, sexo, grau de diferenciação do tumor, estadio, tamanho, localização, profundidade e acometimento de linfonodos. MÉTODOS: Avaliaram-se 29 amostras teciduais de carcinoma epidermóide de esôfago utilizando-se coloração imunoistoquímica com marcador anti-CD34. A quantificação da expressão deste marcador foi realizada por citometria de imagem, pelo sistema SAMBA 4000 nas variáveis índice de marcagem e densidade óptica. A correlação entre subgrupos e análise estatística dos resultados foi realizada com o programa SPSS. RESULTADOS: A expressão média do marcador CD34 foi de 73,40% + 15,20 no índice de marcagem e 56,10 + 23,54 na densidae óptica. O CD34 não apresenta correlação estatisticamente significativa com as características clínico-histopatológicas estudadas (idade, sexo, grau de diferenciação do tumor, estadio, tamanho, localização, profundidade e acometimento de linfonodos. CONCLUSÃO: O marcador CD34 apresenta expressão no carcinoma epidermóide de esôfago, com maior valor no índice de marcagem em relação à densidade óptica. Ele4 não apresenta correlação com as características clínico-histopatológicas estudadas.BACKGROUND: Esophagus carcinoma is rated among the six more common malignant neoplasias in the world. Due to its aggressive

  8. Transformation by Oncogenic Ras Expands the Early Genomic Response to Transforming Growth Factor β in Intestinal Epithelial Cells

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    Carl E. Allen

    2008-10-01

    Full Text Available A substantial body of evidence implicates TGFβ as a tumor promoter in epithelial cells that have become resistant to its tumor suppressor activity. To better understand early, genome-wide TGFβ responses in cells resistant to growth inhibition by TGFβ, we used microarray analysis in a well-defined cell culture system of sensitive and resistant intestinal epithelial cells. TGFβ-regulated gene expression in TGFβ-growth-sensitive, nontransformed rat intestinal epithelial cells (RIE-1 was compared to expression in TGFβ-growth-resistant RIE cells stably transformed by oncogenic Ras(12V. Treatment of RIE-1 cells with 2 ng/ml TGFβ1 for 1 hour increased the expression of eight gene sequences by 2.6-fold or more, whereas eight were down regulated 2.6-fold. In RIE-Ras(12V cells, 42 gene sequences were upregulated and only 3 were down-regulated. Comparison of RIE and RIE-Ras(12V identified 37 gene sequences as unique, Ras-dependent genomic targets of TGFβ1. TGFβ-regulation of connective tissue growth factor and vascular endothelial growth factor, two genes up-regulated in RIE-Ras cells and previously implicated in tumor promotion, was independently confirmed and further characterized by Northern analysis. Our data indicate that overexpression of oncogenic Ras in intestinal epithelial cells confers a significantly expanded repertoire of robust, early transcriptional responses to TGFβ via signaling pathways yet to be fully elucidated but including the canonical Raf-1/MAPK/Erk pathway. Loss of sensitivity to growth inhibition by TGFβ does not abrogate TGFβ signaling and actually expands the early transcriptional response to TGFβ1. Expression of some of these genes may confer to Ras-transformed cells characteristics favorable for tumor promotion.

  9. Protective effects of curcumin on retinal Müller cell in early diabetic rats

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    Zhong-Fu Zuo

    2013-08-01

    Full Text Available AIM: To explore the effects and potential mechanisms of curcumin on retinal Müller cell in early diabetic rats.METHODS: Diabetic rats were induced by a single intraperitoneal injection of streptozotocin (STZ. Male Sprague-Dawley (SD rats were randomly assigned into 4 groups:control group (naïve SD rats administered with a single intraperitoneal injection of citric buffer, diabetic group (STZ-diabetic rats, dimethyl sulfoxide (DMSO group (diabetic rats intraperitoneally administered with mixture of DMSO and normal saline, once a day and curcumin group (diabetic rats intraperitoneally administered with curcumin, 80mg/kg, once a day. Three months after diabetes onset, malondialdehyde (MDA, indication of oxidative stress level and reduced glutathione (GSH in retina were detected with kits, glial fibrillary acidic protein (GFAP in retina was revealed by immunohistochemistry and Western blot, and retinal glutamine synthetase (GS were observed by Western blot.RESULTS: Compared with control group, retinal MDA was increased, and GSH was decreased in diabetic and DMSO groups (P0.05, respectively. While, retinal MDA and GSH in curcumin group showed no difference compared with control group (P>0.05. Furthermore, up-regulation of retinal GFAP and down-regulation of retinal GS were detected in diabetic and DMSO groups, and no alteration could be observed in curcumin group revealed with Western blot. Compared with control group, retinal Müller cells showed significant increase in GFAP immunochemistry staining in diabetic and DMSO groups. Moreover, GFAP-positive staining was decreased in curcumin group compared with diabetic group.CONCLUSION: Curcumin inhibits diabetic retinal oxidative stress, protects Müller cell, and prevents the down-regulation of GS in diabetic retina. Therefore, curcumin has a therapeutic potential in the treatment of diabetic retinopathy (DR.

  10. Mast cell and eosinophil activation during early phase of grass pollen-induced ocular allergic reaction.

    Science.gov (United States)

    Jedrzejczak-Czechowicz, Monika; Lewandowska-Polak, Anna; Jarzebska, Marzanna; Kowalski, Marek L

    2011-01-01

    Both mast cells and eosinophils were implicated in the pathophysiology of allergic conjunctivitis; however, the potential role of eosinophils in an early phase of allergic reaction has not been elucidated. The aim of this study was to assess the relation between clinical symptoms and sequence of mast cells and eosinophils specific mediators release into tear fluid during conjunctival allergen provocation. Patients with grass pollen rhinoconjunctivitis (n = 38) and healthy volunteers (n = 10) were challenged with increasing doses of allergen applied on the conjunctiva. The clinical symptoms were assessed by clinical score. Tear fluid was collected from 12 patients before provocation, at 20 and 40 minutes after positive response. Tryptase and eosinophil cationic protein (ECP) were measured using UniCap and 15-hydroxyeicosanoid acid (15-HETE) with a specific immunoassay. All allergic patients (but no control subjects) had a positive clinical response to the challenge. In 1 patient symptoms appeared after 50 BU/mL of grass allergen administration, in 3 patients symptoms appeared after 500 BU/mL (7.9% of patients), in 14 patients symptoms appeared after 1600 BU/mL (36.8%), and in 20 patients symptoms appeared after 5000 BU/mL (52.6%). The allergen dose was not correlated with the skin-prick test diameter. The mean tryptase concentration increased at 20 minutes from "nondetectable" to 5.89 ± 1.97 micrograms/L and then decreased to 1.77 ± 1.07 micrograms/L (n = 12; p allergic reaction in conjunctiva and activation of eosinophils is preceded by activation of mast cells.

  11. Comparison of the early response of human embryonic stem cells and human induced pluripotent stem cells to ionizing radiation.

    Science.gov (United States)

    Suchorska, Wiktoria Maria; Augustyniak, Ewelina; Łukjanow, Magdalena

    2017-04-01

    Despite the well-demonstrated efficacy of stem cell (SC) therapy, this approach has a number of key drawbacks. One important concern is the response of pluripotent SCs to treatment with ionizing radiation (IR), given that SCs used in regenerative medicine will eventually be exposed to IR for diagnostic or treatment‑associated purposes. Therefore, the aim of the present study was to examine and compare early IR‑induced responses of pluripotent SCs to assess their radioresistance and radiosensitivity. In the present study, 3 cell lines; human embryonic SCs (hESCs), human induced pluripotent SCs (hiPSCs) and primary human dermal fibroblasts (PHDFs); were exposed to IR at doses ranging from 0 to 15 gray (Gy). Double strand breaks (DSBs), and the gene expression of the following DNA repair genes were analyzed: P53; RAD51; BRCA2; PRKDC; and XRCC4. hiPSCs demonstrated greater radioresistance, as fewer DSBs were identified, compared with hESCs. Both pluripotent SC lines exhibited distinct gene expression profiles in the most common DNA repair genes that are involved in homologous recombination, non‑homologous end‑joining and enhanced DNA damage response following IR exposure. Although hESCs and hiPSCs are equivalent in terms of capacity for pluripotency and differentiation into 3 germ layers, the results of the present study indicate that these 2 types of SCs differ in gene expression following exposure to IR. Consequently, further research is required to determine whether hiPSCs and hESCs are equally safe for application in clinical practice. The present study contributes to a greater understanding of DNA damage response (DDR) mechanisms activated in pluripotent SCs and may aid in the future development of safe SC‑based clinical protocols.

  12. Relationship between numerous mast cells and early follicular development in neonatal MRL/MpJ mouse ovaries.

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    Teppei Nakamura

    Full Text Available In the neonatal mouse ovary, clusters of oocytes called nests break into smaller cysts and subsequently form individual follicles. During this period, we found numerous mast cells in the ovary of MRL/MpJ mice and investigated their appearance and morphology with follicular development. The ovarian mast cells, which were already present at postnatal day 0, tended to localize adjacent to the surface epithelium. Among 11 different mouse strains, MRL/MpJ mice possessed the greatest number of ovarian mast cells. Ovarian mast cells were also found in DBA/1, BALB/c, NZW, and DBA/2 mice but rarely in C57BL/6, NZB, AKR, C3H/He, CBA, and ICR mice. The ovarian mast cells expressed connective tissue mast cell markers, although mast cells around the surface epithelium also expressed a mucosal mast cell marker in MRL/MpJ mice. Some ovarian mast cells migrated into the oocyte nests and directly contacted the compressed and degenerated oocytes. In MRL/MpJ mice, the number of oocytes in the nest was significantly lower than in the other strains, and the number of oocytes showed a positive correlation with the number of ovarian mast cells. The gene expression of a mast cell marker also correlated with the expression of an oocyte nest marker, suggesting a link between the appearance of ovarian ? 4mast cells and early follicular development. Furthermore, the expression of follicle developmental markers was significantly higher in MRL/MpJ mice than in C57BL/6 mice. These results indicate that the appearance of ovarian mast cells is a unique phenotype of neonatal MRL/MpJ mice, and that ovarian mast cells participate in early follicular development, especially nest breakdown.

  13. Los manatíes

    OpenAIRE

    2000-01-01

    Los manatíes pueden vivir en ríos de aguas tranquilas, canales, en bahías de agua salada y en el océano. El manatí de las Indias Occidentales, llamado científicamente Trichechus manatus, habita en la cuenca del Caribe, desde la Florida hasta Brasil.

  14. Colombia es una cosa penetrable

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    Gilberto Loaiza Cano

    2007-10-01

    Full Text Available Colombia es una cosa impenetrable. Raíces de la intolerancia y otros ensayos sobre historia política y vida intelectual. Juan Guillermo Gómez García. Bogotá, Diente de León, 2006, 454 pág.

  15. Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation.

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    Marc Crouzet

    Full Text Available Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (pppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of

  16. Early coagulopathy and metabolic acidosis predict transfusion of packed red blood cells in pediatric trauma patients.

    Science.gov (United States)

    Smith, Shane A; Livingston, Michael H; Merritt, Neil H

    2016-05-01

    Severely injured pediatric trauma patients often present to hospital with early coagulopathy and metabolic acidosis. These derangements are associated with poor outcomes, but it is unclear to what degree they predict transfusion of packed red blood cells (pRBC). We retrospectively identified pediatric trauma patients from a level 1 trauma center from 2006 to 2013. Inclusion criteria were age less than 18years, Injury Severity Score greater than 12, and pRBC transfusion within 24h of admission. We identified 96 pediatric trauma patients who underwent pRBC transfusion within 24h of presentation to hospital. On admission, 43% of these patients had one or more signs of coagulopathy, and 81% had metabolic acidosis. Size of pRBC transfusion in the first 24h ranged from 3 to 177mL/kg (mean 29mL/kg), and nineteen patients (20%) underwent massive transfusion (>40ml/kg in 24h). Univariate analysis indicated that size of pRBC transfusion was associated with initial base excess (r=0.46), international normalized ratio (r=0.35), partial thromboplastin time (r=0.41), fibrinogen (r=0.46), and BIG score (Base deficit, INR, Glasgow Coma Scale (GCS), r=0.36). Platelet count, age, GCS, and direct versus referred presentation were not predictive. Multivariable linear regression confirmed that coagulopathy and metabolic acidosis remained predictive after adjusting for direct versus referred presentation (R(2)=0.30). Early coagulopathy and metabolic acidosis predict size of pRBC transfusion among pediatric trauma patients. Further research is needed to develop massive transfusion protocols and guidelines for activation. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. CHLOROPLAST BIOGENESIS Genes Act Cell and Noncell Autonomously in Early Chloroplast Development1

    Science.gov (United States)

    de la Luz Gutiérrez-Nava, María; Gillmor, C. Stewart; Jiménez, Luis F.; Guevara-García, Arturo; León, Patricia

    2004-01-01

    In order to identify nuclear genes required for early chloroplast development, a collection of photosynthetic pigment mutants of Arabidopsis was assembled and screened for lines with extremely low levels of chlorophyll. Nine chloroplast biogenesis (clb) mutants that affect proplastid growth and thylakoid membrane formation and result in an albino seedling phenotype were identified. These mutations identify six new genes as well as a novel allele of cla1. clb mutants have less than 2% of wild-type chlorophyll levels, and little or no expression of nuclear and plastid-encoded genes required for chloroplast development and function. In all but one mutant, proplastids do not differentiate enough to form elongated stroma thylakoid membranes. Analysis of mutants during embryogenesis allows differentiation between CLB genes that act noncell autonomously, where partial maternal complementation of chloroplast development is observed in embryos, and those that act cell autonomously, where complementation during embryogenesis is not observed. Molecular characterization of the noncell autonomous clb4 mutant established that the CLB4 gene encodes for hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), the next to the last enzyme of the methylerythritol 4-phosphate (MEP) pathway for the synthesis of plastidic isoprenoids. The noncell autonomous nature of the clb4 mutant suggests that products of the MEP pathway can travel between tissues, and provides in vivo evidence that some movement of MEP intermediates exists from the cytoplasm to the plastid. The isolation and characterization of clb mutants represents the first systematic study of genes required for early chloroplast development in Arabidopsis. PMID:15133149

  18. Effect of early chemoradiotherapy in patients with limited stage small cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Ha, In Bong; Jeong, Bae Kwon; Jeong, Ho Jin; Choi, Hoon Sik; Chai, Gyu Young; Kang, Myoung Hee; Kim, Hoon Gu; Lee, Gyeong Won; Na, Jae Beom; Kang, Ki Mun [Gyeongsang National University School of Medicine, Jinju (Korea, Republic of)

    2013-12-15

    We evaluated the effect of early chemoradiotherapy on the treatment of patients with limited stage small cell lung cancer (LS-SCLC). Between January 2006 and December 2011, thirty-one patients with histologically proven LS-SCLC who were treated with two cycles of chemotherapy followed by concurrent chemoradiotherapy and consolidation chemotherapy were retrospectively analyzed. The chemotherapy regimen was composed of etoposide and cisplatin. Thoracic radiotherapy consisted of 50 to 60 Gy (median, 54 Gy) given in 5 to 6.5 weeks. The follow-up period ranged from 5 to 53 months (median, 22 months). After chemoradiotherapy, 35.5% of the patients (11 patients) showed complete response, 61.3% (19 patients) showed partial response, 3.2% (one patient) showed progressive disease, resulting in an overall response rate of 96.8% (30 patients). The 1-, 2-, and 3-year overall survival (OS) rates were 66.5%, 41.0%, and 28.1%, respectively, with a median OS of 21.3 months. The 1-, 2-, and 3-year progression free survival (PFS) rates were 49.8%, 22.8%, and 13.7%, respectively, with median PFS of 12 months. The patterns of failure were: locoregional recurrences in 29.0% (nine patients), distant metastasis in 9.7% (three patients), and both locoregional and distant metastasis in 9.7% (three patients). Grade 3 or 4 toxicities of leukopenia, anemia, and thrombocytopenia were observed in 32.2%, 29.0%, and 25.8%, respectively. Grade 3 radiation esophagitis and radiation pneumonitis were shown in 12.9% and 6.4%, respectively. We conclude that early chemoradiotherapy for LS-SCLC provides feasible and acceptable local control and safety.

  19. Quantification of Maternal Serum Cell-Free Fetal DNA in Early-Onset Preeclampsia

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    Mulan Ren

    2013-04-01

    Full Text Available The aim of this study was to determine whether the increased serum cell-free fetal DNA (cffDNA level of gravidas developed into early-onset preeclampsia (EOPE subsequently in the early second trimesters is related to prenatal screening markers. Serum was collected from 1011 gravidas. The level of cffDNA and prenatal screening markers were analyzed in 20 cases with EOPE and 20 controls. All fetuses were male. The maternal serum cffDNA level was assessed by amplification of the Y chromosome specific gene. Correlations between the variables were examined. (Logged cffDNA in EOPE (median, 3.08; interquartile range, 2.93–3.68 was higher than controls (median, 1.79; interquartile range, 1.46–2.53. The increased level of (logged cffDNA was correlated significantly with the increased human chorionic gonadotropin (HCG level (r = 0.628, p < 0.001. Significant reciprocal correlations between cffDNA and babies’ birth weight as well as gestation weeks at delivery were noted (r = −0.516, p = 0.001; r = −0.623, p < 0.001, respectively. The sensitivity and specificity of cffDNA to discriminate between the EOPE cases and the controls were 90% and 85%, respectively. CffDNA is a potential marker for EOPE, which had a significant reciprocal correlation with babies’ birth weight and gestation weeks at delivery. Moreover, it may help in indicating the underlying hypoxic condition in the placenta.

  20. Inhibition of Rho kinase regulates specification of early differentiation events in P19 embryonal carcinoma stem cells.

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    Roman J Krawetz

    Full Text Available The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK play pivotal roles in cell migration, apoptosis (membrane blebbing, cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined.P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-β and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a 'ground' state after the inhibition had been removed.Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the

  1. Methimazole therapy in Graves' disease influences the abnormal expression of CD69 (early activation antigen) on T cells.

    Science.gov (United States)

    Corrales, J J; López, A; Ciudad, J; Mories, M T; Miralles, J M; Orfao, A

    1997-12-01

    At present, the in vivo response of T, B and natural killer (NK) cells to antithyroid drug therapy remains largely unknown. In the present study, we have prospectively analyzed the in vivo effects of methimazole treatment on a large number of circulating T and NK cell subsets, some of them expressing cell surface activation antigens involved in the very early phase of the immune response, in a group of 17 hyperthyroid, untreated patients with Graves' disease (GD). As one of the first events during T cell activation is the expression of interleukin (IL) receptors, we also studied the binding of IL-2 and IL-6 to T cells. Patients with Graves' disease were sequentially studied at diagnosis/before treatment (day 0) and 7, 14, 30, 90 and 180 days after methimazole therapy. The results were compared with both a group of 19 age- and sex-matched control volunteers and a group of 20 untreated/euthyroid patients with Graves' disease in long-term remission. The combination of flow cytometry and three-color immunofluorescence revealed a clear (P effect of the drug. Expression of the low-affinity receptor for IL-2 (CD25)--another early T cell activation marker--was not altered in Graves' disease, but the binding of IL-2 and IL-6 to T cells exhibited a progressive and parallel increase during the first 30 days of therapy, decreasing thereafter. Our results show that methimazole therapy downregulates the abnormally high expression of the CD69 early activation antigen on T cells, being less effective on inducing changes in other T cell activation markers and in NK cells.

  2. mTORC1 is essential for early steps during Schwann cell differentiation of amniotic fluid stem cells and regulates lipogenic gene expression.

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    Andrea Preitschopf

    Full Text Available Schwann cell development is hallmarked by the induction of a lipogenic profile. Here we used amniotic fluid stem (AFS cells and focused on the mechanisms occurring during early steps of differentiation along the Schwann cell lineage. Therefore, we initiated Schwann cell differentiation in AFS cells and monitored as well as modulated the activity of the mechanistic target of rapamycin (mTOR pathway, the major regulator of anabolic processes. Our results show that mTOR complex 1 (mTORC1 activity is essential for glial marker expression and expression of Sterol Regulatory Element-Binding Protein (SREBP target genes. Moreover, SREBP target gene activation by statin treatment promoted lipogenic gene expression, induced mTORC1 activation and stimulated Schwann cell differentiation. To investigate mTORC1 downstream signaling we expressed a mutant S6K1, which subsequently induced the expression of the Schwann cell marker S100b, but did not affect lipogenic gene expression. This suggests that S6K1 dependent and independent pathways downstream of mTORC1 drive AFS cells to early Schwann cell differentiation and lipogenic gene expression. In conclusion our results propose that future strategies for peripheral nervous system regeneration will depend on ways to efficiently induce the mTORC1 pathway.

  3. FDG small animal PET permits early detection of malignant cells in a xenograft murine model

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    Nanni, Cristina; Spinelli, Antonello; Trespidi, Silvia; Ambrosini, Valentina; Castellucci, Paolo; Farsad, Mohsen; Franchi, Roberto; Fanti, Stefano [Azienda Ospedaliera di Bologna Policlinico S.Orsola-Malpighi, Nuclear Medicine, Bologna (Italy); Leo, Korinne di; Tonelli, Roberto; Pession, Andrea [University of Bologna, Sant' Orsola-Malpighi Hospital, Department of Pediatrics, Bologna (Italy); Pettinato, Cinzia [Azienda Ospedaliera di Bologna Policlinico S.Orsola-Malpighi, Physics, Bologna (Italy); Rubello, Domenico [Ospedale S. Maria della Misericordia, Service of Nuclear Medicine and PET, Rovigo (Italy)

    2007-05-15

    The administration of new anticancer drugs in animal models is the first step from in vitro to in vivo pre-clinical protocols. At this stage it is crucial to ensure that cells are in the logarithmic phase of growth and to avoid vascular impairment, which can cause inhomogeneous distribution of the drug within the tumour and thus lead to bias in the final analysis of efficacy. In subcutaneous xenograft murine models, positivity for cancer is visually recognisable 2-3 weeks after inoculation, when a certain amount of necrosis is usually already present. The aim of this study was to evaluate the accuracy of FDG small animal PET for the early detection of malignant masses in a xenograft murine model of human rhabdomyosarcoma. A second goal was to analyse the metabolic behaviour of this xenograft tumour over time. We studied 23 nude mice, in which 7 x 10{sup 6} rhabdomyosarcoma cells (RH-30 cell line) were injected in the dorsal subcutaneous tissues. Each animal underwent four FDG PET scans (GE, eXplore Vista DR) under gas anaesthesia. The animals were studied 2, 5, 14 and 20 days after inoculation. We administered 20 MBq of FDG via the tail vein. Uptake time was 60 min, and acquisition time, 20 min. Images were reconstructed with OSEM 2D iterative reconstruction and the target to background ratio (TBR) was calculated for each tumour. Normal subcutaneous tissue had a TBR of 0.3. Necrosis was diagnosed when one or more cold areas were present within the mass. All the animals were sacrificed and histology was available to verify PET results. PET results were concordant with the findings of necropsy and histology in all cases. The incidence of the tumour was 69.6% (16/23 animals); seven animals did not develop a malignant mass. Ten of the 23 animals had a positive PET scan 2 days after inoculation. Nine of these ten animals developed a tumour; the remaining animal became negative, at the third scan. The positive predictive value of the early PET scan was 90% (9/10 animals

  4. Determination of Early and Late Endothelial Progenitor Cells in Peripheral Circulation and Their Clinical Association with Coronary Artery Disease

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    Shotoku Tagawa

    2015-01-01

    Full Text Available The clinical implications of early and late endothelial progenitor cells (EPCs in coronary artery disease (CAD remain unclear. We investigated endothelial dysfunction in CAD by simultaneously examining early and late EPC colony formation and gene expression of specific surface markers in EPCs. EPCs were extracted from a total of 83 subjects with (n=47 and without (n=36 CAD. Early and late EPC colonies were formed from mononuclear cells extracted from peripheral blood. We found that fewer early EPC colonies were produced in the CAD group (7.2 ± 3.l/well than those in the control group (12.4 ± 1.4/well, p<0.05, and more late EPC colonies were produced in the CAD group (0.8 ± 0.2/well than those in the control group (0.25 ± 0.02/well, p<0.05. In the CAD group, the relative expression of CD31 and KDR of early and late EPCs was lower than in the control group. These results demonstrate that CAD patients could have increased late EPC density and that early and late EPCs in CAD patients exhibited immature endothelial characteristics. We suggest that changes in EPC colony count and gene expression of endothelial markers may have relation with development of CAD.

  5. Serum IGF-1, IGFBP-3 levels and circulating tumor cells (CTCs) in early breast cancer patients.

    Science.gov (United States)

    Papadakis, Georgios Z; Mavroudis, Dimitrios; Georgoulias, Vasilios; Souglakos, John; Alegakis, Athanasios K; Samonis, George; Bagci, Ulas; Makrigiannakis, Antonis; Zoras, Odysseas

    2017-04-01

    Insulin-like growth factor (IGF)-axis is involved in human oncogenesis and metastasis development for various solid tumors including breast cancer. Aim of this study was to assess the association between IGF-1, IGF-binding protein-3 (IGFBP-3) serum levels and the presence of circulating tumor cells (CTCs) in the peripheral blood of women diagnosed with early breast cancer (EBC), before and after adjuvant chemotherapy. 171 patients with early-stage breast adenocarcinomas were retrospectively evaluated. Immunoradiometric (IRMA) assays were employed for the in-vitro determination of IGF-1 and IGFBP-3 serum levels in blood samples collected after surgical treatment and before initiation of adjuvant chemotherapy. CTCs' presence was assessed through detection of cytokeratin-19 (CK-19) mRNA transcripts using quantitative real time reverse transcription polymerase chain reaction (RT-PCR). IGF-1, IGFBP-3 serum levels were correlated with CTCs' presence before and after adjuvant chemotherapy as well as with tumor characteristics including tumor size, axillary lymph node status, oestrogen (ER)/progestorene (PR) and human epidermural growth factor receptor 2 (HER2) receptor status. Log-rank test was applied to investigate possible association between IGF-1, IGFBP-3 serum levels and disease-free interval (DFI) and overall survival (OS). Before initiation of adjuvant therapy IGF-1, IGFBP-3 serum levels were moderately associated (Spearman's rho=0.361, p<0.001) with each other, while presenting significant differences across age groups (all p values<0.05). IGF-1 serum levels did not correlate with the presence of CTCs before initiation (p=0.558) or after completion (p=0.474) of adjuvant chemotherapy. Similarly, IGFBP-3 serum levels did not show significant association with detectable CTCs either before (p=0.487) or after (p=0.134) completion of adjuvant chemotherapy. There was no statistically significant association between the clinical outcome of patients in terms of DFI, OS

  6. Early Increases in Superantigen-Specific Foxp3+ Regulatory T Cells during Mouse Mammary Tumor Virus Infection▿ †

    Science.gov (United States)

    Cabrera, Gabriel; Burzyn, Dalia; Mundiñano, Juliana; Courreges, M. Cecilia; Camicia, Gabriela; Lorenzo, Daniela; Costa, Héctor; Ross, Susan R.; Nepomnaschy, Irene; Piazzon, Isabel

    2008-01-01

    Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Here, we show in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (SAg)-specific Foxp3+ regulatory T cells (Treg) in Peyer's patches (PP). These increases were shown to be dependent on the presence of dendritic cells. CD4+ CD25+ T cells from the PP of infected mice preferentially suppress the proliferative response of T cells to SAg-expressing antigen-presenting cells ex vivo. We investigated the influence of the depletion of CD25+ cells at different stages of the infection. When CD25+ cells were depleted before MMTV infection, an increase in the number of PP SAg-cognate Foxp3− T cells was found at day 6 of infection. Since the SAg response is associated with viral amplification, the possibility exists that Treg cells attenuate the increase in viral load at the beginning of the infection. In contrast, depletion of CD25+ cells once the initial SAg response has developed caused a lower viral load, suggesting that at later stages Treg cells may favor viral persistence. Thus, our results indicated that Treg cells play an important and complex role during MMTV infection. PMID:18495774

  7. Survival after community diagnosis of early-stage non-small cell lung cancer.

    Science.gov (United States)

    Kanarek, Norma F; Hooker, Craig M; Mathieu, Luckson; Tsai, Hua-Ling; Rudin, Charles M; Herman, James G; Brock, Malcolm V

    2014-05-01

    "Rush to surgery" among patients with worse symptoms, delays related to morbidity, and inclusion of patients with advanced disease in study populations have produced a mixed picture of importance of time to treatment to survival of non-small cell lung cancer. Our objective was to assess the contribution of diagnosis to first surgery interval to survival among patients diagnosed in the community with early-stage non-small cell lung cancer. Patients with early-stage lung cancer (N = 174) at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins who were diagnosed and treated from 2003 to 2009 and followed through 2011 made up a prospective study of overall survival. Diagnosis to first surgery interval was examined overall, as 2 segments (referral interval and treatment interval), as short and longer intervals, and as a continuous variable. The majority of patients were female (55%) and aged more than 65 years (61%). The average mean referral and treatment delays were 61.2 and 5.9 days, respectively. Cox method hazard analysis revealed that older age (years) at diagnosis (hazard ratio [HR], 1.02; 95% confidence interval [CI], 1.00-1.05), stage IIB (HR, 2.17; 95% CI, 1.12-4.21), large (>4 cm) (HR, 3.68; 95% CI, 1.05-12.93) or unknown tumor size (HR, 4.45; 95% CI, 1.21-16.38), and weeks from diagnosis to first surgery interval (HR, 1.04; 95% CI, 1.00-1.09) predicted worse overall survival. The threshold period of less than 42 days from diagnosis to surgery did not reach statistical significance. Patients seem to benefit from rapid reduction of tumor burden with surgery. Reasons for delay were not available. Nevertheless, referral delay experienced in the community is unduly long. In addition to patient choices, an unconscious patient or physician bias that lung cancer is untreatable or an inevitable consequence of smoking may be operating and needs further investigation. Copyright © 2014. Published by Elsevier Inc.

  8. Circulating cell-free DNA-based epigenetic assay can detect early breast cancer.

    Science.gov (United States)

    Uehiro, Natsue; Sato, Fumiaki; Pu, Fengling; Tanaka, Sunao; Kawashima, Masahiro; Kawaguchi, Kosuke; Sugimoto, Masahiro; Saji, Shigehira; Toi, Masakazu

    2016-12-19

    Circulating cell-free DNA (cfDNA) has recently been recognized as a resource for biomarkers of cancer progression, treatment response, and drug resistance. However, few have demonstrated the usefulness of cfDNA for early detection of cancer. Although aberrant DNA methylation in cfDNA has been reported for more than a decade, its diagnostic accuracy remains unsatisfactory for cancer screening. Thus, the aim of the present study was to develop a highly sensitive cfDNA-based system for detection of primary breast cancer (BC) using epigenetic biomarkers and digital PCR technology. Array-based genome-wide DNA methylation analysis was performed using 56 microdissected breast tissue specimens, 34 cell lines, and 29 blood samples from healthy volunteers (HVs). Epigenetic markers for BC detection were selected, and a droplet digital methylation-specific PCR (ddMSP) panel with the selected markers was established. The detection model was constructed by support vector machine and evaluated using cfDNA samples. The methylation array analysis identified 12 novel epigenetic markers (JAK3, RASGRF1, CPXM1, SHF, DNM3, CAV2, HOXA10, B3GNT5, ST3GAL6, DACH1, P2RX3, and chr8:23572595) for detecting BC. We also selected four internal control markers (CREM, GLYATL3, ELMOD3, and KLF9) that were identified as infrequently altered genes using a public database. A ddMSP panel using these 16 markers was developed and detection models were constructed with a training dataset containing cfDNA samples from 80 HVs and 87 cancer patients. The best detection model adopted four methylation markers (RASGRF1, CPXM1, HOXA10, and DACH1) and two parameters (cfDNA concentration and the mean of 12 methylation markers), and, and was validated in an independent dataset of 53 HVs and 58 BC patients. The area under the receiver operating characteristic curve for cancer-normal discrimination was 0.916 and 0.876 in the training and validation dataset, respectively. The sensitivity and the specificity of the

  9. SF2/ASF binding region within JC virus NCCR limits early gene transcription in glial cells.

    Science.gov (United States)

    Uleri, Elena; Regan, Patrick; Dolei, Antonina; Sariyer, Ilker Kudret

    2013-05-14

    Patients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the white matter caused by human neurotropic polyomavirus, JC virus. It is now widely accepted that pathologic strains of JCV shows unique rearrangements consist of deletions and insertions within viral NCCR. While these kinds of rearrangements are related to viral tropism and pathology of the disease, their roles in molecular regulation of JCV gene expression and replication are unclear. We have previously identified SF2/ASF as a negative regulator of JCV gene expression in glial cells. This negative impact of SF2/ASF was dependent on its ability to bind a specific region mapped to the tandem repeat within viral promoter. In this report, functional role of SF2/ASF binding region in viral gene expression and replication was investigated by using deletion mutants of viral regulatory sequences. The second 98-base-pair tandem repeat on Mad1 strain was first mutated by deletion and named Mad1-(1X98). In addition to this mutant, the CR3 region which served the binding side for SF2/ASF was also mutated and named Mad1-ΔCR3 (1X73). Both mutations were tested for SF2/ASF binding by ChIP assay. While SF2/ASF was associated with Mad1-WT and Mad1-(1X98), its interaction was completely abolished on Mad1-ΔCR3 (1X73) construct as expected. Surprisingly, reporter gene analysis of Mad1-(1X98) and Mad1-ΔCR3 (1X73) early promoter sequences showed two and three fold increase in promoter activities, respectively. The impact of "CR3" region on JCV propagation was also tested on the viral background. While replication of Mad1-(1X98) strain in glial cells was similar to Mad1-WT strain, propagation of Mad1-ΔCR3 (1X73) was less productive. Further analysis of the

  10. NICOTINAMIDE IN COMPLEX TREATMENT OF LARGE-PLAQUE PARAPSORIASIS AND EARLY STAGES OF MALIGNANT T-CELL SKIN LYMPHOMAS

    Directory of Open Access Journals (Sweden)

    I. V. Khamaganova

    2014-01-01

    Full Text Available Aim: To assess clinical efficacy of nicotinamide in 2 the treatment of patients with early stages of malignant T-cell skin lymphomas and large-plaque parapsoriasis. Materials and methods: 12 patients with erythematous stage of mycosis fungoides and 14 patients with large-plaque parapsoriasis were treated by nicotinamide 15 mg twice daily during 2 weeks. Treatment cycles were repeated 4–5 times per year; topical therapy was also administrated. Results: Nicotinamide demonstrated high therapeutic effect and good tolerability in patients with early stage of mycosis fungoides and large-plaque parapsoriasis. Stable remission was achieved in 1  woman with malignant T-cell lymphoma and 12  patients with large-plaque parapsoriasis; significant clinical improvement was shown in 8 and 12 patients, respectively. Conclusion: Thus, nicotinamide is recommended for comprehensive treatment of large-plaque parapsoriasis and early stages of mycosis fungoides.

  11. Early Transcriptomic Response to LDL and oxLDL in Human Vascular Smooth Muscle Cells.

    Directory of Open Access Journals (Sweden)

    Salvador Damián-Zamacona

    Full Text Available Although nowadays it is well known that the human transcriptome can importantly vary according to external or environmental condition, the reflection of this concept when studying oxidative stress and its direct relationship with gene expression profiling during the process of atherogenesis has not been thoroughly achieved.The ability to analyze genome-wide gene expression through transcriptomics has shown that the genome responds dynamically to diverse stimuli. Here, we describe the transcriptome of human vascular smooth muscle cells (hVSMC stimulated by native and oxidized low-density lipoprotein (nLDL and oxLDL respectively, with the aim of assessing the early molecular changes that induce a response in this cell type resulting in a transcriptomic transformation. This expression has been demonstrated in atherosclerotic plaques in vivo and in vitro, particularly in the light of the oxidative modification hypothesis of atherosclerosis.Total RNA was isolated with TRIzol reagent (Life Technologies and quality estimated using an Agilent 2100 bioanalyzer. The transcriptome of hVSMC under different experimental conditions (1,5 and 24 hours for nLDL and oxLDL was obtained using the GeneChip Human Gene 1.0 ST (Affymetrix designed to measure gene expression of 28,869 well-annotated genes. A fixed fold-change cut-off corresponding to ± 2 was used to identify genes exhibiting the most significant variation and statistical significance (P< 0.05, and 8 genes validated by qPCR using Taqman probes.10 molecular processes were significantly affected in hVSMC: Apoptosis and cell cycle, extracellular matrix remodeling, DNA repair, cholesterol efflux, cGMP biosynthesis, endocytic mechanisms, calcium homeostasis, redox balance, membrane trafficking and finally, the immune response to inflammation. The evidence we present supporting the hypothesis for the involvement of oxidative modification of several processes and metabolic pathways in atherosclerosis is

  12. Cell-associated HIV DNA measured early during infection has prognostic value independent of serum HIV RNA measured concomitantly

    DEFF Research Database (Denmark)

    Katzenstein, Terese L; Oliveri, Roberto S; Benfield, Thomas

    2002-01-01

    Using data from the Danish AIDS Cohort of HIV-infected homosexual men established in the 1980s, the prognostic value of early HIV DNA loads was evaluated. In addition to DNA measurements, concomitant serum HIV RNA levels, CD4 cell counts and CCR5 genotypes were determined. The patients were divided...

  13. SERA Scenarios of Early Market Fuel Cell Electric Vehicle Introductions: Modeling Framework, Regional Markets, and Station Clustering

    Energy Technology Data Exchange (ETDEWEB)

    Bush, B. [National Renewable Energy Lab. (NREL), Golden, CO (United States); Melaina, M. [National Renewable Energy Lab. (NREL), Golden, CO (United States); Penev, M. [National Renewable Energy Lab. (NREL), Golden, CO (United States); Daniel, W. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2013-09-01

    This report describes the development and analysis of detailed temporal and spatial scenarios for early market hydrogen fueling infrastructure clustering and fuel cell electric vehicle rollout using the Scenario Evaluation, Regionalization and Analysis (SERA) model. The report provides an overview of the SERA scenario development framework and discusses the approach used to develop the nationwidescenario.

  14. Dry Olive Leaf Extract in Combination with Methotrexate Reduces Cell Damage in Early Rheumatoid Arthritis Patients-A Pilot Study.

    Science.gov (United States)

    Čabarkapa, Andrea; Živković, Lada; Borozan, Sunčica; Zlatković-Švenda, Mirjana; Dekanski, Dragana; Jančić, Ivan; Radak-Perović, Marija; Bajić, Vladan; Spremo-Potparević, Biljana

    2016-10-01

    The effects of co-administration of dry olive leaf extract (DOLE) with standard methotrexate (MTX) therapy on the parameters of cell damage and inflammation in patients with early and long-term rheumatoid arthritis (RA) were evaluated at baseline, 3 and 6 weeks. Patients were assigned to groups: the early phase RA group on MTX monotherapy (E MTX), and the two RA groups that received co-treatment with DOLE and MTX: early (E MTX + DOLE) and long-term phase patients (L-t MTX+ DOLE). Baseline values indicated increased parameters of cell damage and disruption of redox balance in all groups. After three weeks the E MTX + DOLE group maintained high catalase activity, exhibited decrease of lipid peroxidation and protein damage indicators-thiols and nitrites, while levels of DNA damage and pro-inflammatory interleukin-6 were significantly reduced. In E MTX group catalase activity remained unaltered while significant lipid peroxidation and DNA damage reductions were seen only after six weeks. L-t MTX + DOLE group showed only modest alterations of cell damage parameters during six weeks. Combined administration of DOLE with MTX contributes to faster reduction of cell damage, restores oxidative balance and improves interleukin-6 suppression during high disease activity in early phase RA, but not in long term patients. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Cpt1a gene expression in peripheral blood mononuclear cells as an early biomarker of diet-related metabolic alterations

    KAUST Repository

    Diaz-Rua, Ruben

    2016-11-23

    Background: Research on biomarkers that provide early information about the development of future metabolic alterations is an emerging discipline. Gene expression analysis in peripheral blood mononuclear cells (PBMC) is a promising tool to identify subjects at risk of developing diet-related diseases.

  16. Early hypergravity exposure effects calbindin-D28k and inositol-3-phosphate expression in Purkinje cells

    NARCIS (Netherlands)

    Bouet, [No Value; Dijk, F; Ijkema-Paassen, J; Wubbels, RJ; van der Want, JJ; Gramsbergen, A

    2005-01-01

    In this study the effects of hypergravity were analyzed on cerebellar Purkinje cells during early development in rats. The cerebellum is a key structure in the control and the adaptation of posture and anti-gravity activities. This holds particularly when external conditions are modified. Three

  17. Early transient presence of implanted bone marrow stem cells reduces lesion size after cerebral ischaemia in adult rats

    NARCIS (Netherlands)

    Keimpema, E.; Fokkens, M. R.; Nagy, Z.; Agoston, V.; Luiten, P. G. M.; Nyakas, C.; Boddeke, H. W. G. M.; Copray, J. C. V. M.

    Aims: Previous studies on the therapeutic time window for intravascular administration of bone marrow stem cells (BMSCs) after stroke have shown that early intervention (from 3 h after onset) in the middle cerebral artery occlusion (MCAO) rat model is the most effective approach to reduce ischaemic

  18. Endoscopic radiofrequency ablation for early esophageal squamous cell neoplasia: report of safety and effectiveness from a large prospective trial

    NARCIS (Netherlands)

    He, Shun; Bergman, Jacques; Zhang, Yueming; Weusten, Bas; Xue, Liyan; Qin, Xiumin; Dou, Lizhou; Liu, Yong; Fleischer, David; Lu, Ning; Dawsey, Sanford M.; Wang, Gui-Qi

    2015-01-01

    Endoscopic radiofrequency ablation (RFA) is an established therapy for Barrett's esophagus. Preliminary reports, limited by low patient numbers, also suggest a possible role for RFA in early esophageal squamous cell neoplasia (ESCN). The aim of this study was to evaluate the safety and effectiveness

  19. Physiology: Capacity for Hormone Production of Cultured Trophoblast Cells Obtained from Placentae at Term and in Early Pregnancy

    OpenAIRE

    Malek, Antoine; Willi, Amara; Müller, Jürg; Sager, Ruth; Hänggi, Willy; Bersinger, Nick

    2001-01-01

    Problem: There is an increased doubt about the identity of isolated cytotrophoblast cells at term. Therefore, we compared pregnancy serum levels of three hormones [human placental lactogen (hPL), human chorionic gonadotropin (hCG), and leptin] with the capacity for hormone production of early placentae [EP; 8–13 weeks of gestation (WG)] and term placentae (TP; 38–42 WG).

  20. Early exposure to interleukin-21 limits rapidly generated anti-Epstein-Barr virus T-cell line differentiation.

    Science.gov (United States)

    Orio, Julie; Carli, Cédric; Janelle, Valérie; Giroux, Martin; Taillefer, Julie; Goupil, Mathieu; Richaud, Manon; Roy, Denis-Claude; Delisle, Jean-Sébastien

    2015-04-01

    The adoptive transfer of ex vivo-expanded Epstein-Barr virus (EBV)-specific T-cell lines is an attractive strategy to treat EBV-related neoplasms. Current evidence suggests that for adoptive immunotherapy in general, clinical responses are superior if the transferred cells have not reached a late or terminal effector differentiation phenotype before infusion. The cytokine interleukin (IL)-21 has shown great promise at limiting late T-cell differentiation in vitro, but this remains to be demonstrated in anti-viral T-cell lines. We adapted a clinically validated protocol to rapidly generate EBV-specific T-cell lines in 12 to 14 days and tested whether the addition of IL-21 at the initiation of the culture would affect T-cell expansion and differentiation. We generated clinical-scale EBV-restricted T-cell line expansion with balanced T-cell subset ratios. The addition of IL-21 at the beginning of the culture decreased both T-cell expansion and effector memory T-cell accumulation, with a relative increase in less-differentiated T cells. Within CD4 T-cell subsets, exogenous IL-21 was notably associated with the cell surface expression of CD27 and high KLF2 transcript levels, further arguing for a role of IL-21 in the control of late T-cell differentiation. Our results show that IL-21 has profound effects on T-cell differentiation in a rapid T-cell line generation protocol and as such should be further explored as a novel approach to program anti-viral T cells with features associated with early differentiation and optimal therapeutic efficacy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  1. Flavopiridol causes early mitochondrial damage in chronic lymphocytic leukemia cells with impaired oxygen consumption and mobilization of intracellular calcium.

    Science.gov (United States)

    Hussain, Syed-Rehan A; Lucas, David M; Johnson, Amy J; Lin, Thomas S; Bakaletz, Alan P; Dang, Vinh X; Viatchenko-Karpinski, Serge; Ruppert, Amy S; Byrd, John C; Kuppusamy, Periannan; Crouser, Elliott D; Grever, Michael R

    2008-03-15

    Effective administration of flavopiridol in advanced-stage chronic lymphocytic leukemia (CLL) is often associated with early biochemical evidence of tumor cell lysis. Previous work using other cell types showed that flavopiridol impacts mitochondria, and in CLL cells flavopiridol down-regulates the mitochondrial protein Mcl-1. We therefore investigated mitochondrial structure and function in flavopiridol-treated CLL patient cells and in the lymphoblastic cell line 697 using concentrations and times at which tumor lysis is observed in treated patients. Mitochondrial membrane depolarization was detected in flavopiridol-treated CLL cells by 6 hours, well before the onset of cell death. Flavopiridol-induced mitochondrial depolarization was not blocked by caspase inhibitors or by the calcium chelator EGTA, but was reduced by Bcl-2 overexpression. Intracellular calcium mobilization was noted at early time points using fluorescence microscopy. Furthermore, electron paramagnetic resonance oximetry showed a gradual but significant reduction in cellular oxygen consumption rate by 6 hours, corresponding with ultrastructural mitochondrial damage detected by electron microscopy. These observations suggest that in CLL and 697 cells, flavopiridol mediates its cytotoxic effects via induction of the mitochondrial permeability transition and changes in intracellular calcium.

  2. Cholinergic intrapancreatic neurons induce Ca²+ signaling and early-response gene expression in pancreatic acinar cells.

    Science.gov (United States)

    Turner, D J; cowles, R A; Segura, B J; Romanchuk, G; Barnhart, D C; Mulholland, M W

    2000-01-01

    Pancreatic exocrine function has been demonstrated to be under neuronal regulation. The pathways responsible for this effect, and the long-term consequences of such interactions, are incompletely described. The effects of neuronal depolarization on pancreatic acinar cells were studied to determine whether calcium signaling and c-fos expression were activated. In pancreatic lobules, which contain both neurons and acinar cells, agonists that selectively stimulated neurons increased intracellular calcium in acinar cells. Depolarization also led to the expression of c-fos protein in 24% +/- 4% of the acinar cells. In AR42J pancreatic acinar cells, cholinergic stimulation demonstrated an average increase of 398 +/- 19 nmol/L in intracellular calcium levels, and induced c-fos expression that was time and dose dependent. The data indicate that intrapancreatic neurons induce Ca²+ signaling and early-response gene expression in pancreatic acinar cells.

  3. Transcriptional activation of immediate-early gene ETR101 by human T-cell leukaemia virus type I Tax

    DEFF Research Database (Denmark)

    Chen, Li; Ma, Shiliang; Li, Bo

    2003-01-01

    Human T-cell leukaemia virus type I (HTLV-I) Tax regulates viral and cellular gene expression through interactions with multiple cellular transcription pathways. This study describes the finding of immediate-early gene ETR101 expression in HTLV-I-infected cells and its regulation by Tax. ETR101...... was persistently expressed in HTLV-I-infected cells but not in HTLV-I uninfected cells. Expression of ETR101 was dependent upon Tax expression in the inducible Tax-expressing cell line JPX-9 and also in Jurkat cells transiently transfected with Tax-expressing vectors. Tax transactivated the ETR101 gene promoter...... in a transient transfection assay. A series of deletion and mutation analyses of the ETR101 gene promoter indicated that a 35 bp region immediately upstream of the TATA-box sequence, which contains a consensus cAMP response element (CRE) and a G+C-rich sequence, is the critical responsive element for Tax...

  4. Early apoptosis and cell death induced by ATX-S10Na (II)-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells.

    Science.gov (United States)

    Mitsunaga, Makoto; Tsubota, Akihito; Nariai, Kohichi; Namiki, Yoshihisa; Sumi, Makoto; Yoshikawa, Tetsuya; Fujise, Kiyotaka

    2007-02-07

    To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na (II). HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin V assays, transmission electron microscopy assays, caspase assays and western blotting. Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-x(L), were decreased in Bax- and p53-independent manner. Our results indicate that early apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizing photosensitizer ATX-S10Na (II) are mediated by p53-Bax network and low levels of Bcl-2 and Bcl-x(L) proteins. Our results might help in formulating new therapeutic approaches in photodynamic therapy.

  5. Innate Lymphoid Cells Are the Predominant Source of IL-17A during the Early Pathogenesis of Acute Respiratory Distress Syndrome.

    Science.gov (United States)

    Muir, Roshell; Osbourn, Megan; Dubois, Alice V; Doran, Emma; Small, Donna M; Monahan, Avril; O'Kane, Cecilia M; McAllister, Katherine; Fitzgerald, Denise C; Kissenpfennig, Adrien; McAuley, Daniel F; Ingram, Rebecca J

    2016-02-15

    IL-17A is purported to help drive early pathogenesis in acute respiratory distress syndrome (ARDS) by enhancing neutrophil recruitment. Although IL-17A is the archetypal cytokine of T-helper 17 cells, it is produced by a number of lymphocytes, the source during ARDS being unknown. To identify the cellular source and the role of IL-17A in the early phase of lung injury. Lung injury was induced in wild-type (C57BL/6) and IL-17 knockout (KO) mice with aerosolized LPS (100 μg) or Pseudomonas aeruginosa infection. Detailed phenotyping of the cells expressing RORγt, the transcriptional regulator of IL-17 production, in the mouse lung at 24 hours was performed by flow cytometry. A 100-fold reduction in neutrophil infiltration was observed in the lungs of the IL-17A KO compared with wild-type mice. The majority of RORγt(+) cells in the mouse lung were the recently identified group 3 innate lymphoid cells (ILC3s). Detailed characterization revealed these pulmonary ILC3s (pILC3s) to be discrete from those described in the gut. The critical role of these cells was verified by inducing injury in recombinase-activating gene 2 KO mice, which lack T cells but retain innate lymphoid cells. No amelioration of pathology was observed in the recombinase-activating gene 2 KO mice. IL-17 is rapidly produced during lung injury and significantly contributes to early immunopathogenesis. This is orchestrated largely by a distinct population of pILC3s. Modulation of the activity of pILC3s may potentiate early control of the inflammatory dysregulation seen in ARDS, opening up new therapeutic targets.

  6. Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

    Directory of Open Access Journals (Sweden)

    Vivas Yurena

    2011-12-01

    Full Text Available Abstract Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation.

  7. Visualising impregnated chitosan in Pinus radiata early wood cells using light and scanning electron microscopy.

    Science.gov (United States)

    Singh, Adya P; Singh, Tripti; Rickard, Catherine L

    2010-04-01

    Chitosan, a deacetylated product of an abundant naturally occurring biopolymer chitin, has been used in a range of applications, particularly in food and health areas, as an antimicrobial agent. In the work reported here Pinus radiata wood was impregnated with chitosan as an environmentally compatible organic biocide (Eikenes et al., 2005a,b) to protect wood against wood deteriorating microorganisms and to thus prolong the service life of wooden products. We developed sample preparation techniques targeted to visualise impregnated chitosan within wood tissues using light microscope and field-emission scanning electron microscope (FE-SEM). Sections were viewed with the light microscope without staining with a dye as well as after staining with the dye toluidine blue. Light microscopy was also undertaken on sections that had been stained with 1% aqueous osmium tetroxide (OsO(4)). For SEM observations, the sections were treated with OsO(4) and then examined with the FE-SEM, first in the secondary electron imaging mode (SEI) and then in the backscattered electron imaging (BEI) mode, imaging the same areas of a section in both SEI and BEI modes. The preparation techniques employed and the combined use of light and scanning electron microscopy provided valuable complementary information, revealing that chitosan had penetrated into the cavities (cell lumens, intercellular spaces) of all sizes present within wood tissues and had also impregnated early wood cell walls. The information obtained is discussed in relation to its importance in further development of chitosan formulations and refinement of impregnation technologies to optimise chitosan impregnation into and distribution within wood tissues as well as in assessing chitosan efficacy. Copyright 2009 Elsevier Ltd. All rights reserved.

  8. Peculiarities of death and regeneration of pancreas cells at early stages of alcoholic chronic pancreatitis

    Directory of Open Access Journals (Sweden)

    N. Y. Oshmyanska

    2014-10-01

    Full Text Available The study has been conducted on 39 white laboratory male rats which formed 5 groups: experimental occlusal pancreatitis caused by ligation of the main pancreatic duct (n = 6, experimental alcoholic pancreatitis caused by oral intake of alcohol (n = 6, against the background of an excess (n = 6 or deficiency (n = 6 of nitric oxide, as well as a control group (n = 15. This study provides the detailed description of the processes of death and regeneration in the islets of Langerhans, typical for early stages of the disease. The expression of the proliferation markers (PCNA and Neurogenin-3 has been analyzed using histological and immunohistochemical methods along with the changes of morphological structure, that led to initiation of the alcoholic chronic pancreatitis against the background of imbalance in NO-ergic regulatory system caused by an excess or deficiency of nitric oxide. It has been found that ligation of the pancreatic duct in the experiment reconstructedthe circumstances of chronic pancreatitis in rats and caused the activation of fibrosis and regeneration of endocrine and exocrine tissue. Compared with occlusion, the effects of ethanol on the pancreas also manifested in the activation of fibrogenesis, but the structural changes were negligible and could unlikely lead to advanced fibrosis and chronic pancreatitis in the future. On the other side, an imbalance of NO-system in alcoholic rats leads to disruption of the zymogens secretion in the acinar cells and dilatation of the capillary network in islets. Uneven distribution of zymogen granules may lead to their intracellular activation as evidenced by the deformation of acini and focal apoptosis without inflammatory response. In this case, violation of the key adaptive responses in the pancreas makes it more vulnerable to the effects of ethanol, its metabolites, and other environmental factors, and may increase the probability of chronic pancreatitis development. At the same time

  9. Early lymphocyte recovery as a predictor of outcome, including relapse, after hematopoieticstem cell transplantation

    Directory of Open Access Journals (Sweden)

    Juliane Morando

    2012-01-01

    Full Text Available BACKGROUND: Despite advances in the treatment of acute leukemia, many patients need to undergo hematopoietic stem cell transplantation. Recent studies show that early lymphocyte recovery may be a predictor of relapse and survival in these patients. OBJECTIVE: To analyze the influence of lymphocyte recovery on Days +30 and +100 post-transplant on the occurrence of relapse and survival. METHODS: A descriptive, retrospective study was performed of 137 under 21-year-old patients who were submitted to hematopoietic stem cell transplantation for acute leukemia between 1995 and 2008. A lymphocyte count 0.3 x 10(9/L were considered adequate. Lymphocyte recovery was also analyzed on Day +100 with < 0.75 x 10(9/Land < 0.75 x 10(9/L being considered inadequate and adequate lymphocyte recovery, respectively. RESULTS: There was no significant difference in the occurrence of relapse between patients with inadequate and adequate lymphocyte recovery on Day +30 post-transplant. However, the transplant-related mortality was significantly higher in patients with inadequate recovery on Day +30. Patients with inadequate lymphocyte recovery on Day +30 had worse overall survival and relapse-free survival than patients with adequate recovery. There was no significant difference in the occurrence of infections and acute or chronic graft-versus-host disease. Patients with inadequate lymphocyte recovery on Day +100 had worse overall survival and relapse-free survival and a higher cumulative incidence of relapse. CONCLUSION: The evaluation of lymphocyte recovery on Day +30 is not a good predictor of relapse after transplant however patients with inadequate lymphocyte recovery had worse overall survival and relapse-free survival. Inadequate lymphocyte recovery on Day +100 is correlated with higher cumulative relapse as well as lower overall survival and relapse-free survival.

  10. MF59 Mediates Its B Cell Adjuvanticity by Promoting T Follicular Helper Cells and Thus Germinal Center Responses in Adult and Early Life.

    Science.gov (United States)

    Mastelic Gavillet, Beatris; Eberhardt, Christiane S; Auderset, Floriane; Castellino, Flora; Seubert, Anja; Tregoning, John S; Lambert, Paul-Henri; de Gregorio, Ennio; Del Giudice, Giuseppe; Siegrist, Claire-Anne

    2015-05-15

    The early life influenza disease burden calls for more effective vaccines to protect this vulnerable population. Influenza vaccines including the MF59 oil-in-water adjuvant induce higher, broader, and more persistent Ab responses in adults and particularly in young, through yet undefined mechanisms. In this study, we show that MF59 enhances adult murine IgG responses to influenza hemagglutinin (HA) by promoting a potent T follicular helper cells (TFH) response, which directly controls the magnitude of the germinal center (GC) B cell response. Remarkably, this enhancement of TFH and GC B cells is already fully functional in 3-wk-old infant mice, which were fully protected by HA/MF59 but not HA/PBS immunization against intranasal challenge with the homologous H1N1 (A/California/7/2009) strain. In 1-wk-old neonatal mice, MF59 recruits and activates APCs, efficiently induces CD4(+) effector T cells and primes for enhanced infant responses but induces few fully functional TFH cells, which are mostly follicular regulatory T cells, and poor GC and anti-HA responses. The B cell adjuvanticity of MF59 appears to be mediated by the potent induction of TFH cells which directly controls GC responses both in adult and early life, calling for studies assessing its capacity to enhance the efficacy of influenza immunization in young infants. Copyright © 2015 by The American Association of Immunologists, Inc.

  11. Gastrointestinal dysfunction induced by early weaning is attenuated by delayed weaning and mast cell blockade in pigs.

    Science.gov (United States)

    Moeser, Adam J; Ryan, Kathleen A; Nighot, Prashant K; Blikslager, Anthony T

    2007-08-01

    Our previous work has demonstrated that weaning at 19 days of age has deleterious effects on mucosal barrier function in piglet intestine that are mediated through peripheral CRF receptor signaling pathways. The objectives of the present study were to assess the impact of piglet age on weaning-associated intestinal dysfunction and to determine the role that mast cells play in weaning-induced breakdown of mucosal barrier function. Nursing Yorkshire-cross piglets were either weaned at 19 days of age (early-weaned, n = 8) or 28 days of age (late-weaned, n = 8) and housed in nursery pens. Twenty-four hours postweaning, segments of midjejunum and ascending colon from piglets within each weaning age group were harvested and mounted on Ussing chambers for measurements of transepithelial electrical resistance and serosal-to-mucosal [(3)H]mannitol fluxes. Early weaning resulted in reductions in transepithelial electrical resistance and increases in mucosal permeability to [(3)H]mannitol in the jejunum and colon (P piglets weaned at 28 days of age. Early-weaned piglet intestinal mucosa had increased expression of CRF receptor 1 protein, increased mucosal mast cell tryptase levels, and evidence of enhanced mast cell degranulation compared with late-weaned intestinal mucosa. Pretreatment of piglets with the mast cell stabilizer drug cromolyn, injected intraperitoneally 30 min prior to weaning, abolished the early-weaning-induced intestinal barrier disturbances. Our results indicate that early-weaning stress induces mucosal dysfunction mediated by intestinal mast cell activation and can be prevented by delaying weaning.

  12. FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

    Energy Technology Data Exchange (ETDEWEB)

    Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki, E-mail: yasukiishizaki@gunma-u.ac.jp

    2015-08-07

    The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytes and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture.

  13. Decreased SAP expression in T cells from patients with SLE contributes to early signaling abnormalities and reduced IL-2 production

    Science.gov (United States)

    Karampetsou, Maria P.; Comte, Denis; Kis-Toth, Katalin; Terhorst, Cox; Kyttaris, Vasileios C.; Tsokos, George C.

    2016-01-01

    T cells from patients with systemic lupus erythematosus (SLE) display a number of functions including increased early signaling events following engagement of the T cell receptor (TCR). Signaling lymphocytic activation molecule family (SLAMF) cell surface receptors and the X-chromosome-defined signaling lymphocytic activation molecule-associated protein (SAP) adaptor are important in the development of several immunocyte lineages and modulating immune response. Here we present evidence that SAP protein levels are decreased in T cells and in their main subsets isolated from 32 women and 3 men with SLE independently of disease activity. In SLE T cells the SAP protein is also subject to increased degradation by a caspase-3. Forced expression of SAP in SLE T cells simultaneously heightened IL-2 production, calcium (Ca2+) responses and tyrosine phosphorylation of a number of proteins. Exposure of normal T cells to SLE serum IgG, known to contain anti-CD3/TCR antibodies, resulted in SAP downregulation. We conclude that SLE T cells display reduced levels of the adaptor protein SAP probably as a result of continuous T cell activation and degradation by caspase-3. Restoration of SAP levels in SLE T cells corrects the overexcitable lupus T cell phenotype. PMID:27183584

  14. Targeting of Cancer Stem Cells and Their Microenvironment in Early-Stage MutantK-ras Lung Cancer

    Science.gov (United States)

    2015-10-01

    cancer stem cells of non-small cell lung cancer with IIWR-1, a Wnt and tankyrase inhibitor , and 6-thio-2’deoxyguanosine, a nucleoside-based telomerase ...cyclopamine 200 nM (a potent SMO inhibitor ) and the anti-SHH/IHH 5E1 (2, 3) monoclonal antibody. The inhibitory activity of 5E1 suggests that the... inhibitor in early stage human non-small cell lung cancer. Role: Co-investigator 3. P50-CA70907 (PI: John Minna) 7/1/2014-6/30/2019 0.0 cal months

  15. Long term results of sentinel lymph node biopsy in early oral squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Dequanter D

    2013-06-01

    Full Text Available Didier Dequanter, Mohammad Shahla, Pascal Paulus, Philippe Lothaire Civil Hospital of Charleroi (site Vésale, Montigny le Tilleul, Belgium Abstract: The objective of the study was to evaluate the long term results of the sentinel node (SN biopsy technique in the management of the clinically negative (N0 neck in patients with early oral squamous cell carcinoma (T1–T2. Patients with positive SN underwent neck dissection. A sentinel lymph node (SLN biopsy was performed on 31 consecutive patients. Six of the 31 patients were upstaged by the results of the SLN biopsy. The SLN biopsy allowed the identification of node metastasis in 100% of the cases with a sensitivity of 100%, specificity of 100%, and negative predictive value of 100%. There was a mean follow-up of 59 months. The neck control rate was 100% in the SLN negative group and two SLN positive patients developed subsequent neck disease (neck control rate of 88%. One SLN patient presented at the follow-up with a second primary tumor, 18 months later treated successfully by chemoradiotherapy. The overall survival rate was 100% in both groups. The promising reported short-term results have been sustained by long term follow-up. Patients with negative SLN achieved an excellent neck control rate. The neck control rate in SN negative patients was superior to that in SLN positive patients, but not statistically different. Keywords: sentinel lymph node, characteristics of patients, head and neck cancer

  16. Epigenetic modulations in early endothelial cells and DNA hypermethylation in human skin after sulfur mustard exposure.

    Science.gov (United States)

    Steinritz, Dirk; Schmidt, Annette; Balszuweit, Frank; Thiermann, Horst; Simons, Thilo; Striepling, Enno; Bölck, Birgit; Bloch, Wilhelm

    2016-02-26

    Victims that were exposed to the chemical warfare agent sulfur mustard (SM) suffer from chronic dermal and ocular lesions, severe pulmonary problems and cancer development. It has been proposed that epigenetic perturbations might be involved in that process but this has not been investigated so far. In this study, we investigated epigenetic modulations in vitro using early endothelial cells (EEC) that were exposed to different SM concentrations (0.5, 1.0, 23.5 and 50μM). A comprehensive analysis of 78 genes related to epigenetic pathways (i.e., DNA-methylation and post-translational histone modifications) was performed. Moreover, we analyzed global DNA methylation in vitro in EEC after SM exposure as a maker for epigenetic modulations and in vivo using human skin samples that were obtained from a patient 1 year after an accidently exposure to pure SM. SM exposure resulted in a complex regulation pattern of epigenetic modulators which was accompanied by a global increase of DNA methylation in vitro. Examination of the SM exposed human skin samples also revealed a significant increase of global DNA methylation in vivo, underlining the biological relevance of our findings. Thus, we demonstrated for the first time that SM affects epigenetic pathways and causes epigenetic modulations both in vivo and in vitro. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Induction of cell-mediated immunity during early stages of infection with intracellular protozoa

    Directory of Open Access Journals (Sweden)

    Gazzinelli R.T.

    1998-01-01

    Full Text Available Toxoplasma gondii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI maintained by Th1 lymphocytes and IFN-g. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host. Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.

  18. Has lymphography a role in early stage testicular germ cell tumours?

    Energy Technology Data Exchange (ETDEWEB)

    Stephenson, N.J.H. [Royal Melbourne Hospital, Parkville, VIC (Australia). Dept. of Nuclear Medicine; Sandeman, T.F.; McKenzie, A.F. [Peter MacCallum Cancer Inst., Melbourne, VIC (Australia). Depts. of Radiation Oncology and Diagnostic Imaging

    1995-02-01

    A retrospective study was performed of 183 newly diagnosed seminoma cases and 73 newly diagnosed non-seminomatous germ cell tumours (NSGCT) presenting from 1985 to 1989 to a tertiary referral cancer hospital. The purpose was to assess the contribution of bipedal lymphography (LG) to the management of these patients. As the main value of LG is in detecting small retroperitoneal lymph node (LN) metastases, analysis concentrated upon early stage disease, specifically N{sub 0} and N{sub 1a} LN disease. Comparison between LG results, abdominopelvic computed tomography (APCT), final clinical stage and treatment outcome was performed. We found that with the LG and APCT criteria used (filling defects > 2 mm and LN diameter >20 mm, respectively), LG was much more sensitive in disease detection. However, with modern techniques APCT can reliably detect disease 10 mm or greater. In addition, tumour marker status, primary tumour vascular invasion status and initial clinical examination were each more important in staging NSGCT disease than LG alone. Thus, LG is now rarely used in our institution but we will have to monitor our excellent survival data to confirm that this change in policy is warranted. 14 refs., 5 tabs., 2 figs.

  19. Cost-effectiveness analysis of early vs. late autologous stem cell transplantation in multiple myeloma.

    Science.gov (United States)

    Pandya, Chintan; Hashmi, Shahrukh; Khera, Nandita; Gertz, Morie A; Dispenzieri, Angela; Hogan, William; Siddiqui, Mustaqeem; Noyes, Katia; Kumar, Shaji K

    2014-10-01

    Autologous stem cell transplant (ASCT) is the current standard of care for most patients with multiple myeloma (MM) who are transplant eligible, yet the timing of ASCT is disputed due to a similar overall (OS) and progression-free survival with an early ASCT (eASCT) or a delayed ASCT (dASCT) approach. We developed a decision analytic model to perform cost-effectiveness analysis of the two commonly used treatment strategies for MM. Data on disease progression and treatment effectiveness came from 2001 to 2008 cohort treated at the Mayo Clinic and from published studies. Cost analysis was performed from a third-party payer perspective. The Consumer Price Index adjusted 2012 costs of eASCT and dASCT were $249 236 and $262 610, respectively. eASCT cohort had a benefit of 1.96 quality-adjusted life years (QALYs), 0.23 QALYs more than dASCT, implying that eASCT is preferred (dominant) over dASCT. The most critical variables in one-way sensitivity analysis were treatment-related mortality and OS associated with eASCT strategy. We conclude that eASCT could potentially be a relatively cost-effective treatment option for appropriate patients with MM, and these results would help patients, providers, and payers in decision making for timing of ASCT. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Hematopoietic cell crisis: An early stage of evolving myeloid leukemia following radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Seed, T.M.

    1990-01-01

    Under select radiological conditions, chronic radiation exposure elicits a high incidence of myeloproliferative disease, principally myeloid leukemia (ML), in beagles. Previously we demonstrated that for full ML expression, a four-stage preclinical sequence is required, namely (1) suppression, (2) recovery, (3) accommodation, and (4) preleukemic transition. Within this pathological sequence, a critical early event has been identified as the acquisition of radioresistance by hematopoietic progenitors that serves to mediate a newfound regenerative hematopoietic capacity. As such, this event sets the stage'' for preleukemic progression by initiating progression from preclinical phase 1 to 2. Due to the nature of target cell suppression, the induction of crisis, and the outgrowth of progenitors with altered phenotypes, this preleukemic event resembles the immortalization'' step of the in vitro transformation sequence following induction with either physical and chemical carcinogens. The radiological, temporal, and biological dictates governing this event have been extensively evaluated and will be discussed in light of their role in the induction and progression of chronic radiation leukemia. 35 refs., 2 tabs.

  1. Human herpesvirus 6 major immediate early promoter has strong activity in T cells and is useful for heterologous gene expression

    Directory of Open Access Journals (Sweden)

    Yamanishi Koichi

    2011-01-01

    Full Text Available Abstract Background Human herpesvirus-6 (HHV-6 is a beta-herpesvirus. HHV-6 infects and replicates in T cells. The HHV-6-encoded major immediate early gene (MIE is expressed at the immediate-early infection phase. Human cytomegalovirus major immediate early promoter (CMV MIEp is commercially available for the expression of various heterologous genes. Here we identified the HHV-6 MIE promoter (MIEp and compared its activity with that of CMV MIEp in various cell lines. Methods The HHV-6 MIEp and some HHV-6 MIEp variants were amplified by PCR from HHV-6B strain HST. These fragments and CMV MIEp were subcloned into the pGL-3 luciferase reporter plasmid and subjected to luciferase reporter assay. In addition, to investigate whether the HHV-6 MIEp could be used as the promoter for expression of foreign genes in a recombinant varicella-zoster virus, we inserted HHV-6 MIEp-DsRed expression casette into the varicella-zoster virus genome. Results HHV-6 MIEp showed strong activity in T cells compared with CMV MIEp, and the presence of intron 1 of the MIE gene increased its activity. The NF-κB-binding site, which lies within the R3 repeat, was critical for this activity. Moreover, the HHV-6 MIEp drove heterologous gene expression in recombinant varicella-zoster virus-infected cells. Conclusions These data suggest that HHV-6 MIEp functions more strongly than CMV MIEp in various T-cell lines.

  2. R5-SHIV induces multiple defects in T cell function during early infection of rhesus macaques including accumulation of T reg cells in lymph nodes.

    Directory of Open Access Journals (Sweden)

    Michael Santosuosso

    2011-04-01

    Full Text Available HIV-1 is a pathogen that T cell responses fail to control. HIV-1gp120 is the surface viral envelope glycoprotein that interacts with CD4 T cells and mediates entry. HIV-1gp120 has been implicated in immune dysregulatory functions that may limit anti-HIV antigen-specific T cell responses. We hypothesized that in the context of early SHIV infection, immune dysregulation of antigen-specific T-effector cell and regulatory functions would be detectable and that these would be associated or correlated with measurable concentrations of HIV-1gp120 in lymphoid tissues.Rhesus macaques were intravaginally inoculated with a Clade C CCR5-tropic simian-human immunodeficiency virus, SHIV-1157ipd3N4. HIV-1gp120 levels, antigen-specificity, levels of apoptosis/anergy and frequency and function of Tregs were examined in lymph node and blood derived T cells at 5 and 12 weeks post inoculation.We observed reduced responses to Gag in CD4 and gp120 in CD8 lymph node-derived T cells compared to the peripheral blood at 5 weeks post-inoculation. Reduced antigen-specific responses were associated with higher levels of PD-1 on lymph node-derived CD4 T cells as compared to peripheral blood and uninfected lymph node-derived CD4 T cells. Lymph nodes contained increased numbers of Tregs as compared to peripheral blood, which positively correlated with gp120 levels; T regulatory cell depletion restored CD8 T cell responses to Gag but not to gp120. HIV gp120 was also able to induce T regulatory cell chemotaxis in a dose-dependent, CCR5-mediated manner. These studies contribute to our broader understanding of the ways in which HIV-1 dysregulates T cell function and localization during early infection.

  3. Skeletal muscle cells possess a 'memory' of acute early life TNF-α exposure: role of epigenetic adaptation.

    Science.gov (United States)

    Sharples, Adam P; Polydorou, Ioanna; Hughes, David C; Owens, Daniel J; Hughes, Thomas M; Stewart, Claire E

    2016-06-01

    Sufficient quantity and quality of skeletal muscle is required to maintain lifespan and healthspan into older age. The concept of skeletal muscle programming/memory has been suggested to contribute to accelerated muscle decline in the elderly in association with early life stress such as fetal malnutrition. Further, muscle cells in vitro appear to remember the in vivo environments from which they are derived (e.g. cancer, obesity, type II diabetes, physical inactivity and nutrient restriction). Tumour-necrosis factor alpha (TNF-α) is a pleiotropic cytokine that is chronically elevated in sarcopenia and cancer cachexia. Higher TNF-α levels are strongly correlated with muscle loss, reduced strength and therefore morbidity and earlier mortality. We have extensively shown that TNF-α impairs regenerative capacity in mouse and human muscle derived stem cells [Meadows et al. (J Cell Physiol 183(3):330-337, 2000); Foulstone et al. (J Cell Physiol 189(2):207-215, 2001); Foulstone et al. (Exp Cell Res 294(1):223-235, 2004); Stewart et al. (J Cell Physiol 198(2):237-247, 2004); Al-Shanti et al. (Growth factors (Chur, Switzerland) 26(2):61-73, 2008); Saini et al. (Growth factors (Chur, Switzerland) 26(5):239-253, 2008); Sharples et al. (J Cell Physiol 225(1):240-250, 2010)]. We have also recently established an epigenetically mediated mechanism (SIRT1-histone deacetylase) regulating survival of myoblasts in the presence of TNF-α [Saini et al. (Exp Physiol 97(3):400-418, 2012)]. We therefore wished to extend this work in relation to muscle memory of catabolic stimuli and the potential underlying epigenetic modulation of muscle loss. To enable this aim; C2C12 myoblasts were cultured in the absence or presence of early TNF-α (early proliferative lifespan) followed by 30 population doublings in the absence of TNF-α, prior to the induction of differentiation in low serum media (LSM) in the absence or presence of late TNF-α (late proliferative lifespan). The cells that

  4. The changing dielectric properties of CHO cells can be used to determine early apoptotic events in a bioprocess.

    Science.gov (United States)

    Braasch, Katrin; Nikolic-Jaric, Marija; Cabel, Tim; Salimi, Elham; Bridges, Greg E; Thomson, Doug J; Butler, Michael

    2013-11-01

    To ensure maximum productivity of recombinant proteins it is desirable to prolong cell viability during a mammalian cell bioprocess, and therefore important to carefully monitor cell density and viability. In this study, five different and independent methods of monitoring were applied to Chinese hamster ovary (CHO) cells grown in a batch culture in a controlled bioreactor to determine cell density and/or cell viability. They included: a particle counter, trypan blue exclusion (Cedex), an in situ bulk capacitance probe, an off-line fluorescent flow cytometer, and a prototype dielectrophoretic (DEP) cytometer. These various techniques gave similar values during the exponential growth phase. However, beyond the exponential growth phase the viability measurements diverged. Fluorescent flow cytometry with a range of fluorescent markers was used to investigate this divergence and to establish the progress of cell apoptosis: the cell density estimates by the intermediate stage apoptosis assay agreed with those obtained by the bulk capacitance probe and the early stage apoptosis assay viability measurements correlated well with the DEP cytometer. The trypan blue assay showed higher estimates of viable cell density and viability compared to the capacitance probe or the DEP cytometer. The DEP cytometer measures the dielectric properties of individual cells and identified at least two populations of cells, each with a distinct polarizability. As verified by comparison with the Nexin assay, one population was associated with viable (non-apoptotic) cells and the other with apoptotic cells. From the end of the exponential through the stationary and decline stages there was a gradual shift of cell count from the viable into the apoptotic population. However, the two populations maintained their individual dielectric properties throughout this shift. This leads to the conclusion that changes in bulk dielectric properties of cultures might be better modeled as shifts in cells

  5. Retinal Layers Changes in Human Preclinical and Early Clinical Diabetic Retinopathy Support Early Retinal Neuronal and Müller Cells Alterations

    Directory of Open Access Journals (Sweden)

    Stela Vujosevic

    2013-01-01

    Full Text Available Purpose. To evaluate the changes in thickness of individual inner and outer macular and peripapillary retinal layers in diabetes. Methods. 124 subjects (124 eyes were enrolled: 74 diabetics and 50 controls. Macular edema, proliferative diabetic retinopathy (DR, any intraocular treatment and refractive error >6 diopters were the main exclusion criteria. Full ophthalmic examination, stereoscopic fundus photography, and spectral domain-OCT were performed. After automatic retinal segmentation (layering in 5 layers, the thickness of each layer was calculated, and values compared among groups. Results. Thirty patients had no DR, 44 patients had non proliferative DR. A significant increase of inner plexiform and nuclear layers was found in DR eyes versus controls (P<0.001. A significant decrease (P<0.01 of retinal nerve fiber layer (RNFL and at specific sites of retinal ganglion cell layer (P=0.02 was documented in the macula. In the peripapillary area there were no differences between diabetics and controls. Conclusions. Decreased RNFL thickness and increased INL/OPL thickness in diabetics without DR or with initial DR suggest early alterations in the inner retina. On the contrary, the outer retina seems not to be affected at early stages of DM. Automatic intraretinal layering by SD-OCT may be a useful tool to diagnose and monitor early intraretinal changes in DR.

  6. Expansion and Activation Kinetics of Immune Cells during Early Phase of GVHD in Mouse Model Based on Chemotherapy Conditioning

    Science.gov (United States)

    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

  7. Dietary Glycation Products Regulate Immune Homeostasis: Early Glycation Products Promote Prostate Cancer Cell Proliferation through Modulating Macrophages.

    Science.gov (United States)

    Chen, Yingjia; Filipov, Nikolay M; Guo, Tai L

    2017-10-30

    Well-controlled glycation (generally limited to the early stages) has been proposed as a strategy to improve the physiochemical properties of dietary proteins, but the functional studies of glycation products were mostly on advanced glycation end-products (AGEs) rather than early glycation products (EGPs). Since cytokines are important modulators of various biological processes, this study aimed to determine whether EGPs and AGEs affected immune homeostasis differentially and did so through modulating macrophage-derived factors. Two systems (glycine-glucose and whey protein isolate (WPI)-glucose) were established to generate glycation products. They were applied to human macrophages (PMA-differentiated U937 cells), and cell viability and cytokine production were measured. Furthermore, EGPs, AGEs and their conditioned medium (CM) from macrophages were applied to human prostate cancer (PCa) cells with different etiology (LNCaP and PC-3) and murine PCa cells (TRAMP-C2) to determine their direct and indirect effects on PCa cell proliferation. EGPs enhanced the production of anti-inflammatory and immunosuppressive cytokines, and this enhancement was associated with increased PCa cell proliferation. In contrast, AGEs inhibited macrophages to secret cytokines, but increased PCa cell proliferation directly. Our data suggest that EGPs promote the prostate tumor proliferation indirectly through modulating macrophages, while AGEs have a direct effect. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. Early Transcriptomic Response to LDL and oxLDL in Human Vascular Smooth Muscle Cells.

    Science.gov (United States)

    Damián-Zamacona, Salvador; Toledo-Ibelles, Paola; Ibarra-Abundis, Mabel Z; Uribe-Figueroa, Laura; Hernández-Lemus, Enrique; Macedo-Alcibia, Karla Paola; Delgado-Coello, Blanca; Mas-Oliva, Jaime; Reyes-Grajeda, Juan Pablo

    2016-01-01

    Although nowadays it is well known that the human transcriptome can importantly vary according to external or environmental condition, the reflection of this concept when studying oxidative stress and its direct relationship with gene expression profiling during the process of atherogenesis has not been thoroughly achieved. The ability to analyze genome-wide gene expression through transcriptomics has shown that the genome responds dynamically to diverse stimuli. Here, we describe the transcriptome of human vascular smooth muscle cells (hVSMC) stimulated by native and oxidized low-density lipoprotein (nLDL and oxLDL respectively), with the aim of assessing the early molecular changes that induce a response in this cell type resulting in a transcriptomic transformation. This expression has been demonstrated in atherosclerotic plaques in vivo and in vitro, particularly in the light of the oxidative modification hypothesis of atherosclerosis. Total RNA was isolated with TRIzol reagent (Life Technologies) and quality estimated using an Agilent 2100 bioanalyzer. The transcriptome of hVSMC under different experimental conditions (1,5 and 24 hours for nLDL and oxLDL) was obtained using the GeneChip Human Gene 1.0 ST (Affymetrix) designed to measure gene expression of 28,869 well-annotated genes. A fixed fold-change cut-off corresponding to ± 2 was used to identify genes exhibiting the most significant variation and statistical significance (Pimmune response to inflammation. The evidence we present supporting the hypothesis for the involvement of oxidative modification of several processes and metabolic pathways in atherosclerosis is strengthen by the fact that gene expression patterns obtained when hVSMC are incubated for a long period of time in the presence of nLDL, correspond very much the same as when cells are incubated for a short period of time in the presence of chemically modified oxLDL. Our results indicate that under physiological conditions and directly

  9. Generation of Hprt-disrupted rat through mouse←rat ES chimeras

    OpenAIRE

    Ayako Isotani; Kazuo Yamagata; Masaru Okabe; Masahito Ikawa

    2016-01-01

    We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse?rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction...

  10. The therapeutic T-cell response induced by tumor delivery of TNF and melphalan is dependent on early triggering of natural killer and dendritic cells.

    Science.gov (United States)

    Balza, Enrica; Zanellato, Silvia; Poggi, Alessandro; Reverberi, Daniele; Rubartelli, Anna; Mortara, Lorenzo

    2017-04-01

    The fusion protein L19mTNF (mouse TNF and human antibody fragment L19 directed to fibronectin extra domain B) selectively targets the tumor vasculature, and in combination with melphalan induces a long-lasting T-cell therapeutic response and immune memory in murine models. Increasing evidence suggests that natural killer (NK) cells act to promote effective T-cell-based antitumor responses. We have analyzed the role of NK cells and dendritic cells (DCs) on two different murine tumor models: WEHI-164 fibrosarcoma and C51 colon carcinoma, in which the combined treatment induces high and low rejection rates, respectively. In vivo NK-cell depletion strongly reduced the rejection of WEHI-164 fibrosarcoma and correlated with a decrease in mature DCs, CD4+ , and CD8+ T cells in the tumor-draining LNs and mature DCs and CD4+ T cells in the tumor 40 h after initiation of the therapy. NK-cell depletion also resulted in the impairment of the stimulatory capability of DCs derived from tumor-draining LNs of WEHI-164-treated mice. Moreover, a significant reduction of M2-type infiltrating macrophages was detected in both tumors undergoing therapy. These results suggest that the efficacy of L19mTNF/melphalan therapy is strongly related to the early activation of NK cells and DCs, which are necessary for an effective T-cell response. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Comprehensive single cell-resolution analysis of the role of chromatin regulators in early C. elegans embryogenesis.

    Science.gov (United States)

    Krüger, Angela V; Jelier, Rob; Dzyubachyk, Oleh; Zimmerman, Timo; Meijering, Erik; Lehner, Ben

    2015-02-15

    Chromatin regulators are widely expressed proteins with diverse roles in gene expression, nuclear organization, cell cycle regulation, pluripotency, physiology and development, and are frequently mutated in human diseases such as cancer. Their inhibition often results in pleiotropic effects that are difficult to study using conventional approaches. We have developed a semi-automated nuclear tracking algorithm to quantify the divisions, movements and positions of all nuclei during the early development of Caenorhabditis elegans and have used it to systematically study the effects of inhibiting chromatin regulators. The resulting high dimensional datasets revealed that inhibition of multiple regulators, including F55A3.3 (encoding FACT subunit SUPT16H), lin-53 (RBBP4/7), rba-1 (RBBP4/7), set-16 (MLL2/3), hda-1 (HDAC1/2), swsn-7 (ARID2), and let-526 (ARID1A/1B) affected cell cycle progression and caused chromosome segregation defects. In contrast, inhibition of cir-1 (CIR1) accelerated cell division timing in specific cells of the AB lineage. The inhibition of RNA polymerase II also accelerated these division timings, suggesting that normal gene expression is required to delay cell cycle progression in multiple lineages in the early embryo. Quantitative analyses of the dataset suggested the existence of at least two functionally distinct SWI/SNF chromatin remodeling complex activities in the early embryo, and identified a redundant requirement for the egl-27 and lin-40 MTA orthologs in the development of endoderm and mesoderm lineages. Moreover, our dataset also revealed a characteristic rearrangement of chromatin to the nuclear periphery upon the inhibition of multiple general regulators of gene expression. Our systematic, comprehensive and quantitative datasets illustrate the power of single cell-resolution quantitative tracking and high dimensional phenotyping to investigate gene function. Furthermore, the results provide an overview of the functions of essential

  12. Early Reticulocytosis and Anemia Are Associated with Abnormal and Conditional Transcranial Doppler Velocities in Children with Sickle Cell Anemia.

    Science.gov (United States)

    Meier, Emily Riehm; Fasano, Ross M; Estrada, Monica; He, Jianping; Luban, Naomi L C; McCarter, Robert

    2016-02-01

    To improve prediction of sickle cell anemia severity at an early age, we evaluated whether absolute reticulocyte count (ARC) or hemoglobin (Hb) levels during early infancy (2-6 months of age) in patients with sickle cell anemia predict the risk of later developing an abnormal (abTCD) or conditional (cdTCD) Transcranial Doppler (TCD). We used chart review to identify 121 consecutive patients who underwent TCD screening and had steady state ARC and Hb levels recorded between 2 and 6 months of age. Cox regression analysis was used to determine the relationship between ARC, Hb levels, and risk of developing cdTCD/abTCD over time. Mean ARC in early infancy was highest and mean Hb lowest in those children with abTCDs and cdTCDs. Cox regression analysis revealed that those subjects with an ARC ≥200 K/μL in early infancy had nearly 3 times the risk of having an abTCD/cdTCD than the group with an ARC <200 K/μL, and patients with a Hb <8.5 g/dL had 2.7 times the risk of having an abTCD/cdTCD. These data suggest that both elevated ARC and low baseline Hb during early infancy are associated with an increased risk of developing a cdTCD or abTCD later in childhood. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Early Intervention of Didang Decoction on MLCK Signaling Pathways in Vascular Endothelial Cells of Type 2 Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Shoujiao Ye

    2016-01-01

    Full Text Available In the study, type 2 diabetic rat model was established using streptozotocin (STZ combined with a high-fat diet, and the rats were divided into control and diabetic groups. Diabetic groups were further divided into nonintervening, simvastatin, Didang Decoction (DDD early-phase intervening, DDD mid-phase intervening, and DDD late-phase intervening groups. The expression level of MLCK was detected using Western Blot analysis, and the levels of cyclic adenosine monophosphate (cAMP, protein kinase C (PKC, and protein kinase A (PKA were examined using Real Time PCR. Under the electron microscope, the cells in the early-DDD-intervention group and the simvastatin group were significantly more continuous and compact than those in the diabetic group. Compared with the control group, the expression of cAMP-1 and PKA was decreased in all diabetic groups, whereas the expression of MLCK and PKC was increased in early- and mid-phase DDD-intervening groups (P<0.05; compared with the late-phase DDD-intervening group, the expression of cAMP-1 and PKA was higher, but the level of MLCK and PKC was lower in early-phase DDD-intervening group (P<0.05. In conclusion, the early use of DDD improves the permeability of vascular endothelial cells by regulating the MLCK signaling pathway.

  14. Raman Spectral Dynamics of Single Cells in the Early Stages of Growth Factor Stimulation

    OpenAIRE

    Takanezawa, Sota; Morita, Shin-ichi; Ozaki, Yukihiro; Sako, Yasushi

    2015-01-01

    Cell fates change dynamically in response to various extracellular signals, including growth factors that stimulate differentiation and proliferation. The processes underlying cell-fate decisions are complex and often include large cell-to-cell variations, even within a clonal population in the same environment. To understand the origins of these cell-to-cell variations, we must detect the internal dynamics of single cells that reflect their changing chemical milieu. In this study, we used th...

  15. T-cell chimerism is valuable in predicting early mortality in steroid-resistant acute graft-versus-host disease after myeloablative allogeneic cell transplantation

    DEFF Research Database (Denmark)

    Minculescu, Lia; Madsen, Hans O.; Sengeløv, Henrik

    2014-01-01

    The main aim of this study was to evaluate the impact of early T-cell chimerism status on the incidence and clinical course of acute graft-versus-host disease (aGVHD) in allogeneic transplant recipients after myeloablative conditioning. Of 62 patients, 38 (61%) had complete T-cell donor chimerism...... (DC), and 24 (39%) had mixed T-cell chimerism (MC). The incidence of early death (before day 100) with steroid-resistant aGVHD was significantly higher in patients with complete DC compared to patients with MC (6/38 vs. 0/24, p = 0.04). The time from aGVHD diagnosis to nonrelapse death was markedly...

  16. Sensitive optical detection of an early metastatic tumor using a new cell line with enhanced luminescent and fluorescent signals

    Directory of Open Access Journals (Sweden)

    Yeon Joo Kim

    2011-10-01

    Full Text Available Animal models using cell lines that are dual-labeled with luciferase and green fluorescent protein (GFP are powerful tools for performing simultaneous quantitative and qualitative analysis of metastatic tumors. However, the applications of such dual-labeled tumor models have been limited due to the technical challenges associated with low bioluminescent signaling from tumor cells. Here, we used lentiviral vector (LV encoding firefly luciferase and GFP and engineered a more sensitive and highly metastatic prostate cancer cell line (MLL-Luc/GFP cells, which allows simultaneous fluorescence and luminescence imaging. The light emission of MLL-Luc/GFP cells was 33.5 fold higher than that of PC-3-luc2-GFP prostate cancer cells which showed 750 p/s light emission per cell. Furthermore, the MLL-Luc/GFP cells showed 3.9 fold higher luciferase activities than did 4T1-luc2, which was previously recognized as exhibiting the highest luciferase activity. An in vivo evaluation with optical imaging showed pinpoint localization of GFP-positive cells in a metastatic lung as well as easy detection of early metastatic spreading. The newly engineered MLL-Luc/GFP cells provide an appropriate metastatic animal model system for future studies of metastasis and the testing of anti-metastatic therapies specifically aimed at prostatic cancer.

  17. Clinical value of routine serum squamous cell carcinoma antigen in follow-up of patients with early-stage cervical cancer

    NARCIS (Netherlands)

    Esajas, MD; Duk, JM; de Bruijn, HWA; Aalders, JG; Willemse, PHB; Sluiter, W; Pras, B; ten Hoor, K; Hollema, H; van der Zee, AGJ

    2001-01-01

    Purpose: To investigate the contribution to recurrence detection and survival of serum squamous cell carcinoma antigen (SCC-ag) analysis in the follow-up of early-stage cervical cancer patients. Patients and Methods: Follow-up data were evaluated in patients with early-stage squamous cell cervical

  18. Different effects of resveratrol on early and late passage mesenchymal stem cells through β-catenin regulation

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    Yoon, Dong Suk; Choi, Yoorim; Choi, Seong Mi [Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul (Korea, Republic of); Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul (Korea, Republic of); Park, Kwang Hwan [Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul (Korea, Republic of); Lee, Jin Woo, E-mail: ljwos@yuhs.ac [Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul (Korea, Republic of); Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul (Korea, Republic of)

    2015-11-27

    Resveratrol is a sirtuin 1 (SIRT1) activator and can function as an anti-inflammatory and antioxidant factor. In mesenchymal stem cells (MSCs), resveratrol enhances the proliferation and differentiation potential and has an anti-aging effect. However, contradictory effects of resveratrol on MSC cultures have been reported. In this study, we found that resveratrol had different effects on MSC cultures according to their cell passage and SIRT1 expression. Resveratrol enhanced the self-renewal potential and multipotency of early passage MSCs, but accelerated cellular senescence of late passage MSCs. In early passage MSCs expressing SIRT1, resveratrol decreased ERK and GSK-3β phosphorylation, suppressing β-catenin activity. In contrast, in late passage MSCs, which did not express SIRT1, resveratrol increased ERK and GSK-3β phosphorylation, activating β-catenin. We confirmed that SIRT1-deficient early passage MSCs treated with resveratrol lost their self-renewal potential and multipotency, and became senescent due to increased β-catenin activity. Sustained treatment with resveratrol at early passages maintained the self-renewal potential and multipotency of MSCs up to passage 10. Our findings suggest that resveratrol can be effectively applied to early passage MSC cultures, whereas parameters such as cell passage and SIRT1 expression must be taken into consideration before applying resveratrol to late passage MSCs. - Highlights: • Resveratrol enhances self-renewal potential and multipotency of early passage MSCs. • Resveratrol accelerates the cellular senescence of late passage MSCs. • The effects of resveratrol on MSCs are dependent on the presence of SIRT1. • SIRT1 modulates ERK/GSK-3β/β-catenin signaling. • Sustained resveratrol treatment maintains MSC stemness up to P10.

  19. The early interaction of Leishmania with macrophages and dendritic cells and its influence on the host immune response.

    Science.gov (United States)

    Liu, Dong; Uzonna, Jude E

    2012-01-01

    The complicated interactions between Leishmania and the host antigen-presenting cells (APCs) have fundamental effects on the final outcome of the disease. Two major APCs, macrophages and dendritic cells (DCs), play critical roles in mediating resistance and susceptibility during Leishmania infection. Macrophages are the primary resident cell for Leishmania: they phagocytose and permit parasite proliferation. However, these cells are also the major effector cells to eliminate infection. The effective clearance of parasites by macrophages depends on activation of appropriate immune response, which is usually initiated by DCs. Here, we review the early interaction of APCs with Leishmania parasites and how these interactions profoundly impact on the ensuing adaptive immune response. We also discuss how the current knowledge will allow further refinement of our understanding of the interplay between Leishmania and its hosts that leads to resistance or susceptibility.

  20. Cell fate establishment during early development of cyprinid fishes, with special emphasis on the formation of the primordial germ cells

    NARCIS (Netherlands)

    Gevers, P.

    1992-01-01

    Cell fates can be established either by preformation or by epigenesis. With respect to primordial germ cells (PGCs) it has been shown that the Amphibia exhibit both types of cell fate establishment. Therefore, it is important to study the germ cell origin of the evolutionary lower class of

  1. Selective Loss of Early Differentiated, Highly Functional PD1high CD4 T Cells with HIV Progression.

    Directory of Open Access Journals (Sweden)

    Robert M Paris

    Full Text Available The role of PD-1 expression on CD4 T cells during HIV infection is not well understood. Here, we describe the differential expression of PD-1 in CD127high CD4 T cells within the early/intermediate differentiated (EI (CD27highCD45RAlow T cell population among uninfected and HIV-infected subjects, with higher expression associated with decreased viral replication (HIV-1 viral load. A significant loss of circulating PD-1highCTLA-4low CD4 T cells was found specifically in the CD127highCD27highCD45RAlow compartment, while initiation of antiretroviral treatment, particularly in subjects with advanced disease, reversed these dynamics. Increased HIV-1 Gag DNA was also found in PD-1high compared to PD-1low ED CD4 T cells. In line with an increased susceptibility to HIV infection, PD-1 expression in this CD4 T cell subset was associated with increased activation and expression of the HIV co-receptor, CCR5. Rather than exhaustion, this population produced more IFN-g, MIP1-a, IL-4, IL-10, and IL-17a compared to PD-1low EI CD4 T cells. In line with our previous findings, PD-1high EI CD4 T cells were also characterized by a high expression of CCR7, CXCR5 and CCR6, a phenotype associated with increased in vitro B cell help. Our data show that expression of PD-1 on early-differentiated CD4 T cells may represent a population that is highly functional, more susceptible to HIV infection and selectively lost in chronic HIV infection.

  2. The cytoplasmic domain of Ig alpha is necessary and sufficient to support efficient early B cell development.

    Science.gov (United States)

    Pike, Kelly A; Iacampo, Sandra; Friedmann, Jennifer E; Ratcliffe, Michael J H

    2004-02-15

    The B cell receptor complex (BcR) is essential for normal B lymphocyte function, and surface BcR expression is a crucial checkpoint in B cell development. However, functional requirements for chains of the BcR during development remain controversial. We have used retroviral gene transfer to introduce components of the BcR into chicken B cell precursors during embryonic development. A chimeric heterodimer, in which the cytoplasmic domains of chicken Igalpha and Igbeta are expressed by fusion with the extracellular and transmembrane domains of murine CD8alpha and CD8beta, respectively, targeted the cytoplasmic domains of the BcR to the cell surface in the absence of extracellular BcR domains. Expression of this chimeric heterodimer supported all early stages of embryo B cell development: bursal colonization, clonal expansion, and induction of repertoire diversification by gene conversion. Expression of the cytoplasmic domain of Igalpha, in the absence of the cytoplasmic domain of Igbeta, was not only necessary, but sufficient to support B cell development as efficiently as the endogenous BcR. In contrast, expression of the cytoplasmic domain of Igbeta in the absence of the cytoplasmic domain of Igalpha failed to support B cell development. The ability of the cytoplasmic domain of Igalpha to support early B cell development required a functional Igalpha immunoreceptor tyrosine-based activation motif. These results support a model in which expression of surface IgM following productive V(D)J recombination in developing B cell precursors serves to chaperone the cytoplasmic domain of Igalpha to the B cell surface, thereby initiating subsequent stages of development.

  3. Human VRK1 is an early response gene and its loss causes a block in cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Alberto Valbuena

    Full Text Available BACKGROUND: In mammalian cells regulatory proteins controlling the cell cycle are necessary due to the requirements of living in a heterogeneous environment of cell-interactions and growth factors. VRK1 is a novel serine-threonine kinase that phosphorylates several transcription factors and is associated with proliferation phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In this report VRK1 has been identified as regulated in the cell cycle. VRK1 gene expression is activated by the addition of serum to starved cells, indicating it is required for the exit of G0 phase and entry in G1; a response that parallels the re-expression of MYC, FOS and CCND1 (cyclin D1 genes, suggesting that VRK1 is an early-response gene. VRK1 gene expression is also shutdown by serum withdrawal. The human VRK1 gene promoter cloned in a luciferase reporter responds similarly to serum. In response to serum, the level of VRK1 protein expression has a positive correlation with cell proliferation markers such as phosphorylated-Rb or PCNA, and is inversely correlated with cell cycle inhibitors such as p27. The elimination of VRK1 by siRNA results in a G1 block in cell division, and in loss of phosphorylated-Rb, cyclin D1, and other proliferation markers. Elimination of VRK1 by siRNA induces a reduction of cell proliferation. VRK1 colocalizes with p63 in proliferating areas of squamous epithelium, and identifies a subpopulation in the basal layer. CONCLUSIONS/SIGNIFICANCE: VRK1 is an immediate early response gene required for entry in G1, and due to its implication in normal cell proliferation and division, might be a new target for development of inhibitors of cellular proliferation.

  4. Somatically Hypermutated Plasmodium-Specific IgM(+) Memory B Cells Are Rapid, Plastic, Early Responders upon Malaria Rechallenge.

    Science.gov (United States)

    Krishnamurty, Akshay T; Thouvenel, Christopher D; Portugal, Silvia; Keitany, Gladys J; Kim, Karen S; Holder, Anthony; Crompton, Peter D; Rawlings, David J; Pepper, Marion

    2016-08-16

    Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC heterogeneity. To clarify functional roles for distinct MBC subsets during malaria infection, we generated tetramers that identify Plasmodium-specific MBCs in both humans and mice. Long-lived murine Plasmodium-specific MBCs consisted of three populations: somatically hypermutated immunoglobulin M(+) (IgM(+)) and IgG(+) MBC subsets and an unmutated IgD(+) MBC population. Rechallenge experiments revealed that high affinity, somatically hypermutated Plasmodium-specific IgM(+) MBCs proliferated and gave rise to antibody-secreting cells that dominated the early secondary response to parasite rechallenge. IgM(+) MBCs also gave rise to T cell-dependent IgM(+) and IgG(+)B220(+)CD138(+) plasmablasts or T cell-independent B220(-)CD138(+) IgM(+) plasma cells. Thus, even in competition with IgG(+) MBCs, IgM(+) MBCs are rapid, plastic, early responders to a secondary Plasmodium rechallenge and should be targeted by vaccine strategies. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Radiation Therapy Overcomes Adverse Prognostic Role of Cyclooxygenase-2 Expression on Reed-Sternberg Cells in Early Hodgkin Lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Mestre, Francisco [Service of Radiation Therapy, University Hospital Son Espases, Instituto de Investigación Sanitaria de Palma, Palma de Mallorca (Spain); Gutiérrez, Antonio, E-mail: antoniom.gutierrez@ssib.es [Service of Hematology, University Hospital Son Espases, Instituto de Investigación Sanitaria de Palma, Palma de Mallorca (Spain); Rodriguez, Jose [MD Anderson Cancer Center, Madrid (Spain); Ramos, Rafael [Service of Pathology, University Hospital Son Espases, Instituto de Investigación Sanitaria de Palma, Palma de Mallorca (Spain); Garcia, Juan Fernando [Spanish National Cancer Research Centre, Madrid (Spain); Martinez-Serra, Jordi [Service of Hematology, University Hospital Son Espases, Instituto de Investigación Sanitaria de Palma, Palma de Mallorca (Spain); Casasus, Marta; Nicolau, Cristina [Service of Radiation Therapy, Policlinica Miramar, Palma de Mallorca (Spain); Bento, Leyre; Herraez, Ines [Service of Hematology, University Hospital Son Espases, Instituto de Investigación Sanitaria de Palma, Palma de Mallorca (Spain); Lopez-Perezagua, Paloma [Service of Radiology, IDISPA, Palma de Mallorca (Spain); Daumal, Jaime [Service of Nuclear Medicine, IDISPA, Palma de Mallorca (Spain); Besalduch, Joan [Service of Hematology, University Hospital Son Espases, Instituto de Investigación Sanitaria de Palma, Palma de Mallorca (Spain)

    2015-05-01

    Purpose: To analyze the role of radiation therapy (RT) on the adverse prognostic influence of cyclooxygenase-2 (COX-2) expression on Reed-Sternberg (RS) cells, in the setting of early Hodgkin lymphoma (HL) treated with ABVD (adriamycin, vinblastine, bleomycin, dacarbazine). Methods and Materials: In the present study we retrospectively investigated the prognostic value of COX-2 expression in a large (n=143), uniformly treated e