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Sample records for heterologous liver cells

  1. Liver Cell Culture Devices

    NARCIS (Netherlands)

    Andria, B.; Bracco, A.; Cirino, G.; Chamuleau, R. A. F. M.

    2010-01-01

    In the last 15 years many different liver cell culture devices, consisting of functional liver cells and artificial materials, have been developed. They have been devised for numerous different applications, such as temporary organ replacement (a bridge to liver transplantation or native liver

  2. Polyploidization of liver cells.

    Science.gov (United States)

    Celton-Morizur, Séverine; Desdouets, Chantal

    2010-01-01

    Eukaryotic organisms usually contain a diploid complement of chromosomes. However, there are a number of exceptions. Organisms containing an increase in DNA content by whole number multiples of the entire set of chromosomes are defined as polyploid. Cells that contain more than two sets of chromosomes were first observed in plants about a century ago and it is now recognized that polyploidy cells form in many eukaryotes under a wide variety of circumstance. Although it is less common in mammals, some tissues, including the liver, show a high percentage of polyploid cells. Thus, during postnatal growth, the liver parenchyma undergoes dramatic changes characterized by gradual polyploidization during which hepatocytes of several ploidy classes emerge as a result of modified cell-division cycles. This process generates the successive appearance of tetraploid and octoploid cell classes with one or two nuclei (mononucleated or binucleated). Liver cells polyploidy is generally considered to indicate terminal differentiation and senescence and to lead both to the progressive loss of cell pluripotency and a markedly decreased replication capacity. In adults, liver polyploidization is differentially regulated upon loss of liver mass and liver damage. Interestingly, partial hepatectomy induces marked cell proliferation followed by an increase in liver ploidy. In contrast, during hepatocarcinoma (HCC), growth shifts to a nonpolyploidizing pattern and expansion of the diploid hepatocytes population is observed in neoplastic nodules. Here we review the current state of understanding about how polyploidization is regulated during normal and pathological liver growth and detail by which mechanisms hepatocytes become polyploid.

  3. Prior mucosal exposure to heterologous cells alters the pathogenesis of cell-associated mucosal feline immunodeficiency virus challenge

    Directory of Open Access Journals (Sweden)

    Leavell Sarah

    2010-05-01

    Full Text Available Abstract Background Several lines of research suggest that exposure to cellular material can alter the susceptibility to infection by HIV-1. Because sexual contact often includes exposure to cellular material, we hypothesized that repeated mucosal exposure to heterologous cells would induce an immune response that would alter the susceptibility to mucosal infection. Using the feline immunodeficiency virus (FIV model of HIV-1 mucosal transmission, the cervicovaginal mucosa was exposed once weekly for 12 weeks to 5,000 heterologous cells or media (control and then cats were vaginally challenged with cell-associated or cell-free FIV. Results Exposure to heterologous cells decreased the percentage of lymphocytes in the mucosal and systemic lymph nodes (LN expressing L-selectin as well as the percentage of CD4+ CD25+ T cells. These shifts were associated with enhanced ex-vivo proliferative responses to heterologous cells. Following mucosal challenge with cell-associated, but not cell-free, FIV, proviral burden was reduced by 64% in cats previously exposed to heterologous cells as compared to media exposed controls. Conclusions The pathogenesis and/or the threshold for mucosal infection by infected cells (but not cell-free virus can be modulated by mucosal exposure to uninfected heterologous cells.

  4. Multi-omic profiling of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    2015-01-01

    Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied...

  5. Multi-omic profiling of EPO producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    Heterologous protein production in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied to characterize the physiological impact of erythropoietin production, and discover production bottlenecks, ...

  6. Suppression of adaptive immunity to heterologous antigens during Plasmodium infection through hemozoin-induced failure of dendritic cell function

    Directory of Open Access Journals (Sweden)

    Phillips R

    2006-04-01

    Full Text Available Abstract Background Dendritic cells (DCs are central to the initiation and regulation of the adaptive immune response during infection. Modulation of DC function may therefore allow evasion of the immune system by pathogens. Significant depression of the host's systemic immune response to both concurrent infections and heterologous vaccines has been observed during malaria infection, but the mechanisms underlying this immune hyporesponsiveness are controversial. Results Here, we demonstrate that the blood stages of malaria infection induce a failure of DC function in vitro and in vivo, causing suboptimal activation of T cells involved in heterologous immune responses. This effect on T-cell activation can be transferred to uninfected recipients by DCs isolated from infected mice. Significantly, T cells activated by these DCs subsequently lack effector function, as demonstrated by a failure to migrate to lymphoid-organ follicles, resulting in an absence of B-cell responses to heterologous antigens. Fractionation studies show that hemozoin, rather than infected erythrocyte (red blood cell membranes, reproduces the effect of intact infected red blood cells on DCs. Furthermore, hemozoin-containing DCs could be identified in T-cell areas of the spleen in vivo. Conclusion Plasmodium infection inhibits the induction of adaptive immunity to heterologous antigens by modulating DC function, providing a potential explanation for epidemiological studies linking endemic malaria with secondary infections and reduced vaccine efficacy.

  7. Kupffer Cells in the Liver

    Science.gov (United States)

    Dixon, Laura J.; Barnes, Mark; Tang, Hui; Pritchard, Michele T.; Nagy, Laura E.

    2016-01-01

    Kupffer cells are a critical component of the mononuclear phagocytic system and are central to both the hepatic and systemic response to pathogens. Kupffer cells are reemerging as critical mediators of both liver injury and repair. Kupffer cells exhibit a tremendous plasticity; depending on the local metabolic and immune environment, then can express a range of polarized phenotypes, from the proinflammatory M1 phenotype to the alternative/M2 phenotype. Multiple M2 phenotypes can be distinguished, each involved in the resolution of inflammation and wound healing. Here, we have provided an update on recent research that has contributed to the developing delineation of the contribution of Kupffer cells to different types of liver injury, with an emphasis on alcoholic and nonalcoholic liver diseases. These recent advances in our understanding of Kupffer cell function and regulation will likely provide new insights into the potential for therapeutic manipulation of Kupffer cells to promote the resolution of inflammation and enhance wound healing in liver disease. PMID:23720329

  8. Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells

    International Nuclear Information System (INIS)

    Takahashi, Hideo; Shimada, Ichio

    2010-01-01

    The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.

  9. LCR/MEL: A versatile system for high-level expression of heterologous proteins in erythroid cells.

    NARCIS (Netherlands)

    M. Needham; C. Gooding; K. Hudson; M. Antoniou (Michael); F.G. Grosveld (Frank); M. Hollis

    1992-01-01

    textabstractWe have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin

  10. Combination of heterologous fibrin sealant and bioengineered human embryonic stem cells to improve regeneration following autogenous sciatic nerve grafting repair.

    Science.gov (United States)

    Mozafari, Roghayeh; Kyrylenko, Sergiy; Castro, Mateus Vidigal; Ferreira, Rui Seabra; Barraviera, Benedito; Oliveira, Alexandre Leite Rodrigues

    2018-01-01

    Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F + T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. The experiments indicated that

  11. Liver involvement in Langerhans cell histiocytosis

    International Nuclear Information System (INIS)

    Wong, Adelaine; Ortiz-Neira, Clara L.; Abou Reslan, Walid; Kaura, Deepak; Sharon, Raphael; Anderson, Ronald; Pinto-Rojas, Alfredo

    2006-01-01

    Liver involvement in Langerhans cell histiocytosis (LCH) typically presents with hepatomegaly and other signs of liver dysfunction. We present an 11-month-old child having only minimally elevated liver enzymes as an indication of liver involvement. Using sonography as the initial diagnostic tool followed by MRI, LCH of the liver was revealed. A review of sonographic, CT, MRI and MR cholangiopancreatography findings in liver LCH is presented. We recommend that physicians consider sonography and MRI screening for liver involvement in patients with newly diagnosed LCH, as periportal involvement may be present with little or no liver function abnormality present, as in this patient. (orig.)

  12. P143 proteins from heterologous nucleopolyhedroviruses induce apoptosis in BM-N cells derived from the silkworm Bombyx mori.

    Science.gov (United States)

    Hamajima, Rina; Kobayashi, Michihiro; Ikeda, Motoko

    2017-04-02

    We previously demonstrated that ribosomal RNA (rRNA) of Bombyx mori BM-N cells is rapidly degraded upon infection with heterologous nucleopolyhedroviruses (NPVs), including Autographa californica multiple NPV (AcMNPV), Hyphantria cunea MNPV, Spodoptera exigua MNPV and S. litura MNPV, and that this response is triggered by viral P143 proteins. The transient expression of P143 proteins from heterologous NPVs was also shown to induce apoptosis and caspase-3-like protease activation in BM-N cells. In the present study, we conducted a transient expression assay using BM-N cells expressing mutant AcMNPV P143 (Ac-P143) proteins and demonstrated that five amino acid residues cooperatively participate in Ac-P143 protein-triggered apoptosis of BM-N cells. Notably, these five residues were previously shown to be required for triggering rRNA degradation in BM-N cells. As rRNA degradation in BM-N cells does not result from apoptosis, the present results suggest that Ac-P143-triggered rRNA degradation is the upstream signal for apoptosis induction in BM-N cells. We further showed that P143 protein-triggered apoptosis does not occur in S. frugiperda Sf9 or Lymantria dispar Ld652Y cells, indicating that apoptosis induction by heterologous P143 proteins is a BM-N cell-specific response. In addition, the observed induction of apoptosis in BM-N cells was found to be mediated by activation of the initiator caspase Bm-Dronc. Taken together, these results suggest that BM-N cells evolved a unique antiviral system that recognizes heterologous NPV P143 proteins to induce rRNA degradation and caspase-dependent apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Heterologous prime-boost regimens with a recombinant chimpanzee adenoviral vector and adjuvanted F4 protein elicit polyfunctional HIV-1-specific T-Cell responses in macaques.

    Science.gov (United States)

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN ('A'), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 ('P'), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides

  14. Heterologous Synthesis and Recovery of Advanced Biofuels from Bacterial Cell Factories.

    Science.gov (United States)

    Malik, Sana; Afzal, Ifrah; Mehmood, Muhammad Aamer; Al Doghaither, Huda; Rahimuddin, Sawsan Abdulaziz; Gull, Munazza; Nahid, Nazia

    2018-01-01

    Microbial engineering to produce advanced biofuels is currently the most encouraging approach in renewable energy. Heterologous synthesis of biofuels and other useful industrial chemicals using bacterial cell factories has radically diverted the attentions from the native synthesis of these compounds. However, recovery of biofuels from the media and cellular toxicity are the main hindrances to successful commercialization of advanced biofuels. Therefore, membrane transporter engineering is gaining increasing attentions from all over the world. The main objective of this review is to explore the ways to increase the microbial production of biofuels by counteracting the cellular toxicity and facilitating their easier recovery from media. Microbial synthesis of industrially viable compounds such as biofuels has been increased due to genomic revolution. Moreover, advancements in protein engineering, gene regulation, pathway portability, metabolic engineering and synthetic biology led the focus towards the development of robust and cost-effective systems for biofuel production. The most convenient way to combat cellular toxicity and to secrete biofuels is the use of membrane transport system. The use of membrane transporters is currently a serious oversight as do not involve chemical changes and contribute greatly to efflux biofuels in extracellular milieu. However, overexpression of transport systems can also be detrimental to cell, so, in future, structure-based engineering of transporters can be employed to evaluate optimum expression range, to increase biofuel specificity and transport rate through structural studies of biofuel molecules. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  15. Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels

    Science.gov (United States)

    Jung, Sang-Kyu; Parisutham, Vinuselvi; Jeong, Seong Hun; Lee, Sung Kuk

    2012-01-01

    A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies. PMID:22911272

  16. METHOD FOR THE PRODUCTION OF HETEROLOGOUS POLYPEPTIDES IN TRANSFORMED YEAST CELLS

    DEFF Research Database (Denmark)

    2000-01-01

    The invention describes industrial fermentation of a $i(Saccharomyces) yeast species for production of a heterologous product encoded by a plasmid or DNA contained in said $i(Saccharomyces) yeast species with method utilizes the substrate more efficiently and without fermentative metabolism...... resulting in formation of ethanol and other unwanted primary products of fermentative activity whereby high yields of the heterologous product are obtained. The $i(Saccharomyces) yeast species is preferably a Crabtree negative $i(Saccharomyces species) in particular $i(Saccharomyces kluyveri)....

  17. CXCR4 Is Required by a Nonprimate Lentivirus: Heterologous Expression of Feline Immunodeficiency Virus in Human, Rodent, and Feline Cells

    Science.gov (United States)

    Poeschla, Eric M.; Looney, David J.

    1998-01-01

    A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors. PMID:9658135

  18. TWEAK induces liver progenitor cell proliferation

    Science.gov (United States)

    Jakubowski, Aniela; Ambrose, Christine; Parr, Michael; Lincecum, John M.; Wang, Monica Z.; Zheng, Timothy S.; Browning, Beth; Michaelson, Jennifer S.; Baestcher, Manfred; Wang, Bruce; Bissell, D. Montgomery; Burkly, Linda C.

    2005-01-01

    Progenitor (“oval”) cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors. PMID:16110324

  19. Xenotransplantation of neonatal porcine liver cells.

    Science.gov (United States)

    Garkavenko, O; Emerich, D F; Muzina, M; Muzina, Z; Vasconcellos, A V; Ferguson, A B; Cooper, I J; Elliott, R B

    2005-01-01

    Xenotransplantation of porcine liver cell types may provide a means of overcoming the shortage of suitable donor tissues to treat hepatic diseases characterized by inherited inborn errors of metabolism or protein production. Here we report the successful isolation, culture, and xenotransplantation of liver cells harvested from 7- to 10-day-old piglets. Liver cells were isolated and cultured immediately after harvesting. Cell viability was excellent (>90%) over the duration of the in vitro studies (3 weeks) and the cultured cells continued to significantly proliferate. These cells also retained their normal secretory and metabolic capabilities as determined by continued release of albumin, factor 8, and indocyanin green (ICG) uptake. After 3 weeks in culture, porcine liver cells were loaded into immunoisolatory macro devices (Theracyte devices) and placed into the intraperitoneal cavity of immunocompetant CD1 mice. Eight weeks later, the devices were retrieved and the cells analyzed for posttransplant determinations of survival and function. Post mortem analysis confirmed that the cell-loaded devices were biocompatible, and were well-tolerated without inducing any notable inflammatory reaction in the tissues immediately surrounding the encapsulated cells. Finally, the encapsulated liver cells remained viable and functional as determined by histologic analyses and ICG uptake/release. The successful harvesting, culturing, and xenotransplantation of functional neonatal pig liver cells support the continued development of this approach for treating a range of currently undertreated or intractable hepatic diseases.

  20. Application of the relative activity factor approach in scaling from heterologously expressed cytochromes p450 to human liver microsomes: studies on amitriptyline as a model substrate.

    Science.gov (United States)

    Venkatakrishnan, K; von Moltke, L L; Greenblatt, D J

    2001-04-01

    The relative activity factor (RAF) approach is being increasingly used in the quantitative phenotyping of multienzyme drug biotransformations. Using lymphoblast-expressed cytochromes P450 (CYPs) and the tricyclic antidepressant amitriptyline as a model substrate, we have tested the hypothesis that the human liver microsomal rates of a biotransformation mediated by multiple CYP isoforms can be mathematically reconstructed from the rates of the biotransformation catalyzed by individual recombinant CYPs using the RAF approach, and that the RAF approach can be used for the in vitro-in vivo scaling of pharmacokinetic clearance from in vitro intrinsic clearance measurements in heterologous expression systems. In addition, we have compared the results of two widely used methods of quantitative reaction phenotyping, namely, chemical inhibition studies and the prediction of relative contributions of individual CYP isoforms using the RAF approach. For the pathways of N-demethylation (mediated by CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and E-10 hydroxylation (mediated by CYPs 2B6, 2D6, and 3A4), the model-predicted biotransformation rates in microsomes from a panel of 12 human livers determined from enzyme kinetic parameters of the recombinant CYPs were similar to, and correlated with the observed rates. The model-predicted clearance via N-demethylation was 53% lower than the previously reported in vivo pharmacokinetic estimates. Model-predicted relative contributions of individual CYP isoforms to the net biotransformation rate were similar to, and correlated with the fractional decrement in human liver microsomal reaction rates by chemical inhibitors of the respective CYPs, provided the chemical inhibitors used were specific to their target CYP isoforms.

  1. Natural Killer cells and liver fibrosis

    Directory of Open Access Journals (Sweden)

    Frank eFasbender

    2016-01-01

    Full Text Available In the 40 years since the discovery of Natural Killer (NK cells it has been well established that these innate lymphocytes are important for early and effective immune responses against transformed cells and infections with different pathogens. In addition to these classical functions of NK cells, we now know that they are part of a larger family of innate lymphoid cells and that they can even mediate memory-like responses. Additionally, tissue resident NK cells with distinct phenotypical and functional characteristics have been identified. Here we focus on the phenotype of different NK cell subpopulations that can be found in the liver and summarize the current knowledge about the functional role of these cells with a special emphasis on liver fibrosis. NK cell cytotoxicity can contribute to liver damage in different forms of liver disease. However, NK cells can limit liver fibrosis by killing hepatic stellate cell-derived myofibroblasts, which play a key role in this pathogenic process. Therefore, liver NK cells need to be tightly regulated in order to balance these beneficial and pathological effects.

  2. Cell surface expression system for the display of heterologous gene products using chimeric flagellin fusions of bacillus halodurans isolate

    CSIR Research Space (South Africa)

    Du Plessis, A

    2006-10-01

    Full Text Available system for the display of heterologous gene products using chimeric flagellin fusions of a Bacillus halodurans isolate Slide 2 © CSIR 2006 www.csir.co.za Bacillus halodurans Alk 36 xrhombus Ability to over-produce cell... for functionality of the His-tag for metal binding. Slide 13 © CSIR 2006 www.csir.co.za PAGE gel showing over-production of chimeric poly-His flagellin proteins 66.2 kDa 45.0 kDa 31.0 kDa 1. LMW ladder 2. NC3 3. NHisC3 4. NC6 5...

  3. MAIT cells: new guardians of the liver

    OpenAIRE

    Kurioka, Ayako; Walker, Lucy J; Klenerman, Paul; Willberg, Christian B

    2016-01-01

    The liver is an important immunological organ that remains sterile and tolerogenic in homeostasis, despite continual exposure to non-self food and microbial-derived products from the gut. However, where intestinal mucosal defenses are breached or in the presence of a systemic infection, the liver acts as a second 'firewall', because of its enrichment with innate effector cells able to rapidly respond to infections or tissue dysregulation. One of the largest populations of T cells within the h...

  4. Multi-omic profiling of EPO-producing CHO cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    The Chinese hamster ovary (CHO) cell line is the predominant mammalian cell factory for production of therapeutic glycoproteins. In this work, we aimed to study bottlenecks in the secretory pathway associated with the production of human erythropoietin (EPO) in CHO cells. In connection to this, we...... discovered indications of metabolic adaptation of the amino acid catabolism in favor of heterologous protein production. We established a panel of stably EPO expressing CHO-K1 clones spanning a 25-fold productivity range and characterized the clones in batch and chemostat cultures. For this, we employed...... a multi-omic physiological characterization including metabolic foot printing of amino acids, metabolite fingerprinting of glycolytic intermediates, NAD(P)H-/NAD(P)+ and adenosine nucleotide phosphates. We used qPCR, qRT-PCR, western blots and Affymetrix CHO microarrays to assess EPO gene copy numbers...

  5. Bacillus subtilis MreB paralogues have different filament architectures and lead to shape remodelling of a heterologous cell system.

    Science.gov (United States)

    Soufo, Hervé Joël Defeu; Graumann, Peter L

    2010-12-01

    Like many bacteria, Bacillus subtilis cells contain three actin-like MreB proteins. We show that the three paralogues, MreB, Mbl and MreBH, have different filament architectures in a heterologous cell system, and form straight filaments, helices or ring structures, different from the regular helical arrangement in B. subtilis cells. However, when coexpressed, they colocalize into a single filamentous helical structure, showing that the paralogues influence each other's filament architecture. Ring-like MreBH structures can be converted into MreB-like helical filaments by a single point mutation affecting subunit contacts, showing that MreB paralogues feature flexible filament arrangements. Time-lapse and FRAP experiments show that filaments can extend as well as shrink at both ends, and also show internal rearrangement, suggesting that filaments consist of overlapping bundles of shorter filaments that continuously turn over. Upon induction in Escherichia coli cells, B. subtilis MreB (BsMreB) filaments push the cells into strikingly altered cell morphology, showing that MreB filaments can change cell shape. E. coli cells with a weakened cell wall were ruptured upon induction of BsMreB filaments, suggesting that the bacterial actin orthologue may exert force against the cell membrane and envelope, and thus possibly plays an additional mechanical role in bacteria. © 2010 Blackwell Publishing Ltd.

  6. Chronic inflammation-elicited liver progenitor cell conversion to liver cancer stem cell with clinical significance.

    Science.gov (United States)

    Li, Xiao-Feng; Chen, Cheng; Xiang, Dai-Min; Qu, Le; Sun, Wen; Lu, Xin-Yuan; Zhou, Teng-Fei; Chen, Shu-Zhen; Ning, Bei-Fang; Cheng, Zhuo; Xia, Ming-Yang; Shen, Wei-Feng; Yang, Wen; Wen, Wen; Lee, Terence Kin Wah; Cong, Wen-Ming; Wang, Hong-Yang; Ding, Jin

    2017-12-01

    The substantial heterogeneity and hierarchical organization in liver cancer support the theory of liver cancer stem cells (LCSCs). However, the relationship between chronic hepatic inflammation and LCSC generation remains obscure. Here, we observed a close correlation between aggravated inflammation and liver progenitor cell (LPC) propagation in the cirrhotic liver of rats exposed to diethylnitrosamine. LPCs isolated from the rat cirrhotic liver initiated subcutaneous liver cancers in nonobese diabetic/severe combined immunodeficient mice, suggesting the malignant transformation of LPCs toward LCSCs. Interestingly, depletion of Kupffer cells in vivo attenuated the LCSC properties of transformed LPCs and suppressed cytokeratin 19/Oval cell 6-positive tumor occurrence. Conversely, LPCs cocultured with macrophages exhibited enhanced LCSC properties. We further demonstrated that macrophage-secreted tumor necrosis factor-α triggered chromosomal instability in LPCs through the deregulation of ubiquitin D and checkpoint kinase 2 and enhanced the self-renewal of LPCs through the tumor necrosis factor receptor 1/Src/signal transducer and activator of transcription 3 pathway, which synergistically contributed to the conversion of LPCs to LCSCs. Clinical investigation revealed that cytokeratin 19/Oval cell 6-positive liver cancer patients displayed a worse prognosis and exhibited superior response to sorafenib treatment. Our results not only clarify the cellular and molecular mechanisms underlying the inflammation-mediated LCSC generation but also provide a molecular classification for the individualized treatment of liver cancer. (Hepatology 2017;66:1934-1951). © 2017 by the American Association for the Study of Liver Diseases.

  7. Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

    Directory of Open Access Journals (Sweden)

    Han Lanlan

    2011-10-01

    Full Text Available Abstract Background Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB. In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Results Multiple sequence alignment revealed that the C-terminal region (LcsB of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP or beta-galactosidase (Gal was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. Conclusion The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological

  8. New therapeutic strategies for canine liver disease; Growth factors and liver progenitor cells

    NARCIS (Netherlands)

    Arends, B.

    2008-01-01

    The liver has the unique capacity to regulate its mass after loss of functional liver cells due to liver disease, injury, and/or toxicity. Unfortunately, in the course of chronic liver disease this meticulously regulated regeneration process is imbalanced resulting in a decreased regenerative

  9. The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

    Science.gov (United States)

    de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

    2015-11-01

    In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects.

  10. LIVER AND BONE MARROW STEM/PROGENITOR CELLS AS REGULATORS OF REPARATIVE REGENERATION OF DAMAGED LIVER

    Directory of Open Access Journals (Sweden)

    А. V. Lundup

    2010-01-01

    Full Text Available In this review the modern information about effectiveness of liver insufficiency treatment by stem/ progenitor cells of liver (oval cells and bone marrow (hemopoietic cells and mesenchymal cells was presented. It is shown that medical action of these cells is referred on normalization of liver cell interaction and reorganization of processes of a reparative regeneration in damaged liver. It is believed that application of mesenchymal stromal cells from an autological bone marrow is the most perspective strategy. However, for definitive judgement about regenerative possibilities of the autological bone marrow cells it is necessary to carry out large-scale double blind clinical researches. 

  11. Cellular Mechanisms of Liver Regeneration and Cell-Based Therapies of Liver Diseases

    Directory of Open Access Journals (Sweden)

    Irina V. Kholodenko

    2017-01-01

    Full Text Available The emerging field of regenerative medicine offers innovative methods of cell therapy and tissue/organ engineering as a novel approach to liver disease treatment. The ultimate scientific foundation of both cell therapy of liver diseases and liver tissue and organ engineering is delivered by the in-depth studies of the cellular and molecular mechanisms of liver regeneration. The cellular mechanisms of the homeostatic and injury-induced liver regeneration are unique. Restoration of the mass of liver parenchyma is achieved by compensatory hypertrophy and hyperplasia of the differentiated parenchymal cells, hepatocytes, while expansion and differentiation of the resident stem/progenitor cells play a minor or negligible role. Participation of blood-borne cells of the bone marrow origin in liver parenchyma regeneration has been proven but does not exceed 1-2% of newly formed hepatocytes. Liver regeneration is activated spontaneously after injury and can be further stimulated by cell therapy with hepatocytes, hematopoietic stem cells, or mesenchymal stem cells. Further studies aimed at improving the outcomes of cell therapy of liver diseases are underway. In case of liver failure, transplantation of engineered liver can become the best option in the foreseeable future. Engineering of a transplantable liver or its major part is an enormous challenge, but rapid progress in induced pluripotency, tissue engineering, and bioprinting research shows that it may be doable.

  12. Multigenic DNA vaccine induces protective cross-reactive T cell responses against heterologous influenza virus in nonhuman primates.

    Directory of Open Access Journals (Sweden)

    Merika T Koday

    Full Text Available Recent avian and swine-origin influenza virus outbreaks illustrate the ongoing threat of influenza pandemics. We investigated immunogenicity and protective efficacy of a multi-antigen (MA universal influenza DNA vaccine consisting of HA, M2, and NP antigens in cynomolgus macaques. Following challenge with a heterologous pandemic H1N1 strain, vaccinated animals exhibited significantly lower viral loads and more rapid viral clearance when compared to unvaccinated controls. The MA DNA vaccine induced robust serum and mucosal antibody responses but these high antibody titers were not broadly neutralizing. In contrast, the vaccine induced broadly-reactive NP specific T cell responses that cross-reacted with the challenge virus and inversely correlated with lower viral loads and inflammation. These results demonstrate that a MA DNA vaccine that induces strong cross-reactive T cell responses can, independent of neutralizing antibody, mediate significant cross-protection in a nonhuman primate model and further supports development as an effective approach to induce broad protection against circulating and emerging influenza strains.

  13. Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells

    NARCIS (Netherlands)

    Sinha, B; Francois, P; Que, Y A; Hussain, M; Heilmann, C; Moreillon, P; Lew, D; Krause, K H; Peters, Georg; Herrmann, M

    2000-01-01

    Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell.

  14. Effects of cortisol on the primary response of mouse spleen cell cultures to heterologous erythrocytes

    International Nuclear Information System (INIS)

    Dracott, B.N.

    1974-01-01

    Cell viability and the production of direct PFC were studied in mouse spleen cell cultures after cortisol treatment in vivo or in vitro at various times relative to primary stimulation with SRBC in vitro. Cortisol treatment in vivo reduced spleen cell numbers by 88 percent after 48 hr, but cultures of the remaining cells produced as many PFC in vitro as did cultures of equal numbers of normal spleen cells. In normal spleen cell cultures incubated with cortisol for 4 hr prior to the addition of antigen, peak responses of PFC/culture and PFC/10 6 cells occurred 24 hr later than in controls and averaged, respectively, 27 and 141 percent of control values. Minimum viable cell numbers were observed in cortisol-treated cultures after 3 days; thereafter cell numbers gradually increased. These results were not significantly altered when cultures were treated simultaneously with cortisol and antigen. The response was not suppressed if the addition of antigen preceded that of cortisol by more than 4 hr. Suppression was also considerably reduced if fetal calf serum was used when preparing cells for culture

  15. Liver Progenitor Cell Line HepaRG Differentiated in a Bioartificial Liver Effectively Supplies Liver Support to Rats with Acute Liver Failure

    NARCIS (Netherlands)

    Nibourg, Geert A. A.; Chamuleau, Robert A. F. M.; van der Hoeven, Tessa V.; Maas, Martinus A. W.; Ruiter, An F. C.; Lamers, Wouter H.; Oude Elferink, Ronald P. J.; van Gulik, Thomas M.; Hoekstra, Ruurdtje

    2012-01-01

    A major roadblock to the application of bioartificial livers is the need for a human liver cell line that displays a high and broad level of hepatic functionality. The human bipotent liver progenitor cell line HepaRG is a promising candidate in this respect, for its potential to differentiate into

  16. Cell Patterning for Liver Tissue Engineering via Dielectrophoretic Mechanisms

    Directory of Open Access Journals (Sweden)

    Wan Nurlina Wan Yahya

    2014-07-01

    Full Text Available Liver transplantation is the most common treatment for patients with end-stage liver failure. However, liver transplantation is greatly limited by a shortage of donors. Liver tissue engineering may offer an alternative by providing an implantable engineered liver. Currently, diverse types of engineering approaches for in vitro liver cell culture are available, including scaffold-based methods, microfluidic platforms, and micropatterning techniques. Active cell patterning via dielectrophoretic (DEP force showed some advantages over other methods, including high speed, ease of handling, high precision and being label-free. This article summarizes liver function and regenerative mechanisms for better understanding in developing engineered liver. We then review recent advances in liver tissue engineering techniques and focus on DEP-based cell patterning, including microelectrode design and patterning configuration.

  17. Influence of heterologous MreB proteins on cell morphology of Bacillus subtilis.

    Science.gov (United States)

    Schirner, Kathrin; Errington, Jeff

    2009-11-01

    The prokaryotic cytoskeletal protein MreB is thought to govern cell shape by positioning the cell wall synthetic apparatus at growth sites in the cell. In rod-shaped bacteria it forms helical filaments that run around the periphery of the rod during elongation. Gram-positive bacteria often contain more than one mreB gene. Bacillus subtilis has three mreB-like genes, mreB, mbl and mreBH, the first two of which have been shown to be essential under normal growth conditions. Expression of an mreB homologue from the closely related organism Bacillus licheniformis did not have any effect on cell growth or morphology. In contrast, expression of mreB from the phylogenetically more distant bacterium Clostridium perfringens produced shape defects and ultimately cell death, due to disruption of the endogenous MreB cytoskeleton. However, expression of either mreB(B. licheniformis) (mreB(Bl)) or mreB(C. perfringens) (mreB(Cp)) was sufficient to confer a rod shape to B. subtilis deleted for the three mreB isologues, supporting the idea that the three proteins have largely redundant functions in cell morphogenesis. Expression of mreBCD(Bl) could fully compensate for the loss of mreBCD in B. subtilis and led to the formation of rod-shaped cells. In contrast, expression of mreBCD(Cp) was not sufficient to confer a rod shape to B. subtilis Delta mreBCD, indicating that a complex of these three cell shape determinants is not enough for cell morphogenesis of B. subtilis.

  18. Analytical study of cell liver proliferation and serum AFP in various liver diseases other than hepatomas

    Energy Technology Data Exchange (ETDEWEB)

    Takino, T; Okuda, K; Kitamura, O; Takahashi, T; Ashihara, T [Kyoto Prefectural Univ. of Medicine (Japan)

    1974-12-01

    Cell proliferative activity in the liver tissue obtained in 50 cases by liver biopsy, was analyzed using in vitro labeling of /sup 3/H-thymidine autoradiography. The proliferating cells were found to be located mainly in the periportal areas of the lobules. The mean labeling indices of the liver cells were 0.06 % in chronic hepatitis in its active form, 0.05 % in pre-cirrhosis of the liver, 0.03 % in liver cirrhosis, 0.02 % in chronic hepatitis in an inactive form and 0.018 % in acute hepatitis at the restoractive stage. The labeling indices of the liver parenchymal cells of each specimen studied were very low being at most 0.2 %. On the other hand, when the serum AFP was analyzed by radioimmunoassay technique in 185 patients with various liver diseases, level of the mean serum AFP in each group of the liver diseases was found to correspond to that of the proliferative activity of the liver cells in its respective group. From these data it was suggested that the proliferative activity of the liver cells in various liver diseases, with the exception of hepatomas, was closely related to release of AFP into the serum.

  19. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  20. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-01-01

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

  1. Cutaneous features seen in primary liver cell (Hepatocellular ...

    African Journals Online (AJOL)

    kemrilib

    features associated with the entity as a possible aid to diagnosis cutaneous features being considered a cheap tool that can help ... liver cell cancer (PLCC) and cancer of the breast and ... laboratory based -abdominal ultrasonography, liver.

  2. Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Liu, Zihe; Hou, Jin; Martinez Ruiz, José Luis

    2013-01-01

    .g., metabolic and cellular stresses have a strong impact on recombinant protein production. In this work, we investigated the effect of the specific growth rate on the production of two different recombinant proteins. Our results show that human insulin precursor is produced in a growth-associated manner...... turnover, cell cycle, and global stress response. We also found that there is a shift at a specific growth rate of 0.1 h−1 that influences protein production. Thus, for lower specific growth rates, the α-amylase and insulin precursor-producing strains present similar cell responses and phenotypes, whereas......With the increasing demand for biopharmaceutical proteins and industrial enzymes, it is necessary to optimize the production by microbial fermentation or cell cultures. Yeasts are well established for the production of a wide range of recombinant proteins, but there are also some limitations; e...

  3. Characterization of cell lysis in Pseudomonas putida induced upon expression of heterologous killing genes

    DEFF Research Database (Denmark)

    Ronchel, M.C.; Molina, L.; Witte, A.

    1998-01-01

    Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation agents and biopesticides, The physiological effects that lead to cell dea...... protein was the killing agent. In both cases, cell death occurred as a result of impaired respiration, altered membrane permeability, and the release of some cytoplasmic contents to the extracellular medium.......Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation agents and biopesticides, The physiological effects that lead to cell death......, respectively. Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter. Under induced conditions, killing of P. putida CMC12 cells mediated by phi X174 lysis protein E was faster than that observed for P. putida CMC4, for which the Gef...

  4. Memory NK cells: why do they reside in the liver?

    OpenAIRE

    Jiang, Xiaojun; Chen, Yonglin; Peng, Hui; Tian, Zhigang

    2013-01-01

    Immune memory is the hallmark of adaptive immunity. However, recent studies have shown that natural killer (NK) cells, key components of the innate immune system, also mediate memory responses in mice and humans. Strikingly, memory NK cells were liver-resident in some models, raising the question as to whether the liver is a special organ for the acquisition of NK cell memory. Here, we review the characteristics of NK cell memory by summarizing recent progress and discuss how the liver may ge...

  5. InXy and SeXy, compact heterologous reporter proteins for mammalian cells.

    Science.gov (United States)

    Fluri, David A; Kelm, Jens M; Lesage, Guillaume; Baba, Marie Daoud-El; Fussenegger, Martin

    2007-10-15

    Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream

  6. A unique heterologous fibrin sealant (HFS) as a candidate biological scaffold for mesenchymal stem cells in osteoporotic rats.

    Science.gov (United States)

    Orsi, Patrícia Rodrigues; Landim-Alvarenga, Fernanda Cruz; Justulin, Luis Antônio; Kaneno, Ramon; de Assis Golim, Marjorie; Dos Santos, Daniela Carvalho; Creste, Camila Fernanda Zorzella; Oba, Eunice; Maia, Leandro; Barraviera, Benedito; Ferreira, Rui Seabra

    2017-09-29

    The injection of mesenchymal stem cells (MSCs) directly into the bone of osteoporotic (OP) patients for rapid recovery has been studied worldwide. Scaffolds associated with MSCs are used to maintain and avoid cell loss after application. A unique heterologous fibrin sealant (HFS) derived from snake venom was evaluated for the cytotoxicity of its main components and as a three-dimensional biological scaffold for MSCs to repair a critical femur defect in osteoporotic rats. The cytotoxicity of HFS was assessed using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay and transmission electron microscopy. The cells were cultured, characterized by flow cytometry and differentiated into the osteogenic lineage. Two-month-old rats underwent ovariectomy to induce OP. After 3 months, a 5 mm critical bone defect was made in the distal end of the rat femurs and filled with HFS; HFS + MSCs; and HFS + MSCs D (differentiated into the osteogenic lineage) to evaluate the effects. An injury control group (injury and no treatment) and blank control group (no injury and no treatment) were also included. The animals were observed at days 14 and 28 by microtomographic (micro-CT) analyses, histologic and biochemical analysis, as well as scanning electron microscopy. The results revealed that one of the compounds of HFS, the thrombin-like enzyme extracted from snake venom, had no cytotoxic effects on the MSCs. OP was successfully induced, as demonstrated by the significant differences in the levels of 17β-estradiol, Micro-CT analyses and alkaline phosphatase between the ovariectomized (OVX) and non-ovariectomized (NOVX) groups. The histological data revealed that at 14 days after surgery in both the OVX and NOVX animals, the HFS + CTMs and HFS + CTMsD showed a higher formation of bone cells at the site in relation to the control group (without treatment). Collagen formation was evidenced through bone neoformation in all treated and control groups

  7. A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations

    Science.gov (United States)

    Murphy, Sean C.; Kas, Arnold; Stone, Brad C.; Bevan, Michael J.

    2013-01-01

    Development of an antimalarial subunit vaccine inducing protective cytotoxic T lymphocyte (CTL)-mediated immunity could pave the way for malaria eradication. Experimental immunization with sporozoites induces this type of protective response, but the extremely large number of proteins expressed by Plasmodium parasites has so far prohibited the identification of sufficient discrete T-cell antigens to develop subunit vaccines that produce sterile immunity. Here, using mice singly immunized with Plasmodium yoelii sporozoites and high-throughput screening, we identified a unique CTL response against the parasite ribosomal L3 protein. Unlike CTL responses to the circumsporozoite protein (CSP), the population of L3-specific CTLs was not expanded by multiple sporozoite immunizations. CSP is abundant in the sporozoite itself, whereas L3 expression does not increase until the liver stage. The response induced by a single immunization with sporozoites reduces the parasite load in the liver so greatly during subsequent immunizations that L3-specific responses are only generated during the primary exposure. Functional L3-specific CTLs can, however, be expanded by heterologous prime-boost regimens. Thus, although repeat sporozoite immunization expands responses to preformed antigens like CSP that are present in the sporozoite itself, this immunization strategy may not expand CTLs targeting parasite proteins that are synthesized later. Heterologous strategies may be needed to increase CTL responses across the entire spectrum of Plasmodium liver-stage proteins. PMID:23530242

  8. Heterologous expression of plant cell wall glycosyltransferases in Pichia, pea and tobacco

    DEFF Research Database (Denmark)

    Petersen, Bent Larsen; Damager, Iben; Faber, Kirsten

    Cell). In the present study, Flag-tagged (MDYKDDDD) RGXT2 was expressed in Pichia pastoris as secreted soluble protein, in pea (using the Pea early browning virus as expression vector) as soluble intra-cellular protein and in tobacco as full length membrane bound protein. The amount of expressed...... to participate in plant CW biosynthesis, has been achieved in only a few cases. We have previously reported the characterisation of two highly homologous plant-specific membrane-bound GTs, which when expressed as secreted tagged soluble proteins in the baculo virus system, catalysed the transfer of xylose from...... protein was estimated using anti Flag Ab and corresponding activity monitored. Pros and cons of using the various expression systems are discussed....

  9. The emerging role of mast cells in liver disease.

    Science.gov (United States)

    Jarido, Veronica; Kennedy, Lindsey; Hargrove, Laura; Demieville, Jennifer; Thomson, Joanne; Stephenson, Kristen; Francis, Heather

    2017-08-01

    The depth of our knowledge regarding mast cells has widened exponentially in the last 20 years. Once thought to be only important for allergy-mediated events, mast cells are now recognized to be important regulators of a number of pathological processes. The revelation that mast cells can influence organs, tissues, and cells has increased interest in mast cell research during liver disease. The purpose of this review is to refresh the reader's knowledge of the development, type, and location of mast cells and to review recent work that demonstrates the role of hepatic mast cells during diseased states. This review focuses primarily on liver diseases and mast cells during autoimmune disease, hepatitis, fatty liver disease, liver cancer, and aging in the liver. Overall, these studies demonstrate the potential role of mast cells in disease progression.

  10. Liver cell-targeted delivery of therapeutic molecules.

    Science.gov (United States)

    Kang, Jeong-Hun; Toita, Riki; Murata, Masaharu

    2016-01-01

    The liver is the largest internal organ in mammals and is involved in metabolism, detoxification, synthesis of proteins and lipids, secretion of cytokines and growth factors and immune/inflammatory responses. Hepatitis, alcoholic or non-alcoholic liver disease, hepatocellular carcinoma, hepatic veno-occlusive disease, and liver fibrosis and cirrhosis are the most common liver diseases. Safe and efficient delivery of therapeutic molecules (drugs, genes or proteins) into the liver is very important to increase the clinical efficacy of these molecules and to reduce their side effects in other organs. Several liver cell-targeted delivery systems have been developed and tested in vivo or ex vivo/in vitro. In this review, we discuss the literature concerning liver cell-targeted delivery systems, with a particular emphasis on the results of in vivo studies.

  11. Liver-cell patterning lab chip: mimicking the morphology of liver lobule tissue.

    Science.gov (United States)

    Ho, Chen-Ta; Lin, Ruei-Zeng; Chen, Rong-Jhe; Chin, Chung-Kuang; Gong, Song-En; Chang, Hwan-You; Peng, Hwei-Ling; Hsu, Long; Yew, Tri-Rung; Chang, Shau-Feng; Liu, Cheng-Hsien

    2013-09-21

    A lobule-mimetic cell-patterning technique for on-chip reconstruction of centimetre-scale liver tissue of heterogeneous hepatic and endothelial cells via an enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported. By mimicking the basic morphology of liver tissue, the classic hepatic lobule, the lobule-mimetic-stellate-electrodes array was designed for cell patterning. Through DEP manipulation, well-defined and enhanced spatial electric field gradients were created for in-parallel manipulation of massive individual cells. With this liver-cell patterning labchip design, the original randomly distributed hepatic and endothelial cells inside the microfluidic chamber can be manipulated separately and aligned into the desired pattern that mimicks the morphology of liver lobule tissue. Experimental results showed that both hepatic and endothelial cells were orderly guided, snared, and aligned along the field-induced orientation to form the lobule-mimetic pattern. About 95% cell viability of hepatic and endothelial cells was also observed after cell-patterning demonstration via a fluorescent assay technique. The liver function of CYP450-1A1 enzyme activity showed an 80% enhancement for our engineered liver tissue (HepG2+HUVECs) compared to the non-patterned pure HepG2 for two-day culturing.

  12. CD4 and CD8 T cell responses to the M. tuberculosis Ag85B-TB10.4 promoted by adjuvanted subunit, adenovector or heterologous prime boost vaccination

    DEFF Research Database (Denmark)

    Elvang, Tara; Christensen, Jan P; Billeskov, Rolf

    2009-01-01

    recruited to the site of infection, at the onset of infection. However, compared to CD8 T cells, CD4 T cells showed more extensive recruitment and were the main T cell subset proliferating at the site of infection. CONCLUSIONS/SIGNIFICANCE: Heterologous prime boost based on H4, produced an additive effect...

  13. Bioengineered Liver Models for Drug Testing and Cell Differentiation Studies

    Directory of Open Access Journals (Sweden)

    Gregory H. Underhill

    2018-01-01

    Full Text Available In vitro models of the human liver are important for the following: (1 mitigating the risk of drug-induced liver injury to human beings, (2 modeling human liver diseases, (3 elucidating the role of single and combinatorial microenvironmental cues on liver cell function, and (4 enabling cell-based therapies in the clinic. Methods to isolate and culture primary human hepatocytes (PHHs, the gold standard for building human liver models, were developed several decades ago; however, PHHs show a precipitous decline in phenotypic functions in 2-dimensional extracellular matrix–coated conventional culture formats, which does not allow chronic treatment with drugs and other stimuli. The development of several engineering tools, such as cellular microarrays, protein micropatterning, microfluidics, biomaterial scaffolds, and bioprinting, now allow precise control over the cellular microenvironment for enhancing the function of both PHHs and induced pluripotent stem cell–derived human hepatocyte-like cells; long-term (4+ weeks stabilization of hepatocellular function typically requires co-cultivation with liver-derived or non–liver-derived nonparenchymal cell types. In addition, the recent development of liver organoid culture systems can provide a strategy for the enhanced expansion of therapeutically relevant cell types. Here, we discuss advances in engineering approaches for constructing in vitro human liver models that have utility in drug screening and for determining microenvironmental determinants of liver cell differentiation/function. Design features and validation data of representative models are presented to highlight major trends followed by the discussion of pending issues that need to be addressed. Overall, bioengineered liver models have significantly advanced our understanding of liver function and injury, which will prove useful for drug development and ultimately cell-based therapies.

  14. Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin.

    Science.gov (United States)

    Figueira, Cláudio Pereira; Croda, Julio; Choy, Henry A; Haake, David A; Reis, Mitermayer G; Ko, Albert I; Picardeau, Mathieu

    2011-06-09

    In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.

  15. Liver-resident NK cells and their potential functions.

    Science.gov (United States)

    Peng, Hui; Sun, Rui

    2017-09-18

    Natural killer (NK) cells represent a heterogeneous population of innate lymphocytes with phenotypically and functionally distinct subsets. In particular, recent studies have identified a unique subset of NK cells residing within the liver that are maintained as tissue-resident cells, confer antigen-specific memory responses and exhibit different phenotypical and developmental characteristics compared with conventional NK (cNK) cells. These findings have encouraged researchers to uncover tissue-resident NK cells at other sites, and detailed analyses have revealed that these tissue-resident NK cells share many similarities with liver-resident NK cells and tissue-resident memory T cells. Here, we present a brief historical perspective on the discovery of liver-resident NK cells and discuss their relationship to cNK cells and other emerging NK cell subsets and their potential functions.Cellular &Molecular Immunology advance online publication, 18 September 2017; doi:10.1038/cmi.2017.72.

  16. Memory NK cells: why do they reside in the liver?

    Science.gov (United States)

    Jiang, Xiaojun; Chen, Yonglin; Peng, Hui; Tian, Zhigang

    2013-05-01

    Immune memory is the hallmark of adaptive immunity. However, recent studies have shown that natural killer (NK) cells, key components of the innate immune system, also mediate memory responses in mice and humans. Strikingly, memory NK cells were liver-resident in some models, raising the question as to whether the liver is a special organ for the acquisition of NK cell memory. Here, we review the characteristics of NK cell memory by summarizing recent progress and discuss how the liver may generate both the initiation and the recall phase of memory. We propose that the liver may have unique precursors for memory NK cells, which are developmentally distinct from NK cells derived from bone marrow.

  17. Fraction from human and rat liver which is inhibitory for proliferation of liver cells.

    Science.gov (United States)

    Chen, T S; Ottenweller, J; Luke, A; Santos, S; Keeting, P; Cuy, R; Lea, M A

    1989-01-01

    A comparative study was undertaken with human and rat liver of a fraction reported to have growth inhibitory activity when prepared from rat liver. Fractions which were soluble in 70% ethanol and insoluble in 87% ethanol were prepared from liver cytosols. Electrophoretic analysis under denaturing conditions indicated that there were several quantitative or qualitative differences in the fractions from the two species. Fractions from both human and rat liver were found to be inhibitory for the incorporation of 3H-thymidine into DNA of foetal chick hepatocytes. Under conditions in which the rat fraction inhibited precursor incorporation into DNA of rat liver epithelial cells there was not a significant inhibitory effect with the fraction from human liver. DNA synthesis in a rat hepatoma cell line was not significantly inhibited by preparations from either species. The data suggested that corresponding fractions from both rat and human liver could have inhibitory effects on precursor incorporation into DNA but the magnitude of the effects and target cell specificity may differ.

  18. Generation and characterization of rat liver stem cell lines and their engraftment in a rat model of liver failure

    Science.gov (United States)

    Kuijk, Ewart W.; Rasmussen, Shauna; Blokzijl, Francis; Huch, Meritxell; Gehart, Helmuth; Toonen, Pim; Begthel, Harry; Clevers, Hans; Geurts, Aron M.; Cuppen, Edwin

    2016-01-01

    The rat is an important model for liver regeneration. However, there is no in vitro culture system that can capture the massive proliferation that can be observed after partial hepatectomy in rats. We here describe the generation of rat liver stem cell lines. Rat liver stem cells, which grow as cystic organoids, were characterized by high expression of the stem cell marker Lgr5, by the expression of liver progenitor and duct markers, and by low expression of hepatocyte markers, oval cell markers, and stellate cell markers. Prolonged cultures of rat liver organoids depended on high levels of WNT-signalling and the inhibition of BMP-signaling. Upon transplantation of clonal lines to a Fah−/− Il2rg−/− rat model of liver failure, the rat liver stem cells engrafted into the host liver where they differentiated into areas with FAH and Albumin positive hepatocytes. Rat liver stem cell lines hold potential as consistent reliable cell sources for pharmacological, toxicological or metabolic studies. In addition, rat liver stem cell lines may contribute to the development of regenerative medicine in liver disease. To our knowledge, the here described liver stem cell lines represent the first organoid culture system in the rat. PMID:26915950

  19. On the postradiation effect of cystamine in rat liver cells

    International Nuclear Information System (INIS)

    Gil'yano, N.Ya.; Malinovskij, O.V.

    1979-01-01

    The effect of cystamine, classical radioprotector introduced prior to and after irradiation has been tested. The protector has been paralelly tested on the regenerating liver in a presynthetic phase of a mitotic cycle (G 1 ). White nonbred male mice have been irradiated with 205 and 6O5 rad on the RUM-11 X-ray apparatus prior to (intact liver) and 6 hours after (regenerating liver) partial hepatectomy. Cystamine has been injected to animals 15 min prior to irradiation and in different periods after irradiation in the concentration of 150 mg/kg of weight. The decrease in the share of cells with asymmetic chromosome transformations (bridges and fragments in the anaphase) has been the protector effectiveness index. It is shown that the protector is effective in the cells of a regenerating liver only when introduced before irradiation, while in the cells of an intact liver it produces a protective effect both prior to and 15 mins after irradiation. Cystamine effectiveness for cells of the intact and regenerating liver has been investigated by introducing it after irradiation of animals with 250 and 305 rad. It has been established that the protector makes it possible to modify the irradiation effect within 20 mins after irradiation in the cells of the intact liver (Go). Cystamine postradiation protection in the cells of the regenerating liver (G 1 ) is low if it is introduced immediately after irradiation (1 min) and is absent after 10 min. The dependence of cystamine postradiation protective effect on the moment of liver cell stimulation has been investigated. It has been shown that the modification of irradiation effect is possible within 5 hrs after irradiation if the protector is introduced 15 min after irradiation of animals with 250 rad. The mechanism of the preparation action is discussed

  20. Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

    Science.gov (United States)

    Nair, Nisha R; Chidambareswaren, M; Manjula, S

    2014-09-01

    Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

  1. Hepatic stellate cell and myofibroblast-like cell gene expression in the explanted cirrhotic livers of patients undergoing liver transplantation.

    Science.gov (United States)

    Estep, J Michael; O'Reilly, Linda; Grant, Geraldine; Piper, James; Jonsson, Johann; Afendy, Arian; Chandhoke, Vikas; Younossi, Zobair M

    2010-02-01

    Hepatic stellate cells (HSC) are involved in hepatic fibrogenesis. Cell signaling associated with an insult to the liver affects an HSC transdifferentiation to fibrogenic myofibroblast-like cells. To investigate the transcriptional expression distinguishing HSC and myofibroblast-like cells between livers with and without cirrhosis. Tissue from ten cirrhotic livers (undergoing transplant) and four non-cirrhotic livers from the National Disease Research Interchange underwent cell separation to extract HSC and myofibroblast-like cell populations. Separated cell types as well as LI-90 cells were subjected to microarray analysis. Selected microarray results were verified by quantitative real-time PCR. Differential expression of some genes, such as IL-1beta, IL-1alpha, and IL-6, was associated with both transdifferentiation and disease. Other genes, such as fatty acid 2-hydroxylase only show differential expression in association with disease. Functional analysis supported these findings, indicating some signal transduction pathways (IL-6) are involved in disease and activation, whereas retinoid X receptor signaling in HSC from cirrhotic and non-cirrhotic livers varies in scope and quality. These findings indicate distinct phenotypes for HSC from cirrhotic and non-cirrhotic livers. Furthermore, coordinated differential expression between genes involved in the same signal transduction pathways provides some insight into the mechanisms that may control the balance between fibrogenesis and fibrolysis.

  2. Cell sources for in vitro human liver cell culture models

    Science.gov (United States)

    Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-01-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

  3. Ovarian Sertoli-Leydig cell tumor with heterologous elements of gastrointestinal type associated with elevated serum alpha-fetoprotein level: an unusual case and literature review.

    Science.gov (United States)

    Horta, Mariana; Cunha, Teresa Margarida; Marques, Rita Canas; Félix, Ana

    2014-11-01

    Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alpha-fetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrine-inactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker.

  4. Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

    Directory of Open Access Journals (Sweden)

    Lili Jiang

    Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

  5. Liver sinusoidal endothelial cells induce immunosuppressive IL-10-producing Th1 cells via the Notch pathway

    NARCIS (Netherlands)

    Neumann, Katrin; Rudolph, Christine; Neumann, Christian; Janke, Marko; Amsen, Derk; Scheffold, Alexander

    2015-01-01

    Under homeostasis, liver sinusoidal endothelial cells (LSECs) shift intrahepatic T-cell responses towards tolerance. However, the role of LSECs in the regulation of T-cell-induced liver inflammation is less clear. Here, we studied the capacity of LSECs to modulate pro-inflammatory Th1-cell

  6. Transcriptome atlas of eight liver cell types uncovers effects of ...

    Indian Academy of Sciences (India)

    ... types, and bioinformatic and systems biology approaches were employed to analyse the relationship between above genes and rat liver regeneration. The results showed that the urocanic acid (UA) was degraded from histidine in Kupffer cells, acts on Kupffer cells itself and dendritic cells to generate immune suppression ...

  7. NK Cell Subtypes as Regulators of Autoimmune Liver Disease

    Directory of Open Access Journals (Sweden)

    Guohui Jiao

    2016-01-01

    Full Text Available As major components of innate immunity, NK cells not only exert cell-mediated cytotoxicity to destroy tumors or infected cells, but also act to regulate the functions of other cells in the immune system by secreting cytokines and chemokines. Thus, NK cells provide surveillance in the early defense against viruses, intracellular bacteria, and cancer cells. However, the effecter function of NK cells must be exquisitely controlled to prevent inadvertent attack against normal “self” cells. In an organ such as the liver, where the distinction between immunotolerance and immune defense against routinely processed pathogens is critical, the plethora of NK cells has a unique role in the maintenance of homeostasis. Once self-tolerance is broken, autoimmune liver disease resulted. NK cells act as a “two-edged weapon” and even play opposite roles with both regulatory and inducer activities in the hepatic environment. That is, NK cells act not only to produce inflammatory cytokines and chemokines, but also to alter the proliferation and activation of associated lymphocytes. However, the precise regulatory mechanisms at work in autoimmune liver diseases remain to be identified. In this review, we focus on recent research with NK cells and their potential role in the development of autoimmune liver disease.

  8. Improving L-arabinose utilization of pentose fermenting Saccharomyces cerevisiae cells by heterologous expression of L-arabinose transporting sugar transporters

    Directory of Open Access Journals (Sweden)

    Boles Eckhard

    2011-10-01

    Full Text Available Abstract Background Hydrolysates of plant biomass used for the production of lignocellulosic biofuels typically contain sugar mixtures consisting mainly of D-glucose and D-xylose, and minor amounts of L-arabinose. The yeast Saccharomyces cerevisiae is the preferred microorganism for the fermentative production of ethanol but is not able to ferment pentose sugars. Although D-xylose and L-arabinose fermenting S. cerevisiae strains have been constructed recently, pentose uptake is still a limiting step in mixed sugar fermentations. Results Here we described the cloning and characterization of two sugar transporters, AraT from the yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana, which mediate the uptake of L-arabinose but not of D-glucose into S. cerevisiae cells. A yeast strain lacking all of its endogenous hexose transporter genes and expressing a bacterial L-arabinose utilization pathway could no longer take up and grow with L-arabinose as the only carbon source. Expression of the heterologous transporters supported uptake and utilization of L-arabinose especially at low L-arabinose concentrations but did not, or only very weakly, support D-glucose uptake and utilization. In contrast, the S. cerevisiae D-galactose transporter, Gal2, mediated uptake of both L-arabinose and D-glucose, especially at high concentrations. Conclusions Using a newly developed screening system we have identified two heterologous sugar transporters from a yeast and a plant which can support uptake and utilization of L-arabinose in L-arabinose fermenting S. cerevisiae cells, especially at low L-arabinose concentrations.

  9. T cell progenitors in the mouse fetal liver

    International Nuclear Information System (INIS)

    Rabinowich, H.; Umiel, T.; Globerson, A.

    1983-01-01

    Fourteen-day mouse fetal liver was found to contain cells capable of giving rise to T as well as B cell functions. The experimental system consisted of congenic C3H/DiSn and (C3H/DiSn X C3H.SW)F1 lethally irradiated (900 R) mice reconstituted with C3H/DiSn fetal liver or bone marrow cells. Assays included thyroid allograft rejection as well as in vitro measurement of reactivity to phytohemagglutinin (PHA) and concanavalin A (Con A) and in a mixed lymphocyte culture (MLC) system in spleen, lymph node, and thymus cells. The fetal liver chimeras were found to become as capable as the bone marrow chimeras in responding in these various assays. The T cell responses lagged behind the responses to the B cell mitogens dextran sulfate (DXS) and lipopolysaccharide (LPS) (30 days after reconstitution, as compared with 14 days for DXS and 21 for LPS). The reacting cells were of the donor genotype, as revealed after treatment with C3H/DiSn (H-2k) anti-C3H.SW (H-2b) congenic sera. T cell responses were not manifest in thymectomized (TX) chimeras. Hence, the liver seems to contain cells capable of developing into T cell lineages in a thymus-dependent process

  10. Fetal liver stromal cells promote hematopoietic cell expansion

    International Nuclear Information System (INIS)

    Zhou, Kun; Hu, Caihong; Zhou, Zhigang; Huang, Lifang; Liu, Wenli; Sun, Hanying

    2009-01-01

    Future application of hematopoietic stem and progenitor cells (HSPCs) in clinical therapies largely depends on their successful expansion in vitro. Fetal liver (FL) is a unique hematopoietic organ in which hematopoietic cells markedly expand in number, but the mechanisms involved remain unclear. Stromal cells (StroCs) have been suggested to provide a suitable cellular environment for in vitro expansion of HSPCs. In this study, murine StroCs derived from FL at E14.5, with a high level of Sonic hedgehog (Shh) and Wnt expression, were found to have an increased ability to support the proliferation of HSPCs. This effect was inhibited by blocking Shh signaling. Supplementation with soluble Shh-N promoted the proliferation of hematopoietic cells by activating Wnt signaling. Our findings suggest that FL-derived StroCs support proliferation of HSPCs via Shh inducing an autocrine Wnt signaling loop. The use of FL-derived StroCs and regulation of the Shh pathway might further enhance HPSC expansion.

  11. Hepatocytes polyploidization and cell cycle control in liver physiopathology.

    Science.gov (United States)

    Gentric, Géraldine; Desdouets, Chantal; Celton-Morizur, Séverine

    2012-01-01

    Most cells in mammalian tissues usually contain a diploid complement of chromosomes. However, numerous studies have demonstrated a major role of "diploid-polyploid conversion" during physiopathological processes in several tissues. In the liver parenchyma, progressive polyploidization of hepatocytes takes place during postnatal growth. Indeed, at the suckling-weaning transition, cytokinesis failure events induce the genesis of binucleated tetraploid liver cells. Insulin signalling, through regulation of the PI3K/Akt signalling pathway, is essential in the establishment of liver tetraploidization by controlling cytoskeletal organisation and consequently mitosis progression. Liver cell polyploidy is generally considered to indicate terminal differentiation and senescence, and both lead to a progressive loss of cell pluripotency associated to a markedly decreased replication capacity. Although adult liver is a quiescent organ, it retains a capacity to proliferate and to modulate its ploidy in response to various stimuli or aggression (partial hepatectomy, metabolic overload (i.e., high copper and iron hepatic levels), oxidative stress, toxic insult, and chronic hepatitis etc.). Here we review the mechanisms and functional consequences of hepatocytes polyploidization during normal and pathological liver growth.

  12. Hepatocytes Polyploidization and Cell Cycle Control in Liver Physiopathology

    Directory of Open Access Journals (Sweden)

    Géraldine Gentric

    2012-01-01

    Full Text Available Most cells in mammalian tissues usually contain a diploid complement of chromosomes. However, numerous studies have demonstrated a major role of “diploid-polyploid conversion” during physiopathological processes in several tissues. In the liver parenchyma, progressive polyploidization of hepatocytes takes place during postnatal growth. Indeed, at the suckling-weaning transition, cytokinesis failure events induce the genesis of binucleated tetraploid liver cells. Insulin signalling, through regulation of the PI3K/Akt signalling pathway, is essential in the establishment of liver tetraploidization by controlling cytoskeletal organisation and consequently mitosis progression. Liver cell polyploidy is generally considered to indicate terminal differentiation and senescence, and both lead to a progressive loss of cell pluripotency associated to a markedly decreased replication capacity. Although adult liver is a quiescent organ, it retains a capacity to proliferate and to modulate its ploidy in response to various stimuli or aggression (partial hepatectomy, metabolic overload (i.e., high copper and iron hepatic levels, oxidative stress, toxic insult, and chronic hepatitis etc.. Here we review the mechanisms and functional consequences of hepatocytes polyploidization during normal and pathological liver growth.

  13. TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA enhances CD8+ T Cell responses providing protection against Leishmania (Viannia.

    Directory of Open Access Journals (Sweden)

    Asha Jayakumar

    2011-06-01

    Full Text Available Leishmania (Viannia parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.Using a newly developed mouse model of chronic L. (Viannia panamensis infection and the heterologous DNA prime - modified vaccinia virus Ankara (MVA boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP could provide protection against infection/disease.Heterologous prime - boost (DNA/MVA vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V. panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that

  14. Liver involvement in Langerhans' cell histiocytosis. Case report.

    Science.gov (United States)

    Dina, Ion; Copaescu, Catalin; Herlea, Vlad; Wrba, Fritz; Iacobescu, Claudia

    2006-03-01

    Langerhans'cell histiocytosis (Histiocytosis X) is a rare disease of unknown cause characterized by oligoclonal proliferation of Langerhans cells. It occurs mostly in children and young adults and involves one or more body systems such as bone, hypothalamus, posterior pituitary gland, lymph nodes, liver or various soft tissues. The diagnosis is always made by a histological approach. We report a case of Langerhans'cell histiocytosis in a young patient with clinical signs of diabetes insipidus and hepatic involvement in whom the immunohistochemical analysis of the liver tissue led to the definitive diagnosis.

  15. Heterologous Protein Expression by Lactococcus lactis

    NARCIS (Netherlands)

    Villatoro-Hernández, J.; Kuipers, O.P.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.

    2012-01-01

    This chapter describes the use of Lactococcus lactis as a safe and efficient cell factory to produce heterologous proteins of medical interest. The relevance of the use of this lactic acid bacterium (LAB) is that it is a noncolonizing, nonpathogenic microorganism that can be delivered in vivo at a

  16. A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Lay Teng Ang

    2018-02-01

    Full Text Available How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs. Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-β, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah−/−Rag2−/−Il2rg−/− mouse model of liver failure.

  17. A new liver function test using the asialoglycoprotein-receptor system on the liver cell membrane, 3

    International Nuclear Information System (INIS)

    Hazama, Hiroshi; Kawa, Soukichi; Kubota, Yoshitsugu

    1986-01-01

    We evaluated the vilidity of a new liver function test using liver scintigraphy based on the asialoglycoprotein (ASGP) receptor system on the liver cell membrane in rats with galactosamine-induced acute liver disorder and those with carbon tetra-chloride-induced chronic liver disorder. Neoglycoprotein (GHSA) produced by combining human serum albumin with 32 galactose units was labeled with 99m Tc and administered (50 μg/100 g body weight) to rats with acute or chronic liver disorder. Clearance curves were produced based on liver scintigrams and analysed using the two-compartment model to obtain parameters. In acute liver disorder, the prolongation of 99m Tc-GHSA clearance and the decrease in ASGP receptor activities correlated well to the increase in serum GOT and the decrease in the esterified to total cholesterol ratio (E/T ratio); in chronic liver disorder, they correlated significantly to the increase in the content of liver hydroxyproline (Hyp) which increased in proportion to the severity of liver fibrosis studied histologically, and to the decrease in the contents of cytochrome P-450 and cytochrome b 5 in liver microsomes. Significant correlation was observed between the prolongation of 99m Tc-GHSA clearance and the decrease in ASGP receptor activities in both acute and chronic liver disorders. These findings indicate that the measurement of 99m Tc-GHSA clearance can be a new liver function test sensitively reflecting the severity of liver damage. (author)

  18. Different levels of immunogenicity of two strains of Fowlpox virus as recombinant vaccine vectors eliciting T-cell responses in heterologous prime-boost vaccination strategies.

    Science.gov (United States)

    Cottingham, Matthew G; van Maurik, Andre; Zago, Manola; Newton, Angela T; Anderson, Richard J; Howard, M Keith; Schneider, Jörg; Skinner, Michael A

    2006-07-01

    The FP9 strain of F has been described as a more immunogenic recombinant vaccine vector than the Webster FPV-M (FPW) strain (R. J. Anderson et al., J. Immunol. 172:3094-3100, 2004). This study expands the comparison to include two separate recombinant antigens and multiple, rather than single, independent viral clones derived from the two strains. Dual-poxvirus heterologous prime-boost vaccination regimens using individual clones of recombinant FP9 or FPW in combination with recombinant modified V Ankara expressing the same antigen were evaluated for their ability to elicit T-cell responses against recombinant antigens from Plasmodium berghei (circumsporozoite protein) or human immunodeficiency virus type 1 (a Gag-Pol-Nef fusion protein). Gamma interferon enzyme-linked immunospot assay and fluorescence-activated cell sorting assays of the responses to specific epitopes confirmed the approximately twofold-greater cellular immunogenicity of FP9 compared to FPW, when given as the priming or boosting immunization. Equality of transgene expression in mouse cells infected with the two strains in vitro was verified by Western blotting. Directed partial sequence analysis and PCR analysis of FPW and comparison to available whole-genome sequences revealed that many loci that are mutated in the highly attenuated and culture-adapted FP9 strain are wild type in FPW, including the seven multikilobase deletions. These "passage-specific" alterations are hypothesized to be involved in determining the immunogenicity of fowlpox virus as a recombinant vaccine vector.

  19. Sensitivity of mitochondria of the mouse liver cells to radiation

    International Nuclear Information System (INIS)

    Shima, Akihiro

    1974-01-01

    In order to study the sensitivity of mitochondria (Mt) of the liver cells to radiation, 0.4 mg of riboflavine (RF) was intraperitoneally injected into mice which had been fed RF deficient food for 13 weeks. Three hours later 400 R of X-ray (190 KVP, 25 mA, 0.5 mmCu, 0.5 mmAl filter, FSD 61.5 cm, and HVL 0.80 mmCu) were irradiated to the whole body, and giant Mt of the liver cells were observed. When the liver cells were observed 24 hours after injection, neither giant Mt nor mitotic findings of Mt were found. All Mt observed were small (1.2 μ), although mice received 400 R of X-ray. (Serizawa, K.)

  20. Sensitivity of mitochondria of the mouse liver cells to radiation

    Energy Technology Data Exchange (ETDEWEB)

    Shima, A [Tokyo Univ. (Japan). Faculty of Science

    1974-06-01

    In order to study the sensitivity of mitochondria (Mt) of the liver cells to radiation, 0.4 mg of riboflavine (RF) was intraperitoneally injected into mice which had been fed RF deficient food for 13 weeks. Three hours later 400 R of X-ray (190 KVP, 25 mA, 0.5 mmCu, 0.5 mmAl filter, FSD 61.5 cm, and HVL 0.80 mmCu) were irradiated to the whole body, and giant Mt of the liver cells were observed. When the liver cells were observed 24 hours after injection, neither giant Mt nor mitotic findings of Mt were found. All Mt observed were small (1.2 ..mu..), although mice received 400 R of X-ray.

  1. [Hepatic cell transplantation: a new therapy in liver diseases].

    Science.gov (United States)

    Pareja, Eugenia; Cortés, Miriam; Martínez, Amparo; Vila, Juan José; López, Rafael; Montalvá, Eva; Calzado, Angeles; Mir, José

    2010-07-01

    Liver transplantation has been remarkably effective in the treatment in patients with end-stage liver disease. However, disparity between solid-organ supply and increased demand is the greatest limitation, resulting in longer waiting times and increase in mortality of transplant recipients. This situation creates the need to seek alternatives to orthotopic liver transplantation.Hepatocyte transplantation or liver cell transplantation has been proposed as the best method to support patients. The procedure consists of transplanting individual cells to a recipient organ in sufficient quantity to survive and restore the function. The capacity of hepatic regeneration is the biological basis of hepatocyte transplantation. This therapeutic option is an experimental procedure in some patients with inborn errors of metabolism, fulminant hepatic failure and acute and chronic liver failure, as a bridge to orthotopic liver transplantation. In the Hospital La Fe of Valencia, we performed the first hepatocyte transplantation in Spain creating a new research work on transplant program. Copyright 2009 AEC. Published by Elsevier Espana. All rights reserved.

  2. Characterization of genetically engineered mouse hepatoma cells with inducible liver functions by overexpression of liver-enriched transcription factors.

    Science.gov (United States)

    Yamamoto, Hideaki; Tonello, Jane Marie; Sambuichi, Takanori; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

    2018-01-01

    New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1β, HNF3β [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Immunization of Mice with a Live Transconjugant Shigella Hybrid Strain Induced Th1 and Th17 Cell-Mediated Immune Responses and Confirmed Passive Protection Against Heterologous Shigellae.

    Science.gov (United States)

    Nag, D; Koley, H; Sinha, R; Mukherjee, P; Sarkar, C; Withey, J H; Gachhui, R

    2016-02-01

    An avirulent, live transconjugant Shigella hybrid (LTSHΔstx) strain was constructed in our earlier study by introducing a plasmid vector, pPR1347, into a Shiga toxin gene deleted Shigella dysenteriae 1. Three successive oral administrations of LTSHΔstx to female adult mice produced comprehensive passive heterologous protection in their offspring against challenge with wild-type shigellae. Production of NO and different cytokines such asIL-12p70, IL-1β and IL-23 in peritoneal mice macrophages indicated that LTSHΔstx induced innate and adaptive immunity in mice. Furthermore, production of IFN-γ, IL-10 and IL-17 in LTSH-primed splenic CD4+ T cell suggested that LTSHΔstx may induce Th1 and Th17 cell-mediated immune responses. Exponential increase of the serum IgG and IgA titre against whole shigellae was observed in immunized adult mice during and after the immunization with the highest peak on day 35. Antigen-specific sIgA was also determined from intestinal lavage of immunized mice. The stomach extracts of neonates from immunized mice, mainly containing mother's milk, contained significant levels of anti-LTSHΔstx immunoglobulin. These studies suggest that the LTSHΔstx could be a new live oral vaccine candidate against shigellosis in the near future. © 2015 The Foundation for the Scandinavian Journal of Immunology.

  4. Hypercholesterolemia Induces Differentiation of Regulatory T Cells in the Liver.

    Science.gov (United States)

    Mailer, Reiner K W; Gisterå, Anton; Polyzos, Konstantinos A; Ketelhuth, Daniel F J; Hansson, Göran K

    2017-05-26

    The liver is the central organ that responds to dietary cholesterol intake and facilitates the release and clearance of lipoprotein particles. Persistent hypercholesterolemia leads to immune responses against lipoprotein particles that drive atherosclerosis. However, the effect of hypercholesterolemia on hepatic T-cell differentiation remains unknown. To investigate hepatic T-cell subsets upon hypercholesterolemia. We observed that hypercholesterolemia elevated the intrahepatic regulatory T (Treg) cell population and increased the expression of transforming growth factor-β1 in the liver. Adoptive transfer experiments revealed that intrahepatically differentiated Treg cells relocated to the inflamed aorta in atherosclerosis-prone low-density lipoprotein receptor deficient ( Ldlr -/- ) mice. Moreover, hypercholesterolemia induced the differentiation of intrahepatic, but not intrasplenic, Th17 cells in wild-type mice, whereas the disrupted liver homeostasis in hypercholesterolemic Ldlr -/- mice led to intrahepatic Th1 cell differentiation and CD11b + CD11c + leukocyte accumulation. Our results elucidate a new mechanism that controls intrahepatic T-cell differentiation during atherosclerosis development and indicates that intrahepatically differentiated T cells contribute to the CD4 + T-cell pool in the atherosclerotic aorta. © 2017 American Heart Association, Inc.

  5. Optimizing HIV-1-specific CD8+ T-cell induction by recombinant BCG in prime-boost regimens with heterologous viral vectors.

    Science.gov (United States)

    Hopkins, Richard; Bridgeman, Anne; Bourne, Charles; Mbewe-Mvula, Alice; Sadoff, Jerald C; Both, Gerald W; Joseph, Joan; Fulkerson, John; Hanke, Tomáš

    2011-12-01

    The desire to induce HIV-1-specific responses soon after birth to prevent breast milk transmission of HIV-1 led us to propose a vaccine regimen which primes HIV-1-specific T cells using a recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) vaccine. Because attenuated live bacterial vaccines are typically not sufficiently immunogenic as stand-alone vaccines, rBCG-primed T cells will likely require boost immunization(s). Here, we compared modified Danish (AERAS-401) and Pasteur lysine auxotroph (222) strains of BCG expressing the immunogen HIVA for their potency to prime HIV-1-specific responses in adult BALB/c mice and examined four heterologous boosting HIVA vaccines for their immunogenic synergy. We found that both BCG.HIVA(401) and BCG.HIVA(222) primed HIV-1-specific CD8(+) T-cell-mediated responses. The strongest boosts were delivered by human adenovirus-vectored HAdV5.HIVA and sheep atadenovirus-vectored OAdV7.HIVA vaccines, followed by poxvirus MVA.HIVA; the weakest was plasmid pTH.HIVA DNA. The prime-boost regimens induced T cells capable of efficient in vivo killing of sensitized target cells. We also observed that the BCG.HIVA(401) and BCG.HIVA(222) vaccines have broadly similar immunologic properties, but display a number of differences mainly detected through distinct profiles of soluble intercellular signaling molecules produced by immune splenocytes in response to both HIV-1- and BCG-specific stimuli. These results encourage further development of the rBCG prime-boost regimen. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Adaption of Saccharomyces cerevisiae expressing a heterologous protein

    DEFF Research Database (Denmark)

    Krogh, Astrid Mørkeberg; Beck, Vibe; Højlund Christensen, Lars

    2008-01-01

    Production of the heterologous protein, bovine aprotinin, in Saccharomyces cerevisiae was shown to affect the metabolism of the host cell to various extent depending on the strain genotype. Strains with different genotypes, industrial and laboroatory, respectively, were investigated. The maximal...

  7. Cell Pleomorphism and Cytoskeleton Disorganization in Human Liver Cancer.

    Science.gov (United States)

    Cheng, Chiung-Chi; Lai, Yen-Chang Clark; Lai, Yih-Shyong; Chao, Wei-Ting; Tseng, Yu-Hui; Hsu, Yung-Hsiang; Chen, You-Yin; Liu, Yi-Hsiang

    Nucleoskeleton maintains the framework of a cell nucleus that is required for a variety of nuclear functions. However, the nature of nucleoskeleton structure has not been yet clearly elucidated due to microscopy visualization limitations. Plectin, a nuclear pore-permeable component of cytoskeleton, exhibits a role of cross-linking between cytoplasmic intermediate filaments and nuclear lamins. Presumably, plectin is also a part of nucleoskeleton. Previously, we demonstrated that pleomorphism of hepatoma cells is the consequence of cytoskeletal changes mediated by plectin deficiency. In this study, we applied a variety of technologies to detect the cytoskeletons in liver cells. The images of confocal microscopy did not show the existence of plectin, intermediate filaments, microfilaments and microtubules in hepatic nuclei. However, in the isolated nuclear preparation, immunohistochemical staining revealed positive results for plectin and cytoskeletal proteins that may contribute to the contamination derived from cytoplasmic residues. Therefore, confocal microscopy provides a simple and effective technology to observe the framework of nucleoskeleton. Accordingly, we verified that cytoskeletons are not found in hepatic cell nuclei. Furthermore, the siRNA-mediated knockdown of plectin in liver cells leads to collapsed cytoskeleton, cell transformation and pleomorphic nuclei. Plectin and cytoskeletons were not detected in the nuclei of liver cells compared to the results of confocal microscopy. Despite the absence of nuclear plectin and cytoskeletal filaments, the evidence provided support that nuclear pleomorphism of cancer cells is correlated with the cytoplasmic disorganization of cytoskeleton. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  8. Stem Cells Transplantation in the Treatment of Patients with Liver Failure.

    Science.gov (United States)

    Tao, Ya-Chao; Wang, Meng-Lan; Chen, En-Qiang; Tang, Hong

    2018-02-23

    Liver failure is a life-threatening liver disease encompassing severe acute deterioration of liver function. Emergency liver transplantation is the only curative treatment for liver failure, but is restricted by the severe shortage of organ donors. Stem cell, including embroyonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, hematopoietic stem cells and hepatic progenitor cells, have capacity to proliferate and differentiate and could be used in a variety of liver diseases including hereditary liver diseases, cirrhosis and liver failure. We summarized the basic experimental and clinical advances of stem cell transplantation in liver failure treatment, and also discussed the advantages and disadvantage of different stem cells subtype in this field, aiming to provide a perspective on the stem cell-based therapy for liver failure. Stem cells, especially mesenchymal stem cells (mainly low immunogenicity and paracrine characteristics) and induced pluripotent stem cells (generation of desired cell type from somatic cell), are feasible candidates for cell therapy in the treatment of liver failure, but there are some drawbacks remaining to be resolved, such as low engraftment, cryotpreservation methods and tumorigenesis. Stem cell transplantation is a promising but challenging strategy and paves a new way for curing liver failure. But more efforts need to be made to overcome problems before this new strategy could be safely and effectively applied to humans. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. Steroidogenesis and early response gene expression in MA-10 Leydig tumor cells following heterologous receptor down-regulation and cellular desensitization

    Directory of Open Access Journals (Sweden)

    Tsuey-Ming Chen

    2016-03-01

    Full Text Available The Leydig tumor cell line, MA-10, expresses the luteinizing hormone receptor, a G protein-coupled receptor that, when activated with luteinizing hormone or chorionic gonadotropin (CG, stimulates cAMP production and subsequent steroidogenesis, notably progesterone. These cells also respond to epidermal growth factor (EGF and phorbol esters with increased steroid biosynthesis. In order to probe the intracellular pathways along with heterologous receptor down-regulation and cellular desensitization, cells were preincubated with EGF or phorbol esters and then challenged with CG, EGF, dibutryl-cyclic AMP, and a phorbol ester. Relative receptor numbers, steroid biosynthesis, and expression of the early response genes, JUNB and c-FOS, were measured. It was found that in all cases but one receptor down-regulation and decreased progesterone production were closely coupled under the conditions used; the exception involved preincubation of the cells with EGF followed by addition of CG where the CG-mediated stimulation of steroidogenesis was considerably lower than the level of receptor down-regulation. In a number of instances JUNB and c-FOS expression paralleled the decreases in receptor number and progesterone production, while in some cases these early response genes were affected little if at all by the changes in receptor number. This finding may indicate that even low levels of activated signaling kinases, e.g. protein kinase A, protein kinase C, or receptor tyrosine kinase, may suffice to yield good expression of JUNB and c-FOS, or it may suggest alternative pathways for regulating expression of these two early response genes.

  10. Dendritic cells regulate angiogenesis associated with liver fibrogenesis.

    Science.gov (United States)

    Blois, Sandra M; Piccioni, Flavia; Freitag, Nancy; Tirado-González, Irene; Moschansky, Petra; Lloyd, Rodrigo; Hensel-Wiegel, Karin; Rose, Matthias; Garcia, Mariana G; Alaniz, Laura D; Mazzolini, Guillermo

    2014-01-01

    During liver fibrogenesis the immune response and angiogenesis process are fine-tuned resulting in activation of hepatic stellate cells that produce an excess of extracellular matrix proteins. Dendritic cells (DC) play a central role modulating the liver immunity and have recently been implicated to favour fibrosis regression; although their ability to influence the development of fibrogenesis is unknown. Therefore, we explored whether the depletion of DC during early stages of liver injury has an impact in the development of fibrogenesis. Using the CD11c.DTR transgenic mice, DC were depleted in two experimental models of fibrosis in vivo. The effect of anti-angiogenic therapy was tested during early stages of liver fibrogenesis. DC depletion accelerates the development of fibrosis and as a consequence, the angiogenesis process is boosted. We observed up-regulation of pro-angiogenic factors together with an enhanced vascular endothelial growth factor (VEGF) bioavailability, mainly evidenced by the decrease of anti-angiogenic VEGF receptor 1 (also known as sFlt-1) levels. Interestingly, fibrogenesis process enhanced the expression of Flt-1 on hepatic DC and administration of sFlt-1 was sufficient to abrogate the acceleration of fibrogenesis upon DC depletion. Thus, DC emerge as novel players during the development of liver fibrosis regulating the angiogenesis process and thereby influencing fibrogenesis.

  11. Stem Cells and Liver Disease | Akhter | Internet Journal of Medical ...

    African Journals Online (AJOL)

    Liver transplantation is the primary treatment for various end-stage hepatic diseases but is hindered by the lack of donor organs, complications associated with rejection and immunosuppression. An increasingly unbridgeable gap exists between the supply and demand of transplantable organs. Hence stem cell research ...

  12. Cutaneous features seen in primary liver cell (Hepatocellular ...

    African Journals Online (AJOL)

    Primary liver cell carcinoma (PLCC), predominantly hepatocellular carcinoma is a killer. In the southwestern region of Nigeria it occupies the second position, behind prostate cancer in males. Females account for about a third of diagnosed cases. Children are not spared. Over 80 % of PLCC cases present to the hospital at ...

  13. Liver Function In Patients With Homozygous Sickle Cell Disease ...

    African Journals Online (AJOL)

    Using the sensitive ELISA technique, 213 patients with sickle cell anemia (112 males and 101 females) aged 6 months to 18 years were screened for Hepatitis B infection using Hepatitis B surface antigen (HBsAg) and antibody to Hepatitis B core antigen. A biochemical evaluation of liver function was carried out on all ...

  14. Regenerative medicine using dental pulp stem cells for liver diseases.

    Science.gov (United States)

    Ohkoshi, Shogo; Hara, Hajime; Hirono, Haruka; Watanabe, Kazuhiko; Hasegawa, Katsuhiko

    2017-02-06

    Acute liver failure is a refractory disease and its prognosis, if not treated using liver transplantation, is extremely poor. It is a good candidate for regenerative medicine, where stem cell-based therapies play a central role. Mesenchymal stem cells (MSCs) are known to differentiate into multiple cell lineages including hepatocytes. Autologous cell transplant without any foreign gene induction is feasible using MSCs, thereby avoiding possible risks of tumorigenesis and immune rejection. Dental pulp also contains an MSC population that differentiates into hepatocytes. A point worthy of special mention is that dental pulp can be obtained from deciduous teeth during childhood and can be subsequently harvested when necessary after deposition in a tooth bank. MSCs have not only a regenerative capacity but also act in an anti-inflammatory manner via paracrine mechanisms. Promising efficacies and difficulties with the use of MSC derived from teeth are summarized in this review.

  15. Immunization with Pre-Erythrocytic Antigen CelTOS from Plasmodium falciparum Elicits Cross-Species Protection against Heterologous Challenge with Plasmodium berghei

    Science.gov (United States)

    2010-08-01

    or the early liver-stages of the mammalian life cycle . One of these antigens is the cell-traversal protein for ookinetes and sporozoites (CelTOS...Immunization with Pre-Erythrocytic Antigen CelTOS from Plasmodium falciparum Elicits Cross-Species Protection against Heterologous Challenge with... Plasmodium berghei Elke S. Bergmann-Leitner1*, Ryan M. Mease1, Patricia De La Vega1, Tatyana Savranskaya2, Mark Polhemus1, Christian Ockenhouse1, Evelina

  16. Effect of liver histopathology on islet cell engraftment in the model mimicking autologous islet cell transplantation.

    Science.gov (United States)

    Desai, Chirag S; Khan, Khalid M; Ma, Xiaobo; Li, Henghong; Wang, Juan; Fan, Lijuan; Chen, Guoling; Smith, Jill P; Cui, Wanxing

    2017-11-02

    The inflammatory milieu in the liver as determined by histopathology is different in individual patients undergoing autologous islet cell transplantation. We hypothesized that inflammation related to fatty-liver adversely impacts islet survival. To test this hypothesis, we used a mouse model of fatty-liver to determine the outcome of syngeneic islet transplantation after chemical pancreatectomy. Mice (C57BL/6) were fed a high-fat-diet from 6 weeks of age until attaining a weight of ≥28 grams (6-8 weeks) to produce a fatty liver (histologically > 30% fat);steatosis was confirmed with lipidomic profile of liver tissue. Islets were infused via the intra-portal route in fatty-liver and control mice after streptozotocin induction of diabetes. Outcomes were assessed by the rate of euglycemia, liver histopathology, evaluation of liver inflammation by measuring tissue cytokines IL-1β and TNF-α by RT-PCR and CD31 expression by immunohistochemistry. The difference in the euglycemic fraction between the normal liver group (90%, 9/10) and the fatty-liver group (37.5%, 3/8) was statistically significant at the 18 th day post- transplant and was maintained to the end of the study (day 28) (p = 0.019, X 2 = 5.51). Levels of TNF-α and IL-1β were elevated in fatty-liver mice (p = 0.042, p = 0.037). Compared to controls cytokine levels were elevated after islet cell transplantation and in transplanted fatty-liver mice as compared to either fatty- or islet transplant group alone (p = NS). A difference in the histochemical pattern of CD31 could not be determined. Fatty-liver creates an inflammatory state which adversely affects the outcome of autologous islet cell transplantation.

  17. Uptake and clearance of plutonium-238 from intact liver and liver cells transplanted into fat pads of F344/N rats

    International Nuclear Information System (INIS)

    Brooks, A.L.; Guilmette, R.A.; Hahn, F.F.; Jirtle, R.L.

    1985-01-01

    An understanding of the role of liver cells and the intact liver in plutonium biokinetics is needed. Liver cells were isolated from rats, injected into fat pads of recipient rats, and allowed 21 days to form cell colonies. Rats then received a single intraperitoneal injection of 1 μCi 238 Pu-citrate and were serially sacrificed. Uptake, retention, and distribution of Pu in intact liver and in liver cells growing in fat pads were determined. Intact liver cells took up about twice as much 238 Pu as liver cells transplanted into fat pads. However, the retention kinetics of Pu were similar for both the liver cells in the fat pads and the intact liver cells when the retention was expressed as activity per cell. 4 references, 1 figure, 1 table

  18. Long live the liver: immunohistochemical and stereological study of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells of male and female rats throughout ageing.

    Science.gov (United States)

    Marcos, Ricardo; Correia-Gomes, Carla

    2016-12-01

    Male/female differences in enzyme activity and gene expression in the liver are known to be attenuated with ageing. Nevertheless, the effect of ageing on liver structure and quantitative cell morphology remains unknown. Male and female Wistar rats aged 2, 6, 12 and 18 months were examined by means of stereological techniques and immunohistochemical tagging of hepatocytes (HEP), liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC) in order to assess the total number and number per gram of these cells throughout life. The mean cell volume of HEP and HSC, the lobular position and the collagen content of the liver were also evaluated with stereological techniques. The number per gram of HSC was similar for both genders and was maintained throughout ageing. The mean volume of HSC was also conserved but differences in the cell body and lobular location were observed. Statistically significant gender differences in HEP were noted in young rats (females had smaller and more binucleated HEP) but were attenuated with ageing. The same occurred for KC and LSEC, since the higher number per gram in young females disappeared in older animals. Liver collagen increased with ageing but only in males. Thus, the numbers of these four cell types are related throughout ageing, with well-defined cell ratios. The shape and lobular position of HSC change with ageing in both males and females. Gender dimorphism in HEP, KC and LSEC of young rat liver disappears with ageing.

  19. Role of stellate cells in alcoholic liver fibrosis

    Directory of Open Access Journals (Sweden)

    Krzysztof Plewka

    2009-07-01

    Full Text Available Many different diseases and toxins can cause liver damage, which is diffi cult to treat and often leads to the development of liver fi brosis or even cirrhosis. The key event in this process is the activation of hepatic stellate cells (HSCs. During such activation, HSCs undergo a dramatic transformation in morphology and behavior, changing from a neuronal-like to a fi broblast-like morphology. After activation, HSCs increase their proliferation rate and extracellular matrix (ECM production. Overproduction of ECM, which contains mainly collagen type I, is a direct cause of liver disruption. HSCs also produce substances which inhibit protease activities, such as TIMPs, which enhance ECM deposition in the liver. On the molecular level, HSCs are activated by cytokines, growth factors, and oxidative stress, which are abundant in affl icted liver. These factors induce intracellular signals transmitted by many kinases, the most important of which are JNK, ERK1/2, p38, TAK-1, PKC, FAK, and P3IK. Signals transmitted via these pathways change the activities of transcription factors such as Smad, AP-1, and NF-κβ. This in turn causes changes In gene transcription and ultimately alters the whole cell’s behavior and morphology. The cell begins the production collagen type I, TIMP-1, and aSMA. Activated HSCs can sustain their own activation by producing growth factors such as PDGF and TGF-β. Despite the vast knowledge about the mechanisms causing liver fi brosis and cirrhosis, there is still no effective cure. Further studies are therefore needed to solve this problem.

  20. Heterologous expression of C. elegans fat-1 decreases the n-6/n-3 fatty acid ratio and inhibits adipogenesis in 3T3-L1 cells

    International Nuclear Information System (INIS)

    An, Lei; Pang, Yun-Wei; Gao, Hong-Mei; Tao, Li; Miao, Kai; Wu, Zhong-Hong

    2012-01-01

    Highlights: ► Expression of C. elegans fat-1 reduces the n-6/n-3 PUFA ratio in 3T3-L1 cells. ► fat-1 inhibits the proliferation and differentiation of 3T3-L1 preadipocytes. ► fat-1 reduces lipid deposition in 3T3-L1 adipocytes. ► The lower n-6/n-3 ratio induces apoptosis in 3T3-L1 adipocytes. -- Abstract: In general, a diet enriched in polyunsaturated fatty acids (PUFAs) inhibits the development of obesity and decreases adipose tissue. The specific impacts of n-3 and n-6 PUFAs on adipogenesis, however, have not been definitively determined. Traditional in vivo and in vitro supplementation studies have yielded inconsistent or even contradictory results, which likely reflect insufficiently controlled experimental systems. Caenorhabditiselegans fat-1 gene encodes an n-3 fatty acid desaturase, and its heterologous expression represents an effective method both for altering the n-6/n-3 PUFA ratio and for evaluating the biological effects of n-3 and n-6 PUFAs. We sought to determine whether a reduced n-6/n-3 ratio could influence adipogenesis in 3T3-L1 cells. Lentivirus-mediated introduction of the fat-1 gene into 3T3-L1 preadipocytes significantly reduced the n-6/n-3 ratio and inhibited preadipocyte proliferation and differentiation. In mature adipocytes, fat-1 expression reduced lipid deposition, as measured by Oil Red O staining, and induced apoptosis. Our results indicate that a reduced n-6/n-3 ratio inhibits adipogenesis through several mechanisms and that n-3 PUFAs more effectively inhibit adipogenesis (but not lipogenesis) than do n-6 PUFAs.

  1. Heterologous expression of C. elegans fat-1 decreases the n-6/n-3 fatty acid ratio and inhibits adipogenesis in 3T3-L1 cells

    Energy Technology Data Exchange (ETDEWEB)

    An, Lei, E-mail: anleim@yahoo.com.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Pang, Yun-Wei, E-mail: yunweipang@126.com [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Gao, Hong-Mei, E-mail: Gaohongmei_123@yahoo.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Research Unit for Animal Life Sciences, Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Ibaraki-Iwama 319-0206 (Japan); Tao, Li, E-mail: Eunice8023@yahoo.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin 130118 (China); Miao, Kai, E-mail: miaokai7@163.com [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Wu, Zhong-Hong, E-mail: wuzhh@cau.edu.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); and others

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer Expression of C. elegans fat-1 reduces the n-6/n-3 PUFA ratio in 3T3-L1 cells. Black-Right-Pointing-Pointer fat-1 inhibits the proliferation and differentiation of 3T3-L1 preadipocytes. Black-Right-Pointing-Pointer fat-1 reduces lipid deposition in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer The lower n-6/n-3 ratio induces apoptosis in 3T3-L1 adipocytes. -- Abstract: In general, a diet enriched in polyunsaturated fatty acids (PUFAs) inhibits the development of obesity and decreases adipose tissue. The specific impacts of n-3 and n-6 PUFAs on adipogenesis, however, have not been definitively determined. Traditional in vivo and in vitro supplementation studies have yielded inconsistent or even contradictory results, which likely reflect insufficiently controlled experimental systems. Caenorhabditiselegans fat-1 gene encodes an n-3 fatty acid desaturase, and its heterologous expression represents an effective method both for altering the n-6/n-3 PUFA ratio and for evaluating the biological effects of n-3 and n-6 PUFAs. We sought to determine whether a reduced n-6/n-3 ratio could influence adipogenesis in 3T3-L1 cells. Lentivirus-mediated introduction of the fat-1 gene into 3T3-L1 preadipocytes significantly reduced the n-6/n-3 ratio and inhibited preadipocyte proliferation and differentiation. In mature adipocytes, fat-1 expression reduced lipid deposition, as measured by Oil Red O staining, and induced apoptosis. Our results indicate that a reduced n-6/n-3 ratio inhibits adipogenesis through several mechanisms and that n-3 PUFAs more effectively inhibit adipogenesis (but not lipogenesis) than do n-6 PUFAs.

  2. Stem cell  mediated liver regeneration:

    DEFF Research Database (Denmark)

    Jelnes, Peter

    udformet en strategi som involverede isolering af HPCr vha. Fluorescent Activated Cell Sorting (FACS). Isolerede celler skulle efterfølgende undersøges ved in vitro studier som implicerede tilsætning af specifikke immunrespons relaterede faktorer. Kun få specifikke overflade HPC markører er blevet...

  3. Assay and heterologous expression in Pichia pastoris of plant cell wall type-II membrane anchored glycosyltransferases

    DEFF Research Database (Denmark)

    Petersen, Bent; Egelund, Jack; Damager, Iben

    2009-01-01

    .011 to 0.013 U (1 U = 1 nmol conversion of substrate * min(-1) * microl medium(-1)) similar to those of RGXT1 and RGXT2 expressed in Baculovirus transfected insect Sf9 cells. In summary, the data presented suggest that Pichia is an attractive host candidate for expression of plant glycosyltransferases.......Two Arabidopsis xylosyltransferases, designated RGXT1 and RGXT2, were recently expressed in Baculovirus transfected insect cells and by use of the free sugar assay shown to catalyse transfer of D-xylose from UDP-alpha-D-xylose to L-fucose and derivatives hereof. We have now examined expression...

  4. Cytokines, hepatic cell profiling and cell interactions during bone marrow cell therapy for liver fibrosis in cholestatic mice.

    Directory of Open Access Journals (Sweden)

    Daphne Pinheiro

    Full Text Available Bone marrow cells (BMC migrate to the injured liver after transplantation, contributing to regeneration through multiple pathways, but mechanisms involved are unclear. This work aimed to study BMC migration, characterize cytokine profile, cell populations and proliferation in mice with liver fibrosis transplanted with GFP+ BMC. Confocal microscopy analysis showed GFP+ BMC near regions expressing HGF and SDF-1 in the fibrotic liver. Impaired liver cell proliferation in fibrotic groups was restored after BMC transplantation. Regarding total cell populations, there was a significant reduction in CD68+ cells and increased Ly6G+ cells in transplanted fibrotic group. BMC contributed to the total populations of CD144, CD11b and Ly6G cells in the fibrotic liver, related to an increment of anti-fibrotic cytokines (IL-10, IL-13, IFN-γ and HGF and reduction of pro-inflammatory cytokines (IL-17A and IL-6. Therefore, HGF and SDF-1 may represent important chemoattractants for transplanted BMC in the injured liver, where these cells can give rise to populations of extrahepatic macrophages, neutrophils and endothelial progenitor cells that can interact synergistically with other liver cells towards the modulation of an anti-fibrotic cytokine profile promoting the onset of liver regeneration.

  5. [Effects of three Wenyang Jianpi Tang on cell proliferation and apoptosis of nonalcoholic fatty liver cells].

    Science.gov (United States)

    Yang, Jia-Yao; Tao, Dong-Qing; Liu, Song; Zhang, Shu; Ma, Wei; Shi, Zhao-Hong

    2017-04-01

    To investigate the effects of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang on the cell proliferation and apoptosis of nonalcoholic fatty liver cells through the nonalcoholic fatty liver cell model established by inducing L02 cells with oleic acid. Different concentrations of oleic acid were added into L02 cells to induce the nonalcoholic fatty liver cell model. Oil red O staining was used to observe fatty droplets of fatty liver cells. Automatic biochemical analyzer was used to detect the levels of aspartic transaminase(AST), alanine aminotransferase(ALT), total cholesterol(TC), and triglyceride(TG) in the cell supernatants. There were five groups, namely normal group, model group, model and Sijunzi Tang group, model and Lizhong Tang group, and model and Fuzi Lizhong Tang group. The cell proliferation and apoptosis of the five groups were detected by MTT colorimetry test and flow cytometer. The expressions of PCNA, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax and Bcl-2 proteins of the five groups were detected by Western blot. The oil red O staining results showed that the optimum concentration of oleic acid that was used to induce nonalcoholic fatty liver cell models was 80 mg•L-1. The levels of AST, ALT, TC and TG in the nonalcoholic fatty liver cell supernatants were higher than that in normal liver cell supernatants(PTang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells(PTang showed the best effect. Western blot results showed that Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could down-regulate the expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and Bax proteins, and up-regulate the expressions of PCNA and Bcl-2 proteins of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. In conclusion, all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell

  6. Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment.

    Science.gov (United States)

    Cheng, Chiung-Chi; Chao, Wei-Ting; Liao, Chen-Chun; Tseng, Yu-Hui; Lai, Yen-Chang Clark; Lai, Yih-Shyong; Hsu, Yung-Hsiang; Liu, Yi-Hsiang

    2018-01-02

    Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.

  7. Nano scaffolds and stem cell therapy in liver tissue engineering

    Science.gov (United States)

    Montaser, Laila M.; Fawzy, Sherin M.

    2015-08-01

    Tissue engineering and regenerative medicine have been constantly developing of late due to the major progress in cell and organ transplantation, as well as advances in materials science and engineering. Although stem cells hold great potential for the treatment of many injuries and degenerative diseases, several obstacles must be overcome before their therapeutic application can be realized. These include the development of advanced techniques to understand and control functions of micro environmental signals and novel methods to track and guide transplanted stem cells. A major complication encountered with stem cell therapies has been the failure of injected cells to engraft to target tissues. The application of nanotechnology to stem cell biology would be able to address those challenges. Combinations of stem cell therapy and nanotechnology in tissue engineering and regenerative medicine have achieved significant advances. These combinations allow nanotechnology to engineer scaffolds with various features to control stem cell fate decisions. Fabrication of Nano fiber cell scaffolds onto which stem cells can adhere and spread, forming a niche-like microenvironment which can guide stem cells to proceed to heal damaged tissues. In this paper, current and emergent approach based on stem cells in the field of liver tissue engineering is presented for specific application. The combination of stem cells and tissue engineering opens new perspectives in tissue regeneration for stem cell therapy because of the potential to control stem cell behavior with the physical and chemical characteristics of the engineered scaffold environment.

  8. Macrophages and dendritic cells emerge in the liver during intestinal inflammation and predispose the liver to inflammation.

    Directory of Open Access Journals (Sweden)

    Yohei Mikami

    Full Text Available The liver is a physiological site of immune tolerance, the breakdown of which induces immunity. Liver antigen-presenting cells may be involved in both immune tolerance and activation. Although inflammatory diseases of the liver are frequently associated with inflammatory bowel diseases, the underlying immunological mechanisms remain to be elucidated. Here we report two murine models of inflammatory bowel disease: RAG-2(-/- mice adoptively transferred with CD4(+CD45RB(high T cells; and IL-10(-/- mice, accompanied by the infiltration of mononuclear cells in the liver. Notably, CD11b(-CD11c(lowPDCA-1(+ plasmacytoid dendritic cells (DCs abundantly residing in the liver of normal wild-type mice disappeared in colitic CD4(+CD45RB(high T cell-transferred RAG-2(-/- mice and IL-10(-/- mice in parallel with the emergence of macrophages (Mφs and conventional DCs (cDCs. Furthermore, liver Mφ/cDCs emerging during intestinal inflammation not only promote the proliferation of naïve CD4(+ T cells, but also instruct them to differentiate into IFN-γ-producing Th1 cells in vitro. The emergence of pathological Mφ/cDCs in the liver also occurred in a model of acute dextran sulfate sodium (DSS-induced colitis under specific pathogen-free conditions, but was canceled in germ-free conditions. Last, the Mφ/cDCs that emerged in acute DSS colitis significantly exacerbated Fas-mediated hepatitis. Collectively, intestinal inflammation skews the composition of antigen-presenting cells in the liver through signaling from commensal bacteria and predisposes the liver to inflammation.

  9. Crosstalk between type II NKT cells and T cells leads to spontaneous chronic inflammatory liver disease.

    Science.gov (United States)

    Weng, Xiufang; He, Ying; Visvabharathy, Lavanya; Liao, Chia-Min; Tan, Xiaosheng; Balakumar, Arjun; Wang, Chyung-Ru

    2017-10-01

    Natural killer T (NKT) cells are CD1d-restricted innate-like T cells that modulate innate and adaptive immune responses. Unlike the well-characterized invariant/type I NKT cells, type II NKT cells with a diverse T cell receptor repertoire are poorly understood. This study defines the pathogenic role of type II NKT cells in the etiology of chronic liver inflammation. Transgenic mice with the Lck promoter directing CD1d overexpression on T cells in Jα18 wild-type (Lck-CD1dTgJα18 + ; type I NKT cell sufficient) and Jα18-deficient (Lck-CD1dTgJα18 o , type I NKT cell deficient) mice were analyzed for liver pathology and crosstalk between type II NKT cells and conventional T cells. CD1d expression on T cells in peripheral blood samples and liver sections from autoimmune hepatitis patients and healthy individuals were also examined. Lck-CD1dTgJα18 o and Lck-CD1dTgJα18 + mice developed similar degrees of liver pathology resembling chronic autoimmune hepatitis in humans. Increased CD1d expression on T cells promoted the activation of type II NKT cells and other T cells. This resulted in T h 1-skewing and impaired T h 2 cytokine production in type II NKT cells. Dysfunction of type II NKT cells was accompanied by conventional T cell activation and pro-inflammatory cytokine production, leading to a hepatic T/B lymphocyte infiltration, elevated autoantibodies and hepatic injury in Lck-CD1dTg mice. A similar mechanism could be extended to humans as CD1d expression is upregulated on activated human T cells and increased presence of CD1d-expressing T cells was observed in autoimmune hepatitis patients. Our data reveals enhanced crosstalk between type II NKT cells and conventional T cells, leading to a T h 1-skewed inflammatory milieu, and consequently, to the development of chronic autoimmune liver disease. Lay summary: CD1d overexpression on T cells enhances crosstalk between type II NKT cells and T cells, resulting in their aberrant activation and leading to the

  10. An optimized method for mouse liver sinusoidal endothelial cell isolation

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, Jeremy, E-mail: jeremy.meyer@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Lacotte, Stéphanie, E-mail: stephanie.lacotte@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Morel, Philippe, E-mail: philippe.morel@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Gonelle-Gispert, Carmen, E-mail: carmen.gonelle@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Bühler, Léo, E-mail: leo.buhler@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland)

    2016-12-10

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  11. An optimized method for mouse liver sinusoidal endothelial cell isolation

    International Nuclear Information System (INIS)

    Meyer, Jeremy; Lacotte, Stéphanie; Morel, Philippe; Gonelle-Gispert, Carmen; Bühler, Léo

    2016-01-01

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  12. Bone morphogenetic protein 9 as a key regulator of liver progenitor cells in DDC-induced cholestatic liver injury.

    Science.gov (United States)

    Addante, Annalisa; Roncero, Cesáreo; Almalé, Laura; Lazcanoiturburu, Nerea; García-Álvaro, María; Fernández, Margarita; Sanz, Julián; Hammad, Seddik; Nwosu, Zeribe C; Lee, Se-Jin; Fabregat, Isabel; Dooley, Steven; Ten Dijke, Peter; Herrera, Blanca; Sánchez, Aránzazu

    2018-05-11

    Bone morphogenetic protein 9 (BMP9) interferes with liver regeneration upon acute injury, while promoting fibrosis upon carbon tetrachloride-induced chronic injury. We have now addressed the role of BMP9 in 3,5 diethoxicarbonyl-1,4 dihydrocollidine (DDC)-induced cholestatic liver injury, a model of liver regeneration mediated by hepatic progenitor cell (known as oval cell), exemplified as ductular reaction and oval cell expansion. WT and BMP9KO mice were submitted to DDC diet. Livers were examined for liver injury, fibrosis, inflammation and oval cell expansion by serum biochemistry, histology, RT-qPCR and western blot. BMP9 signalling and effects in oval cells were studied in vitro using western blot and transcriptional assays, plus functional assays of DNA synthesis, cell viability and apoptosis. Crosslinking assays and short hairpin RNA approaches were used to identify the receptors mediating BMP9 effects. Deletion of BMP9 reduces liver damage and fibrosis, but enhances inflammation upon DDC feeding. Molecularly, absence of BMP9 results in overactivation of PI3K/AKT, ERK-MAPKs and c-Met signalling pathways, which together with an enhanced ductular reaction and oval cell expansion evidence an improved regenerative response and decreased damage in response to DDC feeding. Importantly, BMP9 directly targets oval cells, it activates SMAD1,5,8, decreases cell growth and promotes apoptosis, effects that are mediated by Activin Receptor-Like Kinase 2 (ALK2) type I receptor. We identify BMP9 as a negative regulator of oval cell expansion in cholestatic injury, its deletion enhancing liver regeneration. Likewise, our work further supports BMP9 as an attractive therapeutic target for chronic liver diseases. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Dexamethasone-induced haptoglobin release by calf liver parenchymal cells.

    Science.gov (United States)

    Higuchi, H; Katoh, N; Miyamoto, T; Uchida, E; Yuasa, A; Takahashi, K

    1994-08-01

    Parenchymal cells were isolated from the liver of male calves, and monolayer cultures formed were treated with glucocorticoids to examine whether haptoglobin, appearance of which is associated with hepatic lipidosis (fatty liver) in cattle, is induced by steroid hormones. Without addition of dexamethasone, only trace amounts of haptoglobin were detected in culture medium. With addition of dexamethasone (10(-12) to 10(-4) M), considerable amounts of haptoglobin were released into the medium. Maximal release was observed at concentrations of 10(-8) to 10(-6) M dexamethasone. Haptoglobin release was similarly induced by cortisol, although the effect was less potent than that of dexamethasone. Actinomycin D (a known protein synthesis inhibitor) dose-dependently reduced amounts of haptoglobin released in response to 10(-8) M dexamethasone. Dexamethasone also induced annexin I, which is known to be synthesized in response to glucocorticoids. Dexamethasone treatment resulted in reduced protein kinase C activity in the cell cytosol, which has been shown to be an early event in dexamethasone-treated cells. Other than glucocorticoids, estradiol induced haptoglobin release, whereas progesterone was less effective. The association of haptoglobin with hepatic lipidosis can be reasonably explained by the fact that haptoglobin production by the liver is induced by glucocorticoids and estradiol, and these steroid hormones are triggers for development of hepatic lipidosis in cattle.

  14. Adaptive remodeling of the biliary tree: the essence of liver progenitor cell expansion.

    Science.gov (United States)

    Kok, Cindy Yuet-Yin; Miyajima, Atsushi; Itoh, Tohru

    2015-07-01

    The liver progenitor cell population has long been thought to exist within the liver. However, there are no standardized criteria for defining the liver progenitor cells, and there has been intense debate about the origin of these cells in the adult liver. The characteristics of such cells vary depending on the disease model used and also on the method of analysis. Visualization of three-dimensional biliary structures has revealed that the emergence of liver progenitor cells essentially reflects the adaptive remodeling of the hepatic biliary network in response to liver injury. We propose that the progenitor cell exists as a subpopulation in the biliary tree and show that the appearance of liver progenitor cells in injured parenchyma is reflective of extensive remodeling of the biliary structure. © 2015 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

  15. Constitutively internalized dopamine transporter is targeted to late endosomes and lysosomal degradation in heterologous cell lines and dopaminergic neurons

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Madsen, Kenneth; Vægter, Christian Bjerggaard

    and amphetamine, a substrate of the DAT. In antibody feeding experiments we observed that Tac-DAT was constitutively internalized faster than Tac alone and using an ELISA based assay we could quantify time-dependent intracellular accumulation of the transporter. Incubation with inhibitors of lysosomal degradation...... (leupeptin, chloroquine, or ammonium chloride) increased the amount of transporter accumulated intracellularly over time, suggesting that constitutively endocytosed transporter was targeted to lysosomal degradation. This was further supported by expression of Tac-DAT in the immortalized dopaminergic cell...... dopaminergic neurons and visualized the DAT directly in the neurons using the fluorescent cocaine analog JHC 1-064. These data showed pronounced colocalization upon constitutive internalization with Lysotracker, a late endosomal/lysosomal marker; however only little co-lolization was observed with Alexa488...

  16. Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface.

    Directory of Open Access Journals (Sweden)

    Wouter S P Jong

    Full Text Available Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight β-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight β-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or VHH domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the β-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The VHH domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp β-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure.

  17. Isolation of Kupffer Cells and Hepatocytes from a Single Mouse Liver

    DEFF Research Database (Denmark)

    Aparicio-Vergara, Marcela; Tencerova, Michaela; Morgantini, Cecilia

    2017-01-01

    Liver perfusion is a common technique used to isolate parenchymal and non-parenchymal liver cells for in vitro experiments. This method allows hepatic cells to be separated based on their size and weight, by centrifugation using a density gradient. To date, other methods allow the isolation of only...... one viable hepatic cellular fraction from a single mouse; either parenchymal (hepatocytes) or non-parenchymal cells (i.e., Kupffer cells or hepatic stellate cells). Here, we describe a method to isolate both hepatocytes and Kupffer cells from a single mouse liver, thereby providing the unique...... advantage of studying different liver cell types that have been isolated from the same organism....

  18. Tc-99m red blood cells for the study of rapid hemolytic processes associated with heterologous blood transfusions

    International Nuclear Information System (INIS)

    Benedetto, A.R.; Harrison, C.R.; Blumhardt, R.; Trow, L.L.

    1984-01-01

    Chromium-51 labeled erythrocytes (Cr-51 RBC) are suitable for the study of hematologic disorders which involve relatively slow destruction of circulating erythrocytes, taking several days to several weeks. However, Cr-51 RBC are not suitable for investigating rapid hemolytic processes which occur within a matter of a few hours due to the variable and unpredictable elution of Cr-51 from the erythrocytes during the first 24 hours or so. Imaging, which could be useful in identifying organ systems involved in the hemolytic process, cannot be performed with Cr-51 RBC because of the high dose commitment caused by the low yield of gamma rays from Cr-51 (2). A method of labeling RBC with Tc-99m, which results in a radiopharmaceutical that combines the excellent dosimetric and imaging qualities of Tc-99m with an extremely stable bond between the Tc-99m and the RBC, is reported. The successful application of this technique in providing red cell support for a cancer patient with an unusual history of intravascular hemolytic transfusion reactions is also reported

  19. Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells

    Directory of Open Access Journals (Sweden)

    Irit Meivar-Levy

    2011-01-01

    Full Text Available Activation of the pancreatic lineage in the liver has been suggested as a potential autologous cell replacement therapy for diabetic patients. Transcription factors-induced liver-to-pancreas reprogramming has been demonstrated in numerous species both in vivo and in vitro. However, human-derived liver cells capable of acquiring the alternate pancreatic repertoire have never been characterized. It is yet unknown whether hepatic-like stem cells or rather adult liver cells give rise to insulin-producing cells. Using an in vitro experimental system, we demonstrate that proliferating adherent human liver cells acquire mesenchymal-like characteristics and a considerable level of cellular plasticity. However, using a lineage-tracing approach, we demonstrate that insulin-producing cells are primarily generated in cells enriched for adult hepatic markers that coexpress both albumin and mesenchymal markers. Taken together, our data suggest that adult human hepatic tissue retains a substantial level of developmental plasticity, which could be exploited in regenerative medicine approaches.

  20. Establishment and characterization of a unique 1 microm diameter liver-derived progenitor cell line.

    Science.gov (United States)

    Aravalli, Rajagopal N; Behnan Sahin, M; Cressman, Erik N K; Steer, Clifford J

    2010-01-01

    Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 microm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers. Copyright 2009 Elsevier Inc. All rights reserved.

  1. A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory.

    Science.gov (United States)

    Hagström, Åsa K; Wang, Hong-Lei; Liénard, Marjorie A; Lassance, Jean-Marc; Johansson, Tomas; Löfstedt, Christer

    2013-12-13

    Moths (Lepidoptera) are highly dependent on chemical communication to find a mate. Compared to conventional unselective insecticides, synthetic pheromones have successfully served to lure male moths as a specific and environmentally friendly way to control important pest species. However, the chemical synthesis and purification of the sex pheromone components in large amounts is a difficult and costly task. The repertoire of enzymes involved in moth pheromone biosynthesis in insecta can be seen as a library of specific catalysts that can be used to facilitate the synthesis of a particular chemical component. In this study, we present a novel approach to effectively aid in the preparation of semi-synthetic pheromone components using an engineered vector co-expressing two key biosynthetic enzymes in a simple yeast cell factory. We first identified and functionally characterized a ∆11 Fatty-Acyl Desaturase and a Fatty-Acyl Reductase from the Turnip moth, Agrotis segetum. The ∆11-desaturase produced predominantly Z11-16:acyl, a common pheromone component precursor, from the abundant yeast palmitic acid and the FAR transformed a series of saturated and unsaturated fatty acids into their corresponding alcohols which may serve as pheromone components in many moth species. Secondly, when we co-expressed the genes in the Brewer's yeast Saccharomyces cerevisiae, a set of long-chain fatty acids and alcohols that are not naturally occurring in yeast were produced from inherent yeast fatty acids, and the presence of (Z)-11-hexadecenol (Z11-16:OH), demonstrated that both heterologous enzymes were active in concert. A 100 ml batch yeast culture produced on average 19.5 μg Z11-16:OH. Finally, we demonstrated that oxidized extracts from the yeast cells containing (Z)-11-hexadecenal and other aldehyde pheromone compounds elicited specific electrophysiological activity from male antennae of the Tobacco budworm, Heliothis virescens, supporting the idea that genes from different

  2. NKT cells act through third party bone marrow-derived cells to suppress NK cell activity in the liver and exacerbate hepatic melanoma metastases.

    Science.gov (United States)

    Sadegh, Leila; Chen, Peter W; Brown, Joseph R; Han, Zhiqiang; Niederkorn, Jerry Y

    2015-09-01

    Uveal melanoma (UM) is the most common intraocular tumor in adults and liver metastasis is the leading cause of death in UM patients. We have previously shown that NKT cell-deficient mice develop significantly fewer liver metastases from intraocular melanomas than do wild-type (WT) mice. Here, we examine the interplay between liver NKT cells and NK cells in resistance to liver metastases from intraocular melanomas. NKT cell-deficient CD1d(-/-) mice and WT C57BL/6 mice treated with anti-CD1d antibody developed significantly fewer liver metastases than WT mice following either intraocular or intrasplenic injection of B16LS9 melanoma cells. The increased number of metastases in WT mice was associated with reduced liver NK cytotoxicity and decreased production of IFN-γ. However, liver NK cell-mediated cytotoxic activity was identical in non-tumor bearing NKT cell-deficient mice and WT mice, indicating that liver metastases were crucial for the suppression of liver NK cells. Depressed liver NK cytotoxicity in WT mice was associated with production of IL-10 by bone marrow-derived liver cells that were neither Kupffer cells nor myeloid-derived suppressor cells and by increased IL-10 receptor expression on liver NK cells. IL-10(-/-) mice had significantly fewer liver metastases than WT mice, but were not significantly different from NKT cell-deficient mice. Thus, development of melanoma liver metastases is associated with upregulation of IL-10 in the liver and an elevated expression of IL-10 receptor on liver NK cells. This impairment of liver NK activity is NKT cell-dependent and only occurs in hosts with melanoma liver metastases. © 2015 UICC.

  3. Gal-3 regulates the capacity of dendritic cells to promote NKT-cell-induced liver injury.

    Science.gov (United States)

    Volarevic, Vladislav; Markovic, Bojana Simovic; Bojic, Sanja; Stojanovic, Maja; Nilsson, Ulf; Leffler, Hakon; Besra, Gurdyal S; Arsenijevic, Nebojsa; Paunovic, Verica; Trajkovic, Vladimir; Lukic, Miodrag L

    2015-02-01

    Galectin-3 (Gal-3), an endogenous lectin, exhibits pro- and anti-inflammatory effects in various disease conditions. In order to explore the role of Gal-3 in NKT-cell-dependent pathology, we induced hepatitis in C57BL/6 WT and Gal-3-deficient mice by using specific ligand for NKT cells: α-galactosylceramide, glycolipid Ag presented by CD1d. The injection of α-galactosylceramide significantly enhanced expression of Gal-3 in liver NKT and dendritic cells (DCs). Genetic deletion or selective inhibition of Gal-3 (induced by Gal-3-inhibitor TD139) abrogated the susceptibility to NKT-cell-dependent hepatitis. Blood levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-12) and their production by liver DCs and NKT cells were also downregulated. Genetic deletion or selective inhibition of Gal-3 alleviated influx of inflammatory CD11c(+) CD11b(+) DCs in the liver and favored tolerogenic phenotype and IL-10 production of liver NKT and DCs. Deletion of Gal-3 attenuated the capacity of DCs to support liver damage in the passive transfer experiments and to produce pro-inflammatory cytokines in vitro. Gal-3-deficient DCs failed to optimally stimulate production of pro-inflammatory cytokines in NKT cells, in vitro and in vivo. In conclusion, Gal-3 regulates the capacity of DCs to support NKT-cell-mediated liver injury, playing an important pro-inflammatory role in acute liver injury. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Liver

    International Nuclear Information System (INIS)

    Bernardino, M.E.; Sones, P.J. Jr.; Barton Price, R.; Berkman, W.A.

    1984-01-01

    Evaluation of the liver for focal lesions is extremely important because the liver is one of the most common sites for metastatic disease. Most patients with metastatic deposits to the liver have a survival rate of about 6 months. Thus, metastatic disease to the liver has an extremely grave prognosis. In the past patients with hepatic lesions had no therapeutic recourse. However, with recent aggressive surgical advances (such as partial hepatectomies) and hepatic artery embolization, survival of patients with hepatic metastases has increased. Thus it is important for noninvasive imaging not only to detect lesions early in their course, but also to give their true hepatic involvement and the extent of the neoplastic process elsewhere in the body. Recent advances in imaging have been rapidly changing over the past 5 years. These changes have been more rapid in computed tomography (CT) and ultrasound than in radionuclide imaging. Thus, the question addressed in this chapter is: What is the relationship of hepatic ultrasound to the other current diagnostic modalities in detecting metastatic liver disease and other focal liver lesions? Also, what is its possible future relationship to nuclear magnetic resonance?

  5. All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells.

    Directory of Open Access Journals (Sweden)

    Melanie Werner

    Full Text Available Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.Cell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7 hepatocytes, 1.8 ± 0.5 × 10(6 Kupffer cells, 4.3 ± 1.9 × 10(5 liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5 stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7% and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2% and exhibited phagocytic activity, as determined with 1 μm latex beads. Endothelial cells were CD146(+ (97.8 ± 1.1% and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1 ± 1.5%. These cells further exhibited retinol (vitamin A-mediated autofluorescence.Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.

  6. Heterologous human/rat HER2-specific exosome-targeted T cell vaccine stimulates potent humoral and CTL responses leading to enhanced circumvention of HER2 tolerance in double transgenic HLA-A2/HER2 mice.

    Science.gov (United States)

    Xie, Yufeng; Wu, Jie; Xu, Aizhang; Ahmeqd, Shahid; Sami, Amer; Chibbar, Rajni; Freywald, Andrew; Zheng, Changyu; Xiang, Jim

    2018-03-07

    DNA vaccines composed of heterologous human HER2 and rat neu sequences induce stronger antibody response and protective antitumor immunity than either HER2 or neu DNA vaccines in transgenic mice. We previously developed HER2-specific exosome-targeted T-cell vaccine HER2-T EXO capable of stimulating HER2-specific CD8 + T-cell responses, but only leading to partial protective immunity in double-transgenic HLA-A2/HER2 mice with self-immune tolerance to HER2. Here, we constructed an adenoviral vector AdV HuRt expressing HuRt fusion protein composed of NH 2 -HER2 1-407 (Hu) and COOH-neu 408-690 (Rt) fragments, and developed a heterologous human/rat HER2-specific exosome-targeted T-cell vaccine HuRt-T EXO using polyclonal CD4 + T-cells uptaking exosomes released by AdV HuRt -transfected dendritic cells. We found that the HuRt-T EXO vaccine stimulates enhanced CD4 + T-cell responses leading to increased induction of HER2-specific antibody (∼70 µg/ml) compared to that (∼40 µg/ml) triggered by the homologous HER2-T EXO vaccine. By using PE-H-2K d /HER2 23-71 tetramer, we determined that HuRt-T EXO stimulates stronger HER2-specific CD8 + T-cell responses eradicating 90% of HER2-specific target cells, while HER2-T EXO -induced CD8 + T-cell responses only eliminating 53% targets. Furthermore, HuRt-T EXO , but not HER2-T EXO vaccination, is capable of suppressing early stage-established HER2-expressing 4T1 HER2 breast cancer in its lung metastasis or subcutaneous form in BALB/c mice, and of completely protecting transgenic HLA-A2/HER2 mice from growth of HLA-A2/HER2-expressing BL6-10 A2/HER2 melanoma. HuRt-T EXO -stimulated HER2-specific CD8 + T-cells not only are cytolytic to trastuzumab-resistant HLA-A2/HER2-expressing BT474/A2 breast tumor cells in vitro but also eradicates pre-established BT474/A2 tumors in athymic nude mice. Therefore, our novel heterologous human/rat HER2-specific T-cell vaccine HuRt-T EXO, circumventing HER2 tolerance, may provide a new

  7. Proapoptotic Role of Potassium Ions in Liver Cells

    Directory of Open Access Journals (Sweden)

    Zhenglin Xia

    2016-01-01

    Full Text Available Potassium channels are transmembrane proteins that selectively promote the infiltration of potassium ions. The significance of these channels for tumor biology has become obvious. However, the effects of potassium ions on the tumor or normal cells have seldom been studied. To address this problem, we studied the biological effects of L02 and HepG2 cells with ectogenous potassium ions. Cell proliferation, cell cycle, and apoptosis rate were analyzed. Our results indicated that potassium ions inhibited proliferation of L02 and HepG2 cells and promoted their apoptosis. Potassium ions induced apoptosis through regulating Bcl-2 family members and depolarized the mitochondrial membrane, especially for HepG2 cell. These biological effects were associated with channel protein HERG. By facilitating expression of channel protein HERG, potassium ions may prevent it from being shunted to procancerous pathways by inducing apoptosis. These results demonstrated that potassium ions may be a key regulator of liver cell function. Thus, our findings suggest that potassium ions could inhibit tumorigenesis through inducing apoptosis of hepatoma cells by upregulating potassium ions transport channel proteins HERG and VDAC1.

  8. silver nanoparticles on liver cancer cells (HepG2

    Directory of Open Access Journals (Sweden)

    Ahmed I. El-Batal

    2018-01-01

    Full Text Available This study demonstrates a novel approach for the synthesis of silver nanoparticles (AgNPs against human liver cancer cell line (HepG2 using prodigiosin pigment isolated from Serratia marcescens. It further investigates the influence of various parameters such as initial pH, temperature, silver nitrate (AgNO 3 concentration, and prodigiosin concentration on stability and optical properties of synthesized prodigiosin AgNPs. Highly stable, spherical prodigiosin-conjugated AgNPs were synthesized with a mean diameter of 9.98 nm using a rapid one-step method. The cytotoxic activity investigated in the present study indicated that prodigiosin and prodigiosin-conjugated AgNPs possessed a strong cytotoxic potency against human liver cancer. The In silico molecular docking results of prodigiosin and prodigiosin-conjugated AgNPs are congruent with the In vitro studies and these AgNPs can be considered as good inhibitors of mitogen-activated protein kinase 1 (MEK kinases. The study opened the possibility of using prodigiosin-conjugated AgNPs to increase the efficiency of liver cancer treatment.

  9. Dietary macronutrients and the aging liver sinusoidal endothelial cell.

    Science.gov (United States)

    Cogger, Victoria Carroll; Mohamad, Mashani; Solon-Biet, Samantha Marie; Senior, Alistair M; Warren, Alessandra; O'Reilly, Jennifer Nicole; Tung, Bui Thanh; Svistounov, Dmitri; McMahon, Aisling Clare; Fraser, Robin; Raubenheimer, David; Holmes, Andrew J; Simpson, Stephen James; Le Couteur, David George

    2016-05-01

    Fenestrations are pores within the liver sinusoidal endothelial cells (LSECs) that line the sinusoids of the highly vascularized liver. Fenestrations facilitate the transfer of substrates between blood and hepatocytes. With pseudocapillarization of the hepatic sinusoid in old age, there is a loss of fenestrations. LSECs are uniquely exposed to gut-derived dietary and microbial substrates delivered by the portal circulation to the liver. Here we studied the effect of 25 diets varying in content of macronutrients and energy on LSEC fenestrations using the Geometric Framework method in a large cohort of mice aged 15 mo. Macronutrient distribution rather than total food or energy intake was associated with changes in fenestrations. Porosity and frequency were inversely associated with dietary fat intake, while fenestration diameter was inversely associated with protein or carbohydrate intake. Fenestrations were also linked to diet-induced changes in gut microbiome, with increased fenestrations associated with higher abundance of Firmicutes and reduced abundance of Bacteroidetes Diet-induced changes in levels of several fatty acids (C16:0, C19:0, and C20:4) were also significantly inversely associated with fenestrations, suggesting a link between dietary fat and modulation of lipid rafts in the LSECs. Diet influences fenestrations and these data reflect both the key role of the LSECs in clearing gut-derived molecules from the vascular circulation and the impact these molecules have on LSEC morphology. Copyright © 2016 the American Physiological Society.

  10. A new liver function test using the asialoglycoprotein-receptor system on the liver cell membrane, 2

    International Nuclear Information System (INIS)

    Kawa, Soukichi; Hazama, Hiroshi; Kojima, Michimasa

    1986-01-01

    We produced labeled neoglycoprotein (GHSA) that is physiologically equivalent to ASGP, and quantitatively examined whether its uptake by the liver is dose-related using the following methods: 1) binding assay between GHSA and ASGP receptors, 2) measurement of the liver extraction ratio in the initial circulation following administration into the portal vein, and 3) measurement of clearance in normal rats and rats with galacosamine-induced acute liver disorder. The binding assay showed a linear relationship between the concentration of 125 I-GHSA and the amount of ASGP receptors obtained from the rat liver. A membrane assay using 125 I-GHSA and the liver cell membrane revealed similar results. The liver extraction ratio in the initial circulation following the administration into the portal vein of normal rabbits was highly dose-dependent (r = -0.95 in the range of 5 - 100 μg GHSA). Serial imaging of 99m Tc-GHSA during two-hour period after administration into the peripheral blood showed specific accumulation in the liver beginning immediately after the intravenous injection and subsequent transport mainly via the biliary system into the small intestine in the normal rat and mainly into the urine in the bile duct ligated rat. As a dynamic model of 99m Tc-GHSA, its circulation through the heart and liver and inactivated release from the liver was used, and two-compartment analysis was made on measurement curves in the heart and liver to obtain clearance parameters. The concentration of administered 99m Tc-GHSA (50 - 100 μg/100 g body weight) showed a positive linear relationship with clearance. Administration of 50 μg/100 g body weight of 99m Tc-GHSA revealed a significant correlation (p < 0.001) between clearance and ASGP receptor activity in normal rats and rats with galactosamine-induced acute liver disorder. (J.P.N.)

  11. Light microscopical demonstration and zonal distribution of parasinusoidal cells (Ito cells) in normal human liver

    DEFF Research Database (Denmark)

    Horn, T; Junge, Jette; Nielsen, O

    1988-01-01

    The parasinusoidal cells of the liver (Ito cells) were demonstrated light microscopically in autopsy specimens fixed in formalin and stained with Oil red O after dichromate treatment. The method allows examination of large samples containing numerous acini. Quantitative assessment showed a zonal...

  12. Light microscopical demonstration and zonal distribution of parasinusoidal cells (Ito cells) in normal human liver

    DEFF Research Database (Denmark)

    Horn, T; Junge, Jette; Nielsen, O

    1988-01-01

    The parasinusoidal cells of the liver (Ito cells) were demonstrated light microscopically in autopsy specimens fixed in formalin and stained with Oil red O after dichromate treatment. The method allows examination of large samples containing numerous acini. Quantitative assessment showed a zonal ...

  13. A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

    Science.gov (United States)

    Ribera, Jordi; Pauta, Montse; Melgar-Lesmes, Pedro; Córdoba, Bernat; Bosch, Anna; Calvo, Maria; Rodrigo-Torres, Daniel; Sancho-Bru, Pau; Mira, Aurea; Jiménez, Wladimiro; Morales-Ruiz, Manuel

    2017-11-01

    Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl 4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl 4 -treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-β1 (TGF-β1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P livers correlated with a significant decrease in liver fibrosis ( P livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization. NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-β1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization. Copyright © 2017 the American

  14. TRANSPLANTATION OF CRYOPRESERVED FETAL LIVER CELLS SEEDED INTO MACROPOROUS ALGINATE-GELATIN SCAFFOLDS IN RATS WITH LIVER FAILURE

    Directory of Open Access Journals (Sweden)

    D. V. Grizay

    2015-01-01

    Full Text Available Aim. To study the therapeutic potential of cryopreserved fetal liver cells seeded into macroporous alginategelatin scaffolds after implantation to omentum of rats with hepatic failure.Materials and methods.Hepatic failure was simulated by administration of 2-acetyl aminofl uorene followed partial hepatectomy. Macroporous alginate-gelatin scaffolds, seeded with allogenic cryopreserved fetal liver cells (FLCs were implanted into rat omentum. To prevent from colonization of host cells scaffolds were coated with alginate gel shell. Serum transaminase activity, levels of albumin and bilirubin as markers of hepatic function were determined during 4 weeks after failure model formation and scaffold implantation. Morphology of liver and scaffolds after implantation were examined histologically. Results. Macroporous alginate-gelatin scaffolds after implantation to healthy rats were colonized by host cells. Additional formation of alginate gel shell around scaffolds prevented the colonization. Implantation of macroporous scaffolds seeded with cryopreserved rat FLCs and additionally coated with alginate gel shell into omentum of rats with hepatic failure resulted in signifi cant improvement of hepatospecifi c parameters of the blood serum and positive changes of liver morphology. The presence of cells with their extracellular matrix within the scaffolds was confi rmed after 4 weeks post implantation.Conclusion. The data above indicate that macroporous alginate-gelatin scaffolds coated with alginate gel shell are promising cell carriers for the development of bioengineered liver equivalents.

  15. CULTIVATION OF HUMAN LIVER CELLS AND ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS IN PERFUSION BIOREACTOR

    Directory of Open Access Journals (Sweden)

    Yu. В. Basok

    2018-01-01

    Full Text Available Aim: to show the progress of the experiment of cultivation of human liver cells and adipose-derived mesenchymal stromal cells in perfusion bioreactor.Materials and methods. The cultivation of a cell-engineered construct, consisting of a biopolymer microstructured collagen-containing hydrogel, human liver cells, adipose-derived mesenchymal stromal cells, and William’s E Medium, was performed in a perfusion bioreactor.Results. On the 7th day large cells with hepatocyte morphology – of a polygonal shape and a centrally located round nucleus, – were present in the culture chambers of the bioreactor. The metabolic activity of hepatocytes in cell-engineered constructs was confi rmed by the presence of urea in the culture medium on the seventh day of cultivation in the bioreactor and by the resorption of a biopolymer microstructured collagen-containing hydrogel.

  16. ATP Binding cassette transporter gene expression in rat liver progenitor cells

    NARCIS (Netherlands)

    Ros, J.E.; Roskams, T.A.D.; Geuken, M.; Havinga, R.; Splinter, P.L.; Petersen, B.E.; LaRusso, N.F.; Kolk, van der D.M.; Kuipers, F.; Faber, K.N.; Müller, M.R.; Jansen, P.L.M.

    2003-01-01

    Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

  17. ATP binding cassette transporter gene expression in rat liver progenitor cells

    NARCIS (Netherlands)

    Ros, J. E.; Roskams, T. A. D.; Geuken, M.; Havinga, R.; Splinter, P. L.; Petersen, B. E.; LaRusso, N. F.; van der Kolk, D. M.; Kuipers, F.; Faber, K. N.; Müller, M.; Jansen, P. L. M.

    2003-01-01

    BACKGROUND AND AIM: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

  18. ATP binding cassette transporter gene expression in rat liver progenitor cells

    NARCIS (Netherlands)

    Ros, J.E.; Roskams, TAD; Geuken, M; Havinga, R; Splinter, PL; Petersen, BE; LaRusso, NF; van der Kolk, D.M.; Kuipers, F; Faber, KN; Muller, M; Jansen, PLM

    Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

  19. Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

    Science.gov (United States)

    Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

    2009-01-01

    Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

  20. Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques

    International Nuclear Information System (INIS)

    Ahsan, Muhammad H.; Gill, Amy F.; Alvarez, Xavier; Lackner, Andrew A.; Veazey, Ronald S.

    2013-01-01

    Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animals examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. - Highlights: • Kupffer cells increase in the liver of SIV-infected macaques. • Increased proliferation and apoptosis of Kupffer cells occurs in SIV infection. • Productively infected cells are rarely detected in the liver. • The liver is not a major site for SIV replication

  1. Glypican-3 Targeting of Liver Cancer Cells Using Multifunctional Nanoparticles

    Directory of Open Access Journals (Sweden)

    James O. Park

    2011-01-01

    Full Text Available Imaging is essential in accurately detecting, staging, and treating primary liver cancer (hepatocellular carcinoma [HCC], one of the most prevalent and lethal malignancies. We developed a novel multifunctional nanoparticle (NP specifically targeting glypican-3 (GPC3, a proteoglycan implicated in promotion of cell growth that is overexpressed in most HCCs. Quantitative real-time polymerase chain reaction was performed to confirm the differential GPC3 expression in two human HCC cells, Hep G2 (high and HLF (negligible. These cells were treated with biotin-conjugated GPC3 monoclonal antibody (αGPC3 and subsequently targeted using superparamagnetic iron oxide NPs conjugated to streptavidin and Alexa Fluor 647. Flow cytometry demonstrated that only GPC3-expressing Hep G2 cells were specifically targeted using this αGPC3-NP conjugate (fourfold mean fluorescence over nontargeted NP, and magnetic resonance imaging (MRI experiments showed similar findings (threefold R2 relaxivity. Confocal fluorescence microscopy localized the αGPC3 NPs only to the cell surface of GPC3-expressing Hep G2 cells. Further characterization of this construct demonstrated a negatively charged, monodisperse, 50 nm NP, ideally suited for tumor targeting. This GPC3-specific NP system, with dual-modality imaging capability, may enhance pretreatment MRI, enable refined intraoperative HCC visualization by near-infrared fluorescence, and be potentially used as a carrier for delivery of tumor-targeted therapies, improving patient outcomes.

  2. IDH mutations in liver cell plasticity and biliary cancer

    Science.gov (United States)

    Saha, Supriya K; Parachoniak, Christine A; Bardeesy, Nabeel

    2014-01-01

    Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer associated with the bile ducts within the liver. These tumors are characterized by frequent gain-of-function mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes—that are also common in subsets of neural, haematopoietic and bone tumors, but rare or absent in the other types of gastrointestinal malignancy. Mutant IDH acts through a novel mechanism of oncogenesis, producing high levels of the metabolite 2-hydroxyglutarate, which interferes with the function of α-ketoglutarate-dependent enzymes that regulate diverse cellular processes including histone demethylation and DNA modification. Recently, we used in vitro stem cell systems and genetically engineered mouse models (GEMMs) to demonstrate that mutant IDH promotes ICC formation by blocking hepatocyte differentiation and increasing pools of hepatic progenitors that are susceptible to additional oncogenic hits leading to ICC. We found that silencing of HNF4A—encoding a master transcriptional regulator of hepatocyte identity and quiescence—was critical to mutant IDH-mediated inhibition of liver differentiation. In line with these findings, human ICC with IDH mutations are characterized by a hepatic progenitor cell transcriptional signature suggesting that they are a distinct ICC subtype as compared to IDH wild type tumors. The role of mutant IDH in controlling hepatic differentiation state suggests the potential of newly developed inhibitors of the mutant enzyme as a form of differentiation therapy in a solid tumor. PMID:25485496

  3. Photosynthetic fuel for heterologous enzymes

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Vavitsas, Konstantinos; Nielsen, Agnieszka Janina Zygadlo

    2017-01-01

    of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely...... on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However......, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic...

  4. Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.

    Directory of Open Access Journals (Sweden)

    Felix Dempwolff

    Full Text Available Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.

  5. Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.

    Science.gov (United States)

    Dempwolff, Felix; Reimold, Christian; Reth, Michael; Graumann, Peter L

    2011-01-01

    Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.

  6. Hepatocyte transplantation and advancements in alternative cell sources for liver-based regenerative medicine.

    Science.gov (United States)

    Lee, Charlotte A; Sinha, Siddharth; Fitzpatrick, Emer; Dhawan, Anil

    2018-06-01

    Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases.

  7. Liver sinusoidal endothelial cells induce immunosuppressive IL-10-producing Th1 cells via the Notch pathway.

    Science.gov (United States)

    Neumann, Katrin; Rudolph, Christine; Neumann, Christian; Janke, Marko; Amsen, Derk; Scheffold, Alexander

    2015-07-01

    Under homeostasis, liver sinusoidal endothelial cells (LSECs) shift intrahepatic T-cell responses towards tolerance. However, the role of LSECs in the regulation of T-cell-induced liver inflammation is less clear. Here, we studied the capacity of LSECs to modulate pro-inflammatory Th1-cell differentiation in mice. Using in vitro co-culture systems and subsequent cytokine analysis, we showed that LSECs induced high amounts of the anti-inflammatory cytokine IL-10 in developing Th1 cells. These LSEC-stimulated Th1 cells had no pro-inflammatory capacity in vivo but instead actively suppressed an inflammatory Th1-cell-induced delayed-type hypersensitivity reaction. Blockage of IL-10 signaling in vivo inhibited immunosuppressive activity of LSEC-stimulated Th1 cells. We identified the Notch pathway as a mechanism how LSECs trigger IL-10 expression in Th1 cells. LSECs expressed high levels of the Delta-like and Jagged family of Notch ligands and induced expression of the Notch target genes hes-1 and deltex-1 in Th1 cells. Blockade of Notch signaling selectively inhibited IL-10 induction in Th1 cells by LSECs. Our findings suggest that LSEC-induced IL-10 expression in Th1 cells via the Notch pathway may contribute to the control of hepatic inflammatory immune responses by induction of a self-regulatory mechanism in pro-inflammatory Th1 cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

    International Nuclear Information System (INIS)

    Shikanai, Mima; Asahina, Kinji; Iseki, Sachiko; Teramoto, Kenichi; Nishida, Tomohiro; Shimizu-Saito, Keiko; Ota, Masato; Eto, Kazuhiro; Teraoka, Hirobumi

    2009-01-01

    Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or α-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

  9. Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongxia; Cui, Ruina [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Guo, Xuejiang [State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029 (China); Hu, Jiayue [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Dai, Jiayin, E-mail: daijy@ioz.ac.cn [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China)

    2016-08-05

    Highlights: • Differential expression of proteins induced by PFOA in HL-7702 was identified. • Most of the differentially expressed proteins are related to cell proliferation. • A low dose of PFOA stimulates HL-7702 cell proliferation. • A high dose of PFOA inhibits HL-7702 cell proliferation. - Abstract: Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50 μM PFOA for 48 h and 96 h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50–100 μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200–400 μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

  10. Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

    International Nuclear Information System (INIS)

    Zhang, Hongxia; Cui, Ruina; Guo, Xuejiang; Hu, Jiayue; Dai, Jiayin

    2016-01-01

    Highlights: • Differential expression of proteins induced by PFOA in HL-7702 was identified. • Most of the differentially expressed proteins are related to cell proliferation. • A low dose of PFOA stimulates HL-7702 cell proliferation. • A high dose of PFOA inhibits HL-7702 cell proliferation. - Abstract: Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50 μM PFOA for 48 h and 96 h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50–100 μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200–400 μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

  11. Priming T-cell responses with recombinant measles vaccine vector in a heterologous prime-boost setting in non-human primates.

    Science.gov (United States)

    Bolton, Diane L; Santra, Sampa; Swett-Tapia, Cindy; Custers, Jerome; Song, Kaimei; Balachandran, Harikrishnan; Mach, Linh; Naim, Hussein; Kozlowski, Pamela A; Lifton, Michelle; Goudsmit, Jaap; Letvin, Norman; Roederer, Mario; Radošević, Katarina

    2012-09-07

    Licensed live attenuated virus vaccines capable of expressing transgenes from other pathogens have the potential to reduce the number of childhood immunizations by eliciting robust immunity to multiple pathogens simultaneously. Recombinant attenuated measles virus (rMV) derived from the Edmonston Zagreb vaccine strain was engineered to express simian immunodeficiency virus (SIV) Gag protein for the purpose of evaluating the immunogenicity of rMV as a vaccine vector in rhesus macaques. rMV-Gag immunization alone elicited robust measles-specific humoral and cellular responses, but failed to elicit transgene (Gag)-specific immune responses, following aerosol or intratracheal/intramuscular delivery. However, when administered as a priming vaccine to a heterologous boost with recombinant adenovirus serotype 5 expressing the same transgene, rMV-Gag significantly enhanced Gag-specific T lymphocyte responses following rAd5 immunization. Gag-specific humoral responses were not enhanced, however, which may be due to either the transgene or the vector. Cellular response priming by rMV against the transgene was highly effective even when using a suboptimal dose of rAd5 for the boost. These data demonstrate feasibility of using rMV as a priming component of heterologous prime-boost vaccine regimens for pathogens requiring strong cellular responses. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Propranolol inhibits the in vitro conversion of thyroxine into triiodothyronine by isolated rat liver parenchymal cells

    NARCIS (Netherlands)

    van Noorden, C. J.; Wiersinga, W. M.; Touber, J. L.

    1979-01-01

    A model for the in vitro study of the conversion of thyroxine into triiodothyronine using isolated rat liver parenchymal cells is described. Isolated liver cells (mean protein content 18 mg/ml) convert approximately 0.8% of 1.3 microM exogenously added T4 into T3 during thirty minutes incubation.

  13. Modeling Dynamics and Function of Bone Marrow Cells in Mouse Liver Regeneration

    NARCIS (Netherlands)

    Pedone, Elisa; Olteanu, Vlad-Aris; Marucci, Lucia; Muñoz-Martin, Maria Isabel; Youssef, Sameh A; de Bruin, Alain; Cosma, Maria Pia

    2017-01-01

    In rodents and humans, the liver can efficiently restore its mass after hepatectomy. This is largely attributed to the proliferation and cell cycle re-entry of hepatocytes. On the other hand, bone marrow cells (BMCs) migrate into the liver after resection. Here, we find that a block of BMC

  14. Synthesis of erythrocyte membrane proteins in dispersed cells from fetal rat liver

    International Nuclear Information System (INIS)

    Kitagawa, Yasuo; Murakami, Akihiko; Sugimoto, Etsuro

    1984-01-01

    Protein synthesis in dispersed cells from fetal liver was studied by fluorography of SDS-polyacrylamide gel electrophoresis of a [ 35 S] methionine labeled cell lysate. Synthesis of several proteins with molecular weights ranging from 45,000 to 220,000 was observed during erythropoiesis in fetal liver. Some of these proteins were demonstrated to be erythrocyte membrane proteins because they were immunoprecipitated with antiserum against rat red blood cells and the immunoprecipitation was competitive with non-radioactive proteins solubilized from erythrocyte ghosts. The same antiserum caused agglutination of dispered cells from fetal liver. This supported the possibility that these proteins are translocated onto plasma membranes of the dispersed cells. (author)

  15. Human Mesenchymal Stem Cell Transfusion Is Safe and Improves Liver Function in Acute-on-Chronic Liver Failure Patients

    Science.gov (United States)

    Shi, Ming; Zhang, Zheng; Xu, Ruonan; Lin, Hu; Fu, Junliang; Zou, Zhengsheng; Zhang, Aimin; Shi, Jianfei; Chen, Liming; Lv, Sa; He, Weiping; Geng, Hua; Jin, Lei; Liu, Zhenwen

    2012-01-01

    Acute-on-chronic liver failure (ACLF) is a severe, life-threatening complication, and new and efficient therapeutic strategies for liver failure are urgently needed. Mesenchymal stem cell (MSC) transfusions have been shown to reverse fulminant hepatic failure in mice and to improve liver function in patients with end-stage liver diseases. We assessed the safety and initial efficacy of umbilical cord-derived MSC (UC-MSC) transfusions for ACLF patients associated with hepatitis B virus (HBV) infection. A total of 43 ACLF patients were enrolled for this open-labeled and controlled study; 24 patients were treated with UC-MSCs, and 19 patients were treated with saline as controls. UC-MSC therapy was given three times at 4-week intervals. The liver function, adverse events, and survival rates were evaluated during the 48-week or 72-week follow-up period. No significant side effects were observed during the trial. The UC-MSC transfusions significantly increased the survival rates in ACLF patients; reduced the model for end-stage liver disease scores; increased serum albumin, cholinesterase, and prothrombin activity; and increased platelet counts. Serum total bilirubin and alanine aminotransferase levels were significantly decreased after the UC-MSC transfusions. UC-MSC transfusions are safe in the clinic and may serve as a novel therapeutic approach for HBV-associated ACLF patients. PMID:23197664

  16. Biodistribution of Liver-Derived Mesenchymal Stem Cells After Peripheral Injection in a Hemophilia A Patient.

    Science.gov (United States)

    Sokal, Etienne M; Lombard, Catherine Anne; Roelants, Véronique; Najimi, Mustapha; Varma, Sharat; Sargiacomo, Camillo; Ravau, Joachim; Mazza, Giuseppe; Jamar, François; Versavau, Julia; Jacobs, Vanessa; Jacquemin, Marc; Eeckhoudt, Stéphane; Lambert, Catherine; Stéphenne, Xavier; Smets, Françoise; Hermans, Cédric

    2017-08-01

    With the exception of liver transplantation, there is no cure for hemophilia, which is currently managed by preemptive replacement therapy. Liver-derived stem cells are in clinical development for inborn and acquired liver diseases and could represent a curative treatment for hemophilia A. The liver is a major factor VIII (FVIII) synthesis site, and mesenchymal stem cells have been shown to control joint bleeding in animal models of hemophilia. Adult-derived human liver stem cells (ADHLSCs) have mesenchymal characteristics and have been shown able to engraft in and repopulate both animal and human livers. Thus, the objectives were to evaluate the potency of ADHLSCs to control bleeding in a hemophilia A patient and assess the biodistribution of the cells after intravenous injection. A patient suffering from hemophilia A was injected with repeated doses of ADHLSCs via a peripheral vein (35 million In-oxine-labeled cells, followed by 125 million cells the next day, and 3 infusions of 250 million cells every 2 weeks thereafter; total infusion period, 50 days). After cell therapy, we found a temporary (15 weeks) decrease in the patient's FVIII requirements and severe bleeding complications, despite a lack of increase in circulating FVIII. The cells were safely administered to the patient via a peripheral vein. Biodistribution analysis revealed an initial temporary entrapment of the cells in the lungs, followed by homing to the liver and to a joint afflicted with hemarthrosis. These results suggest the potential use of ADHLSCs in the treatment of hemophilia A.

  17. Cation Homeostasis in Red Cells From Patients With Sickle Cell Disease Heterologous for HbS and HbC (HbSC Genotype

    Directory of Open Access Journals (Sweden)

    A. Hannemann

    2015-11-01

    Full Text Available Sickle cell disease (SCD in patients of HbSC genotype is considered similar, albeit milder, to that in homozygous HbSS individuals — but with little justification. In SCD, elevated red cell cation permeability is critical as increased solute loss causes dehydration and encourages sickling. Recently, we showed that the KCl cotransporter (KCC activity in red cells from HbSC patients correlated significantly with disease severity, but that in HbSS patients did not. Two transporters involved in red cell dehydration, the conductive channels Psickle and the Gardos channel, behaved similarly in red cells from the two genotypes, but were significantly less active in HbSC patients. By contrast, KCC activity was quantitatively greater in HbSC red cells. Results suggest that KCC is likely to have greater involvement in red cell dehydration in HbSC patients, which could explain its association with disease severity in this genotype. This work supports the hypothesis that SCD in HbSC patients is a distinct disease entity to that in HbSS patients. Results suggest the possibility of designing specific treatments of particular benefit to HbSC patients and a rationale for the development of prognostic markers, to inform early treatment of children likely to develop more severe complications of the disease.

  18. Mesenchymal Stem Cells Enhance Liver Regeneration via Improving Lipid Accumulation and Hippo Signaling

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2018-01-01

    Full Text Available The liver has the potential to regenerate after injury. It is a challenge to improve liver regeneration (LR after liver resection in clinical practice. Bone morrow-derived mesenchymal stem cells (MSCs have shown to have a role in various liver diseases. To explore the effects of MSCs on LR, we established a model of 70% partial hepatectomy (PHx. Results revealed that infusion of MSCs could improve LR through enhancing cell proliferation and cell growth during the first 2 days after PHx, and MSCs could also restore liver synthesis function. Infusion of MSCs also improved liver lipid accumulation partly via mechanistic target of rapamycin (mTOR signaling and enhanced lipid β-oxidation support energy for LR. Rapamycin-induced inhibition of mTOR decreased liver lipid accumulation at 24 h after PHx, leading to impaired LR. And after infusion of MSCs, a proinflammatory environment formed in the liver, evidenced by increased expression of IL-6 and IL-1β, and thus the STAT3 and Hippo-YAP pathways were activated to improve cell proliferation. Our results demonstrated the function of MSCs on LR after PHx and provided new evidence for stem cell therapy of liver diseases.

  19. The role of Foxp3+ regulatory T cells in liver transplant tolerance.

    Science.gov (United States)

    Li, W; Carper, K; Zheng, X X; Kuhr, C S; Reyes, J D; Liang, Y; Perkins, D L; Thomson, A W; Perkins, J D

    2006-12-01

    The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.

  20. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Österlund, Tobias; Liu, Zihe

    2013-01-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular...... stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent...... the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high...

  1. Human liver cell trafficking mutants: characterization and whole exome sequencing.

    Directory of Open Access Journals (Sweden)

    Fei Yuan

    Full Text Available The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

  2. Autologous Bone Marrow Stem Cell Infusion (AMBI therapy for Chronic Liver Diseases

    Directory of Open Access Journals (Sweden)

    Rajkumar JS

    2007-01-01

    Full Text Available Liver Cirrhosis is the end stage of chronic liver disease which may happen due to alcoholism, viral infections due to Hepatitis B, Hepatitis C viruses and is difficult to treat. Liver transplantation is the only available definitive treatment which is marred by lack of donors, post operative complications such as rejection and high cost. Autologous bone marrow stem cells have shown a lot of promise in earlier reported animal studies and clinical trials. We have in this study administered in 22 patients with chronic liver disease, autologous bone marrow stem cell whose results are presented herewith.

  3. Subnormothermic ex vivo liver perfusion reduces endothelial cell and bile duct injury after donation after cardiac death pig liver transplantation.

    Science.gov (United States)

    Knaak, Jan M; Spetzler, Vinzent N; Goldaracena, Nicolas; Boehnert, Markus U; Bazerbachi, Fateh; Louis, Kristine S; Adeyi, Oyedele A; Minkovich, Leonid; Yip, Paul M; Keshavjee, Shaf; Levy, Gary A; Grant, David R; Selzner, Nazia; Selzner, Markus

    2014-11-01

    An ischemic-type biliary stricture (ITBS) is a common feature after liver transplantation using donation after cardiac death (DCD) grafts. We compared sequential subnormothermic ex vivo liver perfusion (SNEVLP; 33°C) with cold storage (CS) for the prevention of ITBS in DCD liver grafts in pig liver transplantation (n = 5 for each group). Liver grafts were stored for 10 hours at 4°C (CS) or preserved with combined 7-hour CS and 3-hour SNEVLP. Parameters of hepatocyte [aspartate aminotransferase (AST), international normalized ratio (INR), factor V, and caspase 3 immunohistochemistry], endothelial cell (EC; CD31 immunohistochemistry and hyaluronic acid), and biliary injury and function [alkaline phosphatase (ALP), total bilirubin, and bile lactate dehydrogenase (LDH)] were determined. Long-term survival (7 days) after transplantation was similar between the SNEVLP and CS groups (60% versus 40%, P = 0.13). No difference was observed between SNEVLP- and CS-treated animals with respect to the peak of serum INR, factor V, or AST levels within 24 hours. CD31 staining 8 hours after transplantation demonstrated intact EC lining in SNEVLP-treated livers (7.3 × 10(-4) ± 2.6 × 10(-4) cells/μm(2)) but not in CS-treated livers (3.7 × 10(-4) ± 1.3 × 10(-4) cells/μm(2) , P = 0.03). Posttransplant SNEVLP animals had decreased serum ALP and serum bilirubin levels in comparison with CS animals. In addition, LDH in bile fluid was lower in SNEVLP pigs versus CS pigs (14 ± 10 versus 60 ± 18 μmol/L, P = 0.02). Bile duct histology revealed severe bile duct necrosis in 3 of 5 animals in the CS group but none in the SNEVLP group (P = 0.03). Sequential SNEVLP preservation of DCD grafts reduces bile duct and EC injury after liver transplantation. © 2014 American Association for the Study of Liver Diseases.

  4. In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

    Science.gov (United States)

    Ramachandran, Sarada Devi; Schirmer, Katharina; Münst, Bernhard; Heinz, Stefan; Ghafoory, Shahrouz; Wölfl, Stefan; Simon-Keller, Katja; Marx, Alexander; Øie, Cristina Ionica; Ebert, Matthias P; Walles, Heike; Braspenning, Joris; Breitkopf-Heinlein, Katja

    2015-01-01

    In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

  5. Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins

    International Nuclear Information System (INIS)

    Corcelle, V.; Stieger, B.; Gjinovci, A.; Wollheim, C.B.; Gauthier, B.R.

    2006-01-01

    Despite extensive studies, the hematopoietic versus hepatic origin of liver progenitor oval cells remains controversial. The aim of this study was to determine the origin of such cells after liver injury and to establish an oval cell line. Rat liver injury was induced by subcutaneous insertion of 2-AAF pellets for 7 days with subsequent injection of CCl 4 . Livers were removed 9 to 13 days post-CCl 4 treatment. Immunohistochemistry was performed using anti-c-kit, OV6, Thy1, CK19, AFP, vWF and Rab3b. Isolated non-parenchymal cells were grown on mouse embryonic fibroblast, and their gene expression profile was characterized by RT-PCR. We identified a subpopulation of OV6/CK19/Rab3b-expressing cells that was activated in the periportal region of traumatized livers. We also characterized a second subpopulation that expressed the HSCs marker c-kit but not Thy1. Although we successfully isolated both cell types, OV6/CK19/Rab3b + cells fail to propagate while c-kit + -HSCs appeared to proliferate for up to 7 weeks. Cells formed clusters which expressed c-kit, Thy1 and albumin. Our results indicate that a bona fide oval progenitor cell population resides within the liver and is distinct from c-kit + -HSCs. Oval cells require the hepatic niche to proliferate, while cells mobilized from the circulation proliferate and transdifferentiate into hepatocytes without evidence of cell fusion

  6. Comparative Peripheral Blood T Cells Analysis Between Adult Deceased Donor Liver Transplantation (DDLT) and Living Donor Liver Transplantation (LDLT).

    Science.gov (United States)

    Kim, Jong Man; Kwon, Choon Hyuck David; Joh, Jae-Won; Choi, Gyu-Seong; Kang, Eun-Suk; Lee, Suk-Koo

    2017-08-08

    BACKGROUND T lymphocytes are an essential component of allograft rejection and tolerance. The aim of the present study was to analyze and compare the characteristics of T cell subsets in patients who underwent deceased donor liver transplantation (DDLT) versus living donor liver transplantation (LDLT). MATERIAL AND METHODS Between April 2013 and June 2014, 64 patients underwent adult liver transplantation. The distribution of peripheral blood T lymphocyte subsets before transplantation and at 4, 8, 12, and 24 weeks post-transplantation were monitored serially. RESULTS In the serial peripheral blood samples, the absolute CD3+ T cell counts in the LDLT group were higher than those in the DDLT group (p=0.037). The CD4+, CD8+, CD4/CD8, Vδ1, Vδ2, and γδ T cell counts did not change significantly over time in either group. The Vδ1/Vδ2 ratio was higher in patients with cytomegalovirus (CMV) infection than in patients without CMV infection (0.12 versus 0.26; p=0.033). The median absolute CD3+ and CD8+ T cell counts in patients with biopsy-proven acute rejection (BPAR) were 884 (range, 305-1,320) and 316 (range, 271-1,077), respectively, whereas they were 320 (range, 8-1,167) and 257 (range, 58-1,472) in patients without BPAR. The absolute CD3+ and CD8 T cell counts were higher in patients with BPAR than in patients without BPAR (p=0.007 and p=0.039, respectively). CONCLUSIONS With the exception of CD3+ T cells, T cell populations did not differ significantly between patients who received DDLT versus LDLT. In liver transplantation patients, CMV infection and BPAR were closely associated with T cell population changes.

  7. Potential and Challenges of Induced Pluripotent Stem Cells in Liver Diseases Treatment

    Directory of Open Access Journals (Sweden)

    Yue Yu

    2014-09-01

    Full Text Available Tens of millions of patients are affected by liver disease worldwide. Many of these patients can benefit from cell therapy involving living metabolically active cells, either by treatment of their liver disease, or by prevention of their disease phenotype. Cell therapies, including hepatocyte transplantation and bioartificial liver (BAL devices, have been proposed as therapeutic alternatives to the shortage of transplantable livers. Both BAL and hepatocyte transplantation are cellular therapies that avoid use of a whole liver. Hepatocytes are also widely used in drug screening and liver disease modelling. However, the demand for human hepatocytes, heavily outweighs their availability by conventional means. Induced pluripotent stem cells (iPSCs technology brings together the potential benefits of embryonic stem cells (ESCs (i.e., self-renewal, pluripotency and addresses the major ethical and scientific concerns of ESCs: embryo destruction and immune-incompatibility. It has been shown that hepatocyte-like cells (HLCs can be generated from iPSCs. Furthermore, human iPSCs (hiPSCs can provide an unlimited source of human hepatocytes and hold great promise for applications in regenerative medicine, drug screening and liver diseases modelling. Despite steady progress, there are still several major obstacles that need to be overcome before iPSCs will reach the bedside. This review will focus on the current state of efforts to derive hiPSCs for potential use in modelling and treatment of liver disease.

  8. The Role of Dendritic Cells in Fibrosis Progression in Nonalcoholic Fatty Liver Disease

    Directory of Open Access Journals (Sweden)

    Paloma Almeda-Valdes

    2015-01-01

    Full Text Available Nonalcoholic fatty liver disease (NAFLD is the most frequent cause of chronic liver disease. NAFLD encompasses a wide range of pathologies, from simple steatosis to steatosis with inflammation to fibrosis. The pathogenesis of NAFLD progression has not been completely elucidated, and different liver cells could be implicated. This review focuses on the current evidence of the role of liver dendritic cells (DCs in the progression from NAFLD to fibrosis. Liver DCs are a heterogeneous population of hepatic antigen-presenting cells; their main function is to induce T-cell mediated immunity by antigen processing and presentation to T cells. During the steady state liver DCs are immature and tolerogenic. However, in an environment of chronic inflammation, DCs are transformed to potent inducers of immune responses. There is evidence about the role of DC in liver fibrosis, but it is not clearly understood. Interestingly, there might be a link between lipid metabolism and DC function, suggesting that immunogenic DCs are associated with liver lipid storage, representing a possible pathophysiological mechanism in NAFLD development. A better understanding of the interaction between inflammatory pathways and the different cell types and the effect on the progression of NAFLD is of great relevance.

  9. Comparison of Species and Cell-Type Differences in Fraction Unbound of Liver Tissues, Hepatocytes, and Cell Lines.

    Science.gov (United States)

    Riccardi, Keith; Ryu, Sangwoo; Lin, Jian; Yates, Phillip; Tess, David; Li, Rui; Singh, Dhirender; Holder, Brian R; Kapinos, Brendon; Chang, George; Di, Li

    2018-04-01

    Fraction unbound ( f u ) of liver tissue, hepatocytes, and other cell types is an essential parameter used to estimate unbound liver drug concentration and intracellular free drug concentration. f u,liver and f u,cell are frequently measured in multiple species and cell types in drug discovery and development for various applications. A comparison study of 12 matrices for f u,liver and f u,cell of hepatocytes in five different species (mouse, rat, dog, monkey, and human), as well as f u,cell of Huh7 and human embryonic kidney 293 cell lines, was conducted for 22 structurally diverse compounds with the equilibrium dialysis method. Using an average bioequivalence approach, our results show that the average difference in binding to liver tissue, hepatocytes, or different cell types was within 2-fold of that of the rat f u,liver Therefore, we recommend using rat f u,liver as a surrogate for liver binding in other species and cell types in drug discovery. This strategy offers the potential to simplify binding studies and reduce cost, thereby enabling a more effective and practical determination of f u for liver tissues, hepatocytes, and other cell types. In addition, f u under hepatocyte stability incubation conditions should not be confused with f u,cell , as one is a diluted f u and the other is an undiluted f u Cell density also plays a critical role in the accurate measurement of f u,cell . Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  10. Uptake and clearance of plutonium-238 from liver cells transplanted into fat pads of F344 rats

    International Nuclear Information System (INIS)

    Brooks, A.L.; Guilmette, R.A.; Hahn, F.F.

    1986-01-01

    Animals injected with liver cells and control animals received a single intraperitoneal injection of 37 kBq (1 μCi) 238 Pu citrate and were serially sacrificed. It was observed that the cells of the intact liver took up about twice as much 238 Pu as liver cells transplanted into the fat pads of the same animal. The retention half-life was 8.3 days for the total activity in the liver, 20 days using tracks/cell measurements in the liver and 16 days for the tracks/cell measurements in the liver cells translocated to fat pads. When the data on tracks/cell were standardized relative to the amount of Pu present at 5 days after injection, there was no significant difference between the retention of Pu in liver cells from intact animals and liver cells transplanted into the fat pads. About 20% of the 5-day Pu liver burden in both liver cells and liver cells transplanted into fat pads was retained at 70 days. The smaller retention and clearance for liver cells in different environments indicate that uptake and clearance of Pu from the body is dependent, to a major extent, upon hepatocyte function. (author)

  11. From the Cover: Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells

    Science.gov (United States)

    Sapir, Tamar; Shternhall, Keren; Meivar-Levy, Irit; Blumenfeld, Tamar; Cohen, Hamutal; Skutelsky, Ehud; Eventov-Friedman, Smadar; Barshack, Iris; Goldberg, Iris; Pri-Chen, Sarah; Ben-Dor, Lya; Polak-Charcon, Sylvie; Karasik, Avraham; Shimon, Ilan; Mor, Eytan; Ferber, Sarah

    2005-05-01

    Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue. pancreas | transdifferentiation

  12. Extracellular Vesicles from Human Liver Stem Cells Reduce Injury in an Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model.

    Science.gov (United States)

    Rigo, Federica; De Stefano, Nicola; Navarro-Tableros, Victor; David, Ezio; Rizza, Giorgia; Catalano, Giorgia; Gilbo, Nicholas; Maione, Francesca; Gonella, Federica; Roggio, Dorotea; Martini, Silvia; Patrono, Damiano; Salizzoni, Mauro; Camussi, Giovanni; Romagnoli, Renato

    2018-05-01

    The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. Here, we aimed to explore the feasibility of a pharmacological intervention on isolated rat livers by using a combination of NMP and human liver stem cells-derived extracellular vesicles (HLSC-EV). We established an ex vivo murine model of NMP capable to maintain liver function despite an ongoing hypoxic injury induced by hemodilution. Livers were perfused for 4 hours without (control group, n = 10) or with HLSC-EV (treated group, n = 9). Bile production was quantified; perfusate samples were collected hourly to measure metabolic (pH, pO2, pCO2) and cytolysis parameters (AST, alanine aminotransferase, lactate dehydrogenase). At the end of perfusion, we assessed HLSC-EV engraftment by immunofluorescence, tissue injury by histology, apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, tissue hypoxia-inducible factor 1-α, and transforming growth factor-beta 1 RNA expression by quantitative reverse transcription-polymerase chain reaction. During hypoxic NMP, livers were able to maintain homeostasis and produce bile. In the treated group, AST (P = 0.018) and lactate dehydrogenase (P = 0.032) levels were significantly lower than those of the control group at 3 hours of perfusion, and AST levels persisted lower at 4 hours (P = 0.003). By the end of NMP, HLSC-EV had been uptaken by hepatocytes, and EV treatment significantly reduced histological damage (P = 0.030), apoptosis (P = 0.049), and RNA overexpression of hypoxia-inducible factor 1-α (P < 0.0001) and transforming growth factor-beta 1 (P = 0.014). HLSC-EV treatment, even in a short-duration model, was feasible and effectively reduced liver injury during hypoxic NMP.

  13. A Pilot Study of Mesenchymal Stem Cell Therapy for Acute Liver Allograft Rejection

    OpenAIRE

    Shi, Ming; Liu, Zhenwen; Wang, Ying; Xu, Rounan; Sun, Yanling; Zhang, Min; Yu, Xi; Wang, Hongbo; Meng, Lingzhan; Su, Haibin; Jin, Lei; Wang, Fu‐Sheng

    2017-01-01

    Abstract Acute allograft rejection remains common after liver transplantation despite modern immunosuppressive agents. In addition, the long‐term side effects of these regimens, including opportunistic infections, are challenging. This study evaluated the safety and clinical feasibility of umbilical cord‐derived mesenchymal stem cell (UC‐MSC) therapy in liver transplant patients with acute graft rejection. Twenty‐seven liver allograft recipients with acute rejection were randomly assigned int...

  14. [Toxic effect of trichloroethylene on liver cells with CYP3A4 gene defect].

    Science.gov (United States)

    Liao, R Y; Liu, S

    2016-06-20

    To investigate the toxic effect of trichloroethylene on liver cells with CYP3A4 gene defect. The normal human liver cells (L02 cells) and liver cells with CYP3A4 gene defect were exposed to trichloroethylene at different doses (0.0, 0.4, 0.8, 1.6, 3.2, and 6.4 mmol/L). CCK8 assay and RT-qPCR were used to measure cell viability and changes in the expression of apoptosis genes and oncogenes. After being exposed to trichloroethylene at doses of 1.6, 3.2, and 6.4 mmol/L, the liver cells with CYP3A4 gene defect showed significantly higher cell viability than L02 cells (0.91±0.06/0.89±0.05/0.85±0.07 vs 0.80±0.04/0.73±0.06/0.67±0.07, Ptrichloroethylene groups showed significant increases in the expression of the apoptosis genes caspase-3, caspase-8, and caspase-9 (PTrichloroethylene exposure has a less effect on the expression of apoptosis genes and oncogenes in liver cells with CYP3A4 gene defect than in normal human liver cells, suggesting that CYP3A4 gene defect reduces the inductive effect of trichloroethylene on apoptosis genes and oncogenes.

  15. Evaluation of rat liver apoptotic and necrotic cell death after cold storage using UW, HTK, and Celsior

    NARCIS (Netherlands)

    Straatsburg, Irene H.; Abrahamse, Salomon L.; Song, Shao W.; Hartman, Robin J.; van Gulik, Thomas M.

    2002-01-01

    Background. The benefit of Celsior in liver graft preservation is controversial. In the isolated perfused rat liver model, we compared the effects of Celsior, University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) preservation solutions on liver cell death. Methods. Rat livers

  16. Acute Liver Injury Is Independent of B Cells or Immunoglobulin M.

    Directory of Open Access Journals (Sweden)

    James A Richards

    Full Text Available Acute liver injury is a clinically important pathology and results in the release of Danger Associated Molecular Patterns, which initiate an immune response. Withdrawal of the injurious agent and curtailing any pathogenic secondary immune response may allow spontaneous resolution of injury. The role B cells and Immunoglobulin M (IgM play in acute liver injury is largely unknown and it was proposed that B cells and/or IgM would play a significant role in its pathogenesis.Tissue from 3 models of experimental liver injury (ischemia-reperfusion injury, concanavalin A hepatitis and paracetamol-induced liver injury and patients transplanted following paracetamol overdose were stained for evidence of IgM deposition. Mice deficient in B cells (and IgM were used to dissect out the role B cells and/or IgM played in the development or resolution of injury. Serum transfer into mice lacking IgM was used to establish the role IgM plays in injury.Significant deposition of IgM was seen in the explanted livers of patients transplanted following paracetamol overdose as well as in 3 experimental models of acute liver injury (ischemia-reperfusion injury, concanavalin A hepatitis and paracetamol-induced liver injury. Serum transfer into IgM-deficient mice failed to reconstitute injury (p = 0.66, despite successful engraftment of IgM. Mice deficient in both T and B cells (RAG1-/- mice (p<0.001, but not B cell deficient (μMT mice (p = 0.93, were significantly protected from injury. Further interrogation with T cell deficient (CD3εKO mice confirmed that the T cell component is a key mediator of sterile liver injury. Mice deficient in B cells and IgM mice did not have a significant delay in resolution following acute liver injury.IgM deposition appears to be common feature of both human and murine sterile liver injury. However, neither IgM nor B cells, play a significant role in the development of or resolution from acute liver injury. T cells appear to be key

  17. Human amnion epithelial cells expressing HLA-G as novel cell-based treatment for liver disease.

    Science.gov (United States)

    Strom, Stephen C; Gramignoli, Roberto

    2016-09-01

    Despite routine liver transplantation and supporting medical therapies, thousands of patients currently wait for an organ and there is an unmet need for more refined and widely available regenerative strategies to treat liver diseases. Cell transplants attempt to maximize the potential for repair and/or regeneration in liver and other organs. Over 40years of laboratory pre-clinical research and 25years of clinical procedures have shown that certain liver diseases can be treated by the infusion of isolated cells (hepatocyte transplant). However, like organ transplants, hepatocyte transplant suffers from a paucity of tissues useful for cell production. Alternative sources have been investigated, yet with limited success. The tumorigenic potential of pluripotent stem cells together with their primitive level of hepatic differentiation, have limited the use of stem cell populations. Stem cell sources from human placenta, and the amnion tissue in particular are receiving renewed interest in the field of regenerative medicine. Unlike pluripotent stem cells, human amnion epithelial (AE) cells are easily available without ethical or religious concerns; they do not express telomerase and are not immortal or tumorigenic when transplanted. In addition, AE cells have been reported to express genes normally expressed in mature liver, when transplanted into the liver. Moreover, because of the possibility of an immune-privileged status related to their expression of HLA-G, it might be possible to transplant human AE cells without immunosuppression of the recipient. Copyright © 2016. Published by Elsevier Inc.

  18. Copper uptake and retention in liver parenchymal cells isolated from nutritionally copper-deficient rats

    NARCIS (Netherlands)

    Berg, van den G.J.; de Goeij, J.J.M.; Bock, I.; Gijbels, M.J.J.; Brouwer, A.; Lei, K.Y.; Hendriks, H.F.J.

    1991-01-01

    Copper uptake and retention were studied in primary cultures of liver parenchymal cells isolated from copper-deficient rats. Male Sprague-Dawley rats were fed a copper-deficient diet (<1 mg Cu/kg) for 10 wk. Copper-deficient rats were characterized by low copper concentrations in plasma and liver,

  19. Copper uptake and retention in liver parenchymal cells isolated from nutritionally copper-deficient rats

    NARCIS (Netherlands)

    Berg, G.J. van den; Goeij, J.J.M. de; Bock, I.; Gijbels, M.J.J.; Brouwer, A.; Lei, K.Y.; Hendruiks, H.F.J.

    1991-01-01

    Copper uptake and retention were studied in primary cultures of liver parenchymal cells isolated from copper-deficient rats. Male Sprague-Dawley rats were fed a copper-deficient diet (< 1 mg Cu/kg) for 10 wk. Copper-deficient rats were characterized by low copper concentrations in plasma and liver,

  20. Carcinoma-associated perisinusoidal laminin may signal tumour cell metastasis to the liver

    DEFF Research Database (Denmark)

    Wewer, U M; Albrechtsen, R

    1992-01-01

    using chain-specific monoclonal antibodies against B2 laminin. In an ex vivo assay, viable tumour cells (Panc-1 and clone A) were found to bind with remarkable specificity to frozen sections of liver tissue containing perisinusoidal laminin as opposed to liver tissues without laminin. We suggest...

  1. Gastrointestinal toxicity, systemic inflammation, and liver biochemistry in allogeneic hematopoietic stem cell transplantation

    DEFF Research Database (Denmark)

    Jordan, Karina; Pontoppidan, Peter; Uhlving, Hilde Hylland

    2017-01-01

    Liver toxicity is frequently seen in relation to allogeneic hematopoietic stem cell transplantation (HSCT), but pathogenesis and the risk factors are poorly understood. The purpose of this study was to investigate associations between liver toxicity, gastrointestinal toxicity, and levels of immun...

  2. Gastrointestinal toxicity, systemic inflammation, and liver biochemistry in allogeneic hematopoietic stem cell transplantation

    Science.gov (United States)

    Liver toxicity is frequently seen in relation to allogeneic hematopoietic stem cell transplantation (HSCT), but pathogenesis and the risk factors are poorly understood. The purpose of this study was to investigate associations between liver toxicity, gastrointestinal toxicity, and levels of immune-r...

  3. Cell-swelling-induced taurine release from isolated perfused rat liver

    NARCIS (Netherlands)

    Brand, H. S.; Meijer, A. J.; Gustafson, L. A.; Jörning, G. G.; Leegwater, A. C.; Maas, M. A.; Chamuleau, R. A.

    1994-01-01

    Astrocytes and lymphocytes are able to release significant amounts of taurine during periods of hypotonicity to reduce the increase in cell volume. To investigate this mechanism in the liver, we studied the release of free amino acids from isolated perfused rat liver during hypotonicity. The

  4. Mangosteen peel extract reduces formalin-induced liver cell death in rats

    Directory of Open Access Journals (Sweden)

    Afiana Rohmani

    2014-08-01

    Full Text Available Background Formalin is a xenobiotic that is now commonly used as a preservative in the food industry. The liver is an organ that has the highest metabolic capacity as compared to other organs. Mangosteen or Garcinia mangostana Linn (GML peel contains xanthones, which are a source of natural antioxidants. The purpose of this study was to evaluate the effect of mangosteen peel extract on formalin-induced liver cell mortality rate and p53 protein expression in Wistar rats. Methods Eighteen rats received formalin orally for 2 weeks, and were subsequently divided into 3 groups, consisting of the formalin-control group receiving a placebo and treatment groups 1 and 2, which were treated with mangosteen peel extract at doses of 200 and 400 mg/kgBW/day, respectively. The treatment was carried out for 1 week, and finally the rats were terminated. The differences in liver cell mortality rate and p53 protein expression were analyzed. Results One-way ANOVA analysis showed significant differences in liver cell mortality rate among the three groups (p=0.004. The liver cell mortality rate in the treatment group receiving 400 mg/kgBW/day extract was lower than that in the formalin-control group. There was no p53 expression in all groups. Conclusions Garcinia mangostana Linn peel extract reduced the mortality rate of liver cells in rats receiving oral formalin. Involvement of p53 expression in liver cell mortality in rats exposed to oral formalin is presumably negligible.

  5. Liver restores immune homeostasis after local inflammation despite the presence of autoreactive T cells.

    Science.gov (United States)

    Béland, Kathie; Lapierre, Pascal; Djilali-Saiah, Idriss; Alvarez, Fernando

    2012-01-01

    The liver must keep equilibrium between immune tolerance and immunity in order to protect itself from pathogens while maintaining tolerance to food antigens. An imbalance between these two states could result in an inflammatory liver disease. The aims of this study were to identify factors responsible for a break of tolerance and characterize the subsequent restoration of liver immune homeostasis. A pro-inflammatory environment was created in the liver by the co-administration of TLR ligands CpG and Poly(I:C) in presence or absence of activated liver-specific autoreactive CD8(+) T cells. Regardless of autoreactive CD8(+) T cells, mice injected with CpG and Poly(I:C) showed elevated serum ALT levels and a transient liver inflammation. Both CpG/Poly(I:C) and autoreactive CD8(+)T cells induced expression of TLR9 and INF-γ by the liver, and an up-regulation of homing and adhesion molecules CXCL9, CXCL10, CXCL16, ICAM-1 and VCAM-1. Transferred CFSE-labeled autoreactive CD8(+) T cells, in presence of TLR3 and 9 ligands, were recruited by the liver and spleen and proliferated. This population then contracted by apoptosis through intrinsic and extrinsic pathways. Up-regulation of FasL and PD-L1 in the liver was observed. In conclusion, TLR-mediated activation of the innate immune system results in a pro-inflammatory environment that promotes the recruitment of lymphocytes resulting in bystander hepatitis. Despite this pro-inflammatory environment, the presence of autoreactive CD8(+) T cells is not sufficient to sustain an autoimmune response against the liver and immune homeostasis is rapidly restored through the apoptosis of T cells.

  6. Liver restores immune homeostasis after local inflammation despite the presence of autoreactive T cells.

    Directory of Open Access Journals (Sweden)

    Kathie Béland

    Full Text Available The liver must keep equilibrium between immune tolerance and immunity in order to protect itself from pathogens while maintaining tolerance to food antigens. An imbalance between these two states could result in an inflammatory liver disease. The aims of this study were to identify factors responsible for a break of tolerance and characterize the subsequent restoration of liver immune homeostasis. A pro-inflammatory environment was created in the liver by the co-administration of TLR ligands CpG and Poly(I:C in presence or absence of activated liver-specific autoreactive CD8(+ T cells. Regardless of autoreactive CD8(+ T cells, mice injected with CpG and Poly(I:C showed elevated serum ALT levels and a transient liver inflammation. Both CpG/Poly(I:C and autoreactive CD8(+T cells induced expression of TLR9 and INF-γ by the liver, and an up-regulation of homing and adhesion molecules CXCL9, CXCL10, CXCL16, ICAM-1 and VCAM-1. Transferred CFSE-labeled autoreactive CD8(+ T cells, in presence of TLR3 and 9 ligands, were recruited by the liver and spleen and proliferated. This population then contracted by apoptosis through intrinsic and extrinsic pathways. Up-regulation of FasL and PD-L1 in the liver was observed. In conclusion, TLR-mediated activation of the innate immune system results in a pro-inflammatory environment that promotes the recruitment of lymphocytes resulting in bystander hepatitis. Despite this pro-inflammatory environment, the presence of autoreactive CD8(+ T cells is not sufficient to sustain an autoimmune response against the liver and immune homeostasis is rapidly restored through the apoptosis of T cells.

  7. Clinical potential of regulatory T cell therapy in liver diseases: An overview and current perspectives

    Directory of Open Access Journals (Sweden)

    Hannah Claire Jeffery

    2016-09-01

    Full Text Available The increasing demand for liver transplantation and the decline in donor organs has highlighted the need for alternative novel therapies to prevent chronic active hepatitis, which eventually leads to liver cirrhosis and liver cancer. Liver histology of chronic hepatitis is composed of both effector and regulatory lymphocytes. The human liver contains different subsets of effector lymphocytes, that are kept in check by a subpopulation of T cells known as Regulatory T cells (Treg. The balance of effector and regulatory lymphocytes generally determines the outcome of hepatic inflammation: resolution, fulminant hepatitis or chronic active hepatitis. Thus, maintaining and adjusting this balance is crucial in immunological manipulation of liver diseases. One of the options to restore this balance is to enrich Treg in the liver disease patients.Advances in the knowledge of Treg biology and development of clinical grade isolation reagents, cell sorting equipment and Good Manufacturing Practice (GMP facilities have paved the way to apply Treg cells as a potential therapy to restore peripheral self-tolerance in autoimmune liver diseases, chronic rejection and post-transplantation. Past and on-going studies have applied Treg in type-1 diabetes mellitus, systemic lupus erythematosus, graft versus host diseases (GVHD and solid organ transplantations. There have not been any new therapies for the autoimmune liver diseases for more than three decades; thus the clinical potential for the application of autologous Treg cell therapy to treat autoimmune liver disease is an attractive and novel option. However, it is fundamental to understand the deep immunology, genetic profiles, biology, homing behavior and microenvironment of Treg before applying the cells to the patients.

  8. Addressing liver fibrosis with Liposomes targeted to hepatic stellate cells

    NARCIS (Netherlands)

    Adrian, Joanna E.; Poelstra, Klaas; Kamps, Jan A. A. M.

    2007-01-01

    Liver fibrosis is a chronic disease that results from hepatitis B and C infections, alcohol abuse or metabolic and genetic disorders. Ultimately, progression of fibrosis leads to cirrhosis, a stage of the disease characterized by failure of the normal liver functions. Currently, the treatment of

  9. Continuous cell injury promotes hepatic tumorigenesis in cdc42-deficient mouse liver

    DEFF Research Database (Denmark)

    van Hengel, Jolanda; D'Hooge, Petra; Hooghe, Bart

    2008-01-01

    be required for liver function. METHODS: Mice in which Cdc42 was ablated in hepatocytes and bile duct cells were generated by Cre-loxP technology. Livers were examined by histologic, immunohistochemical, ultrastructural, and serum analysis to define the effect of loss of Cdc42 on liver structure. RESULTS...... of 2 months, the canaliculi between hepatocytes were greatly enlarged, although the tight junctions flanking the canaliculi appeared normal. Regular liver plates were absent. E-cadherin expression pattern and gap junction localization were distorted. Analysis of serum samples indicated cholestasis...

  10. Intravascular Immune Surveillance by CXCR6+ NKT Cells Patrolling Liver Sinusoids

    Directory of Open Access Journals (Sweden)

    Geissmann Frederic

    2005-01-01

    Full Text Available We examined the in vivo behavior of liver natural killer T cells (NKT cells by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6+ cells in liver, were found to crawl within hepatic sinusoids at 10-20 µm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance.

  11. Surgery-induced reactive oxygen species enhance colon carcinoma cell binding by disrupting the liver endothelial cell lining

    NARCIS (Netherlands)

    Gül, Nuray; Bögels, Marijn; Grewal, Simran; van der Meer, Anne Jan; Rojas, Lucy Baldeon; Fluitsma, Donna M.; van den Tol, M. Petrousjka; Hoeben, Kees A.; van Marle, Jan; de Vries, Helga E.; Beelen, Robert H. J.; van Egmond, Marjolein

    2011-01-01

    Resection of primary colorectal cancer is associated with enhanced risk of development of liver metastases. It was previously demonstrated that surgery initiated an early inflammatory response resulting in elevated tumour cell adhesion in the liver. Because reactive oxygen species (ROS) are shown to

  12. Surgery-induced reactive oxygen species enhance colon carcinoma cell binding by disrupting the liver endothelial cell lining

    NARCIS (Netherlands)

    Gül, N.; Bögels, M.; Grewal, S.; van der Meer, A.J.; Rojas, L.B.; Fluitsma, D.M.; van den Tol, M.P.; Hoeben, K.A.; van Marle, J.; de Vries, H.E.; Beelen, R.H.J.; van Egmond, M.

    2011-01-01

    Objective: Resection of primary colorectal cancer is associated with enhanced risk of development of liver metastases. It was previously demonstrated that surgery initiated an early inflammatory response resulting in elevated tumour cell adhesion in the liver. Because reactive oxygen species (ROS)

  13. Surgery-induced reactive oxygen species enhance colon carcinoma cell binding by disrupting the liver endothelial cell lining

    NARCIS (Netherlands)

    Gul, N.; Bogels, M.; Grewal, S.; van der Meer, A.J.; Rojas, L.B.; Fluitsma, D.M.; van den Tol, M.P.; Hoeben, K.A.; van Marle, J.; de Vries, H.E.; Beelen, R.H.J.; van Egmond, M.

    2011-01-01

    Objective Resection of primary colorectal cancer is associated with enhanced risk of development of liver metastases. It was previously demonstrated that surgery initiated an early inflammatory response resulting in elevated tumour cell adhesion in the liver. Because reactive oxygen species (ROS)

  14. T cells but not NK cells are associated with a favourable outcome for resected colorectal liver metastases

    International Nuclear Information System (INIS)

    Pugh, Siân A; Harrison, Rebecca J; Primrose, John N; Khakoo, Salim I

    2014-01-01

    The adaptive immune response to colorectal cancer is important for survival. Less is understood about the role of innate lymphocytes, such as Natural Killer (NK) cells, which are abundant in human liver. Samples of fresh liver (n = 21) and tumour (n = 11) tissue were obtained from patients undergoing surgical resection of colorectal liver metastases. Flow cytometry was used to analyse the presence and phenotype of NK cells, as compared to T cells, in the tumour and liver tissue. Results were correlated with survival. NK cells were poorly recruited to the tumours (distant liver tissue 38.3%, peritumoural liver 34.2%, tumour 12.9%, p = 0.0068). Intrahepatic and intratumoural NK cells were KIR (killer immunoglobulin-like receptor) lo NKG2A hi whereas circulating NK cells were KIR hi NKG2A lo . By contrast T cells represented 65.7% of the tumour infiltrating lymphocytes. Overall survival was 43% at 5 years, with the 5-year survival for individuals with a T cell rich infiltrate being 60% (95% CI 17-93%) and for those with a low T cell infiltrate being 0% (95% CI 0-48%). Conversely individuals with higher levels of NK cells in the tumour had an inferior outcome, although there were insufficient numbers to reach significance (median survivals: NK Hi 1.63 years vs NK Lo 3.92 years). T cells, but not NK cells, are preferentially recruited to colorectal liver metastases. NK cells within colorectal metastases have an intrahepatic and potentially tolerogenic, rather than a peripheral, phenotype. Similar to primary tumours, the magnitude of the T cell infiltrate in colorectal metastases is positively associated with survival

  15. Fasting enhances TRAIL-mediated liver natural killer cell activity via HSP70 upregulation.

    Directory of Open Access Journals (Sweden)

    Vu T A Dang

    Full Text Available Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL+ and CD69+ natural killer cells were significantly elevated (n = 7, p <0.01, as determined by flow cytometric analysis. Furthermore, we found that TRAIL- natural killer cells that were adoptively transferred into Rag-2-/- γ chain-/- mice could convert into TRAIL+ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05 in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05. In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05. These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.

  16. Impact of NKT Cells and LFA-1 on Liver Regeneration under Subseptic Conditions.

    Directory of Open Access Journals (Sweden)

    Ann-Kathrin Jörger

    Full Text Available Activation of the immune system in terms of subseptic conditions during liver regeneration is of paramount clinical importance. However, little is known about molecular mechanisms and their mediators that control hepatocyte proliferation. We sought to determine the functional role of immune cells, especially NKT cells, in response to partial hepatectomy (PH, and to uncover the impact of the integrin lymphocyte function-associated antigen-1 (LFA-1 on liver regeneration in a subseptic setting.Wild-type (WT and LFA-1-/- mice underwent a 2/3 PH and low-dose lipopolysaccharid (LPS application. Hepatocyte proliferation, immune cell infiltration, and cytokine profile in the liver parenchyma were determined.Low-dose LPS application after PH results in a significant delay of liver regeneration between 48h and 72h, which is associated with a reduced number of CD3+ cells within the regenerating liver. In absence of LFA-1, an impaired regenerative capacity was observed under low-dose LPS application. Analysis of different leukocyte subpopulations showed less CD3+NK1.1+ NKT cells in the liver parenchyma of LFA-1-/- mice after PH and LPS application compared to WT controls, while CD3-NK1.1+ NK cells markedly increased. Concordantly with this observation, lower levels of NKT cell related cytokines IL-12 and IL-23 were expressed in the regenerating liver of LFA-1-/- mice, while the expression of NK cell-associated CCL5 and IL-10 was increased compared to WT mice.A subseptic situation negatively alters hepatocyte proliferation. Within this scenario, we suggest an important impact of NKT cells and postulate a critical function for LFA-1 during processes of liver regeneration.

  17. Human Mesenchymal Stem Cell Transfusion Is Safe and Improves Liver Function in Acute-on-Chronic Liver Failure Patients

    OpenAIRE

    Shi, Ming; Zhang, Zheng; Xu, Ruonan; Lin, Hu; Fu, Junliang; Zou, Zhengsheng; Zhang, Aimin; Shi, Jianfei; Chen, Liming; Lv, Sa; He, Weiping; Geng, Hua; Jin, Lei; Liu, Zhenwen; Wang, Fu-Sheng

    2012-01-01

    This study assessed the safety and initial efficacy of umbilical cord-derived mesenchymal stem cell (UC-MSC) transfusions for acute-on-chronic liver failure (ACLF) patients associated with hepatitis B virus (HBV) infection. No significant side effects were observed, and the UC-MSC transfusions significantly increased the survival rates in ACLF patients. It was found that UC-MSC transfusions are safe in the clinic and may serve as a novel therapeutic approach for HBV-associated ACLF patients.

  18. Magnetic targeting of iron-oxide-labeled fluorescent hepatoma cells to the liver

    Energy Technology Data Exchange (ETDEWEB)

    Luciani, Alain [Universite Rene Descartes, Hopital Europeen Georges Pompidou, Laboratoire de Recherche en Imagerie, EA 4062, Paris (France); Imagerie Medicale, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France); Wilhelm, Claire; Gazeau, Florence [Universite Paris Diderot, Batiment Condorcet, Laboratoire Matiere et Systemes Complexes, CNRS-UMR 7057, Paris Cedex (France); Bruneval, Patrick [Anatomopathologie, Hopital Europeen Georges Pompidou, Paris (France); Cunin, Patrick [Unite de Recherche Clinique, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France); Autret, Gwennhael; Clement, Olivier [Universite Rene Descartes, Hopital Europeen Georges Pompidou, Laboratoire de Recherche en Imagerie, EA 4062, Paris (France); Rahmouni, Alain [Imagerie Medicale, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France)

    2009-05-15

    The purpose of this study was to determine whether an external magnet field can induce preferential trafficking of magnetically labeled Huh7 hepatoma cells to the liver following liver cell transplantation. Huh7 hepatoma cells were labeled with anionic magnetic nanoparticles (AMNP) and tagged with a fluorescent membrane marker (PKH67). Iron-uptake was measured by magnetophoresis. Twenty C57Bl6 mice received an intrasplenic injection of 2 x 10{sup 6} labeled cells. An external magnet (0.29 T; 25 T/m) was placed over the liver of 13 randomly selected animals (magnet group), while the remaining 7 animals served as controls. MRI (1.5 T) and confocal fluorescence microscopy (CFM) were performed 10 days post-transplantation. The presence and location of labeled cells within the livers were compared in the magnet group and controls, and confronted with histological analysis representing the standard of reference. Mean iron content per cell was 6 pg. Based on histology, labeled cells were more frequently present within recipient livers in the magnet group (p < 0.01) where their distribution was preferentially peri-vascular (p<0.05). MRI and CFM gave similar results for the overall detection of transplanted cells (kappa=0.828) and for the identification of peri-vascular cells (kappa=0.78). Application of an external magnet can modify the trafficking of transplanted cells, especially by promoting the formation of perivascular aggregates. (orig.)

  19. Magnetic targeting of iron-oxide-labeled fluorescent hepatoma cells to the liver

    International Nuclear Information System (INIS)

    Luciani, Alain; Wilhelm, Claire; Gazeau, Florence; Bruneval, Patrick; Cunin, Patrick; Autret, Gwennhael; Clement, Olivier; Rahmouni, Alain

    2009-01-01

    The purpose of this study was to determine whether an external magnet field can induce preferential trafficking of magnetically labeled Huh7 hepatoma cells to the liver following liver cell transplantation. Huh7 hepatoma cells were labeled with anionic magnetic nanoparticles (AMNP) and tagged with a fluorescent membrane marker (PKH67). Iron-uptake was measured by magnetophoresis. Twenty C57Bl6 mice received an intrasplenic injection of 2 x 10 6 labeled cells. An external magnet (0.29 T; 25 T/m) was placed over the liver of 13 randomly selected animals (magnet group), while the remaining 7 animals served as controls. MRI (1.5 T) and confocal fluorescence microscopy (CFM) were performed 10 days post-transplantation. The presence and location of labeled cells within the livers were compared in the magnet group and controls, and confronted with histological analysis representing the standard of reference. Mean iron content per cell was 6 pg. Based on histology, labeled cells were more frequently present within recipient livers in the magnet group (p < 0.01) where their distribution was preferentially peri-vascular (p<0.05). MRI and CFM gave similar results for the overall detection of transplanted cells (kappa=0.828) and for the identification of peri-vascular cells (kappa=0.78). Application of an external magnet can modify the trafficking of transplanted cells, especially by promoting the formation of perivascular aggregates. (orig.)

  20. Behavior of HepG2 liver cancer cells using microfluidic-microscopy: a preliminary study

    Science.gov (United States)

    Karamahmutoglu, Hande; ćetin, Metin; Yaǧcı, Tamer; Elitaş, Meltem

    2018-02-01

    Hepatocellular carcinoma is one of the most common types of liver cancer causing death all over the world. Although early-stage liver cancer can sometimes be treated with partial hepatectomy, liver transplantation, ablation, and embolization, sorafenib treatment is the only approved systemic therapy for advanced HCC. The aim of this research is to develop tools and methods to understand the individuality of hepatocellular carcinoma. Microfluidic cell-culture platform has been developed to observe behavior of single-cells; fluorescence microscopy has been implemented to investigate phenotypic changes of cells. Our preliminary data proved high-level heterogeneity of hepatocellular carcinoma while verifying limited growth of liver cancer cell lines on the silicon wafer.

  1. Use of heterologous expressed polyketide synthase and small molecule foldases to make aromatic and cyclic compounds

    DEFF Research Database (Denmark)

    2016-01-01

    A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell.......A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell....

  2. Effect of New Water-Soluble Dendritic Phthalocyanines on Human Colorectal and Liver Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Ebru YABAŞ

    2017-08-01

    Full Text Available Human hepatocellular carcinoma (HepG2 cells and colorectal adenocarcinoma (DLD-1 cells were treated with the synthesized water soluble phthalocyanine derivatives to understand the effect of the compounds both on colorectal and liver cancer cells. The compounds inhibited cell proliferation and displayed cytotoxic effect on these cancer cell lines however; the effect of the compounds on healthy control fibroblast cell line was comparatively lower. The compounds can be employed for cancer treatment as anticancer agents.

  3. Treatment with 4-methylpyrazole modulated stellate cells and natural killer cells and ameliorated liver fibrosis in mice.

    Directory of Open Access Journals (Sweden)

    Hyon-Seung Yi

    Full Text Available Accumulating evidence suggests that retinol and its metabolites are closely associated with liver fibrogenesis. Recently, we demonstrated that genetic ablation of alcohol dehydrogenase 3 (ADH3, a retinol metabolizing gene that is expressed in hepatic stellate cells (HSCs and natural killer (NK cells, attenuated liver fibrosis in mice. In the current study, we investigated whether pharmacological ablation of ADH3 has therapeutic effects on experimentally induced liver fibrosis in mice.Liver fibrosis was induced by intraperitoneal injections of carbon tetrachloride (CCl4 or bile duct ligation (BDL for two weeks. To inhibit ADH3-mediated retinol metabolism, 10 μg 4-methylpyrazole (4-MP/g of body weight was administered to mice treated with CCl4 or subjected to BDL. The mice were sacrificed at week 2 to evaluate the regression of liver fibrosis. Liver sections were stained for collagen and α-smooth muscle actin (α-SMA. In addition, HSCs and NK cells were isolated from control and treated mice livers for molecular and immunological studies.Treatment with 4-MP attenuated CCl4- and BDL-induced liver fibrosis in mice, without any adverse effects. HSCs from 4-MP treated mice depicted decreased levels of retinoic acids and increased retinol content than HSCs from control mice. In addition, the expression of α-SMA, transforming growth factor-β1 (TGF-β1, and type I collagen α1 was significantly reduced in the HSCs of 4-MP treated mice compared to the HSCs from control mice. Furthermore, inhibition of retinol metabolism by 4-MP increased interferon-γ production in NK cells, resulting in increased apoptosis of activated HSCs.Based on our data, we conclude that inhibition of retinol metabolism by 4-MP ameliorates liver fibrosis in mice through activation of NK cells and suppression of HSCs. Therefore, retinol and its metabolizing enzyme, ADH3, might be potential targets for therapeutic intervention of liver fibrosis.

  4. Liver-primed memory T cells generated under noninflammatory conditions provide anti-infectious immunity.

    Science.gov (United States)

    Böttcher, Jan P; Schanz, Oliver; Wohlleber, Dirk; Abdullah, Zeinab; Debey-Pascher, Svenja; Staratschek-Jox, Andrea; Höchst, Bastian; Hegenbarth, Silke; Grell, Jessica; Limmer, Andreas; Atreya, Imke; Neurath, Markus F; Busch, Dirk H; Schmitt, Edgar; van Endert, Peter; Kolanus, Waldemar; Kurts, Christian; Schultze, Joachim L; Diehl, Linda; Knolle, Percy A

    2013-03-28

    Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

    Directory of Open Access Journals (Sweden)

    Jan P. Böttcher

    2013-03-01

    Full Text Available Development of CD8+ T cell (CTL immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1+ memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire.

  6. Human Adipose Tissue Derived Stem Cells Promote Liver Regeneration in a Rat Model of Toxic Injury

    Directory of Open Access Journals (Sweden)

    Eva Koellensperger

    2013-01-01

    Full Text Available In the light of the persisting lack of donor organs and the risks of allotransplantations, the possibility of liver regeneration with autologous stem cells from adipose tissue (ADSC is an intriguing alternative. Using a model of a toxic liver damage in Sprague Dawley rats, generated by repetitive intraperitoneal application of retrorsine and allyl alcohol, the ability of human ADSC to support the restoration of liver function was investigated. A two-thirds hepatectomy was performed, and human ADSC were injected into one remaining liver lobe in group 1 (n = 20. Injection of cell culture medium performed in group 2 (n = 20 served as control. Cyclosporine was applied to achieve immunotolerance. Blood samples were drawn weekly after surgery to determine liver-correlated blood values. Six and twelve weeks after surgery, animals were sacrificed and histological sections were analyzed. ADSC significantly raised postoperative albumin (P < 0.017, total protein (P < 0.031, glutamic oxaloacetic transaminase (P < 0.001, and lactate dehydrogenase (P < 0.04 levels compared to injection of cell culture medium alone. Transplanted cells could be found up to twelve weeks after surgery in histological sections. This study points towards ADSC being a promising alternative to hepatocyte or liver organ transplantation in patients with severe liver failure.

  7. Gene expression profiling of liver cancer stem cells by RNA-sequencing.

    Directory of Open Access Journals (Sweden)

    David W Y Ho

    Full Text Available BACKGROUND: Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90(+ liver cancer stem cells (CSCs in hepatocellular carcinoma (HCC. Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq to compare the gene expression profiling of CD90(+ cells sorted from tumor (CD90(+CSCs with parallel non-tumorous liver tissues (CD90(+NTSCs and elucidate the roles of putative target genes in hepatocarcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: CD90(+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90(+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90(+CSCs and CD90(+NTSCs, and validated by quantitative real-time PCR (qRT-PCR on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes between CD90(+CSCs and CD90(+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90(+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3, a member of glypican family, was markedly elevated in CD90(+CSCs compared to CD90(+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90(+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90(+CSCs in liver tumor tissues. CONCLUSIONS

  8. Gene Expression Profiling of Liver Cancer Stem Cells by RNA-Sequencing

    Science.gov (United States)

    Lam, Chi Tat; Ng, Michael N. P.; Yu, Wan Ching; Lau, Joyce; Wan, Timothy; Wang, Xiaoqi; Yan, Zhixiang; Liu, Hang; Fan, Sheung Tat

    2012-01-01

    Background Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90+ liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90+ cells sorted from tumor (CD90+CSCs) with parallel non-tumorous liver tissues (CD90+NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis. Methodology/Principal Findings CD90+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90+CSCs and CD90+NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90+CSCs and CD90+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90+CSCs compared to CD90+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90+CSCs in liver tumor tissues. Conclusions/Significance The identified genes

  9. Two sides of one coin: massive hepatic necrosis and progenitor cell-mediated regeneration in acute liver failure

    Directory of Open Access Journals (Sweden)

    Honglei eWeng

    2015-06-01

    Full Text Available Massive hepatic necrosis is a key event underlying acute liver failure, a serious clinical syndrome with high mortality. Massive hepatic necrosis in acute liver failure has unique pathophysiological characteristics including extremely rapid parenchymal cell death and removal. On the other hand, massive necrosis rapidly induces the activation of liver progenitor cells, the so-called second pathway of liver regeneration. The final clinical outcome of acute liver failure depends on whether liver progenitor cell-mediated regeneration can efficiently restore parenchymal mass and function within a short time. This review summarizes the current knowledge regarding massive hepatic necrosis and liver progenitor cell-mediated regeneration in patients with acute liver failure, the two sides of one coin.

  10. Two sides of one coin: massive hepatic necrosis and progenitor cell-mediated regeneration in acute liver failure.

    Science.gov (United States)

    Weng, Hong-Lei; Cai, Xiaobo; Yuan, Xiaodong; Liebe, Roman; Dooley, Steven; Li, Hai; Wang, Tai-Ling

    2015-01-01

    Massive hepatic necrosis is a key event underlying acute liver failure, a serious clinical syndrome with high mortality. Massive hepatic necrosis in acute liver failure has unique pathophysiological characteristics including extremely rapid parenchymal cell death and removal. On the other hand, massive necrosis rapidly induces the activation of liver progenitor cells, the so-called "second pathway of liver regeneration." The final clinical outcome of acute liver failure depends on whether liver progenitor cell-mediated regeneration can efficiently restore parenchymal mass and function within a short time. This review summarizes the current knowledge regarding massive hepatic necrosis and liver progenitor cell-mediated regeneration in patients with acute liver failure, the two sides of one coin.

  11. Three-dimentional growth of liver / stem cells in vitro under simulated microgravity

    Science.gov (United States)

    Feng, Mei Fu

    Liver is a important and largest parenchymatous organ in vivo, and have complex and diverse structures and functions. In the world, there are many peoples suffers from liver injury and dis-ease, especially in Asia, but serious shortage of donor organ, especially for organic pathological changes, is a big problem in the world. Stem cells have the capabilities to self-renew and differ-entiate into multiple lineages, and are very significant in both theoretical research and clinical applications. Compared with traditional cell culture, cells of 3D growth are more close to their situation in vivo. The specific physics environment in space provides a great opportunity for 3D growth of cells and tissues. Due to the chance for entering into the space is so scarce, to mimic microgravity effects using a rotating cell culture system (RCCS) designed by NASA, and some other methods were studied for cellular 3D growth in vitro. Neonatal mouse liver Cells, hepatic progenitor/stem cells from fetal liver and WB-F344 cells were cultured in a 1:1 mixture of DMEM and F-12 supplemented with 10 % FCS and several factors, and seeded into the RCCS, 6-well and 24-well plates. Their growth characteristic, metabolism, differentiation and gene expression were studied by SEM, Histochemistry, Flow Cytometry, RT-PCR and so on. The results showed: 1. Neonatal mouse liver Cells (1day after birth) seem easy to grow for a three-dimentional-like structure, when the cells were cultured in the RCCS, a cell aggregate formed after 1 day of culture and were kept during 10 days culture. The size of aggregate was about 1 2 mm in diameter. 2. Hepatic progenitor/stem cells from fetal liver seem a good cell resource for liver disease'cell therapy. They expressed AFP and CKs, and no mature hepato-cytes marker and bile duct epithelial cells marker were detected. When were transplanted into Nod-Scid mice, they had multi-potential differentiation. 3. WB-F344 cells, a liver epithelial cell line, could grew well on

  12. Establishment and characterization of a unique 1 μm diameter liver-derived progenitor cell line

    International Nuclear Information System (INIS)

    Aravalli, Rajagopal N.; Behnan Sahin, M.; Cressman, Erik N.K.; Steer, Clifford J.

    2010-01-01

    Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 μm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

  13. Establishment and characterization of a unique 1 {mu}m diameter liver-derived progenitor cell line

    Energy Technology Data Exchange (ETDEWEB)

    Aravalli, Rajagopal N., E-mail: arava001@umn.edu [Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Behnan Sahin, M. [Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Cressman, Erik N.K. [Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Steer, Clifford J., E-mail: steer001@umn.edu [Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN 55455 (United States)

    2010-01-01

    Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 {mu}m in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

  14. Remarkable heterogeneity displayed by oval cells in rat and mouse models of stem cell-mediated liver regeneration

    DEFF Research Database (Denmark)

    Jelnes, Peter; Santoni-Rugiu, Eric; Rasmussen, Morten

    2007-01-01

    The experimental protocols used in the investigation of stem cell-mediated liver regeneration in rodents are characterized by activation of the hepatic stem cell compartment in the canals of Hering followed by transit amplification of oval cells and their subsequent differentiation along hepatic...... the molecular phenotypes of oval cells in several of the most commonly used protocols of stem cell-mediated liver regeneration-namely, treatment with 2-acetylaminofluorene and partial (70%) hepatectomy (AAF/PHx); a choline-deficient, ethionine-supplemented (CDE) diet; a 3,5-diethoxycarbonyl-1,4-dihydro...... remarkable phenotypic discrepancies exhibited by oval cells in stem cell-mediated liver regeneration between rats and mice and underline the importance of careful extrapolation between individual species....

  15. Procalcitonin Impairs Liver Cell Viability and Function In Vitro: A Potential New Mechanism of Liver Dysfunction and Failure during Sepsis?

    Directory of Open Access Journals (Sweden)

    Martin Sauer

    2017-01-01

    Full Text Available Purpose. Liver dysfunction and failure are severe complications of sepsis and result in poor outcome and increased mortality. The underlying pathologic mechanisms of hepatocyte dysfunction and necrosis during sepsis are only incompletely understood. Here, we investigated whether procalcitonin, a biomarker of sepsis, modulates liver cell function and viability. Materials and Methods. Employing a previously characterized and patented biosensor system evaluating hepatocyte toxicity in vitro, human hepatocellular carcinoma cells (HepG2/C3A were exposed to 0.01–50 ng/mL procalcitonin for 2×72 h and evaluated for proliferation, necrosis, metabolic activity, cellular integrity, microalbumin synthesis, and detoxification capacity. Acetaminophen served as positive control. For further standardization, procalcitonin effects were confirmed in a cellular toxicology assay panel employing L929 fibroblasts. Data were analyzed using ANOVA/Tukey’s test. Results. Already at concentrations as low as 0.25 ng/mL, procalcitonin induced HepG2/C3A necrosis (P<0.05 and reduced metabolic activity, cellular integrity, synthesis, and detoxification capacity (all P<0.001. Comparable effects were obtained employing L929 fibroblasts. Conclusion. We provide evidence for procalcitonin to directly impair function and viability of human hepatocytes and exert general cytotoxicity in vitro. Therapeutical targeting of procalcitonin could thus display a novel approach to reduce incidence of liver dysfunction and failure during sepsis and lower morbidity and mortality of septic patients.

  16. Replicative stress and alterations in cell cycle checkpoint controls following acetaminophen hepatotoxicity restrict liver regeneration.

    Science.gov (United States)

    Viswanathan, Preeti; Sharma, Yogeshwar; Gupta, Priya; Gupta, Sanjeev

    2018-03-05

    Acetaminophen hepatotoxicity is a leading cause of hepatic failure with impairments in liver regeneration producing significant mortality. Multiple intracellular events, including oxidative stress, mitochondrial damage, inflammation, etc., signify acetaminophen toxicity, although how these may alter cell cycle controls has been unknown and was studied for its significance in liver regeneration. Assays were performed in HuH-7 human hepatocellular carcinoma cells, primary human hepatocytes and tissue samples from people with acetaminophen-induced acute liver failure. Cellular oxidative stress, DNA damage and cell proliferation events were investigated by mitochondrial membrane potential assays, flow cytometry, fluorescence staining, comet assays and spotted arrays for protein expression after acetaminophen exposures. In experimental groups with acetaminophen toxicity, impaired mitochondrial viability and substantial DNA damage were observed with rapid loss of cells in S and G2/M and cell cycle restrictions or even exit in the remainder. This resulted from altered expression of the DNA damage regulator, ATM and downstream transducers, which imposed G1/S checkpoint arrest, delayed entry into S and restricted G2 transit. Tissues from people with acute liver failure confirmed hepatic DNA damage and cell cycle-related lesions, including restrictions of hepatocytes in aneuploid states. Remarkably, treatment of cells with a cytoprotective cytokine reversed acetaminophen-induced restrictions to restore cycling. Cell cycle lesions following mitochondrial and DNA damage led to failure of hepatic regeneration in acetaminophen toxicity but their reversibility offers molecular targets for treating acute liver failure. © 2018 John Wiley & Sons Ltd.

  17. The Role of Innate Lymphoid Cells in Immune-Mediated Liver Diseases

    Science.gov (United States)

    Liu, Meifang; Zhang, Cai

    2017-01-01

    Innate lymphoid cells (ILCs) are a recently identified group of innate immune cells lacking antigen-specific receptors that can mediate immune responses and regulate tissue homeostasis and inflammation. ILCs comprise group 1 ILCs, group 2 ILCs, and group 3 ILCs. These ILCs usually localize at mucosal surfaces and combat pathogens by the rapid release of certain cytokines. However, the uncontrolled activation of ILCs can also lead to damaging inflammation, especially in the gut, lung, and skin. Although the physiological and pathogenic roles of ILCs in liver diseases have been attracting increasing attention recently, there has been no systematic review regarding the roles of ILCs in immune-mediated liver diseases. Here, we review the relationships between the ILC subsets and their functions in immune-mediated liver diseases, and discuss their therapeutic potential based on current knowledge about the functional roles of these cells in liver diseases. PMID:28659927

  18. Kupffer cells hasten resolution of liver immunopathology in mouse models of viral hepatitis.

    Directory of Open Access Journals (Sweden)

    Giovanni Sitia

    2011-06-01

    Full Text Available Kupffer cells (KCs are widely considered important contributors to liver injury during viral hepatitis due to their pro-inflammatory activity. Herein we utilized hepatitis B virus (HBV-replication competent transgenic mice and wild-type mice infected with a hepatotropic adenovirus to demonstrate that KCs do not directly induce hepatocellular injury nor do they affect the pathogenic potential of virus-specific CD8 T cells. Instead, KCs limit the severity of liver immunopathology. Mechanistically, our results are most compatible with the hypothesis that KCs contain liver immunopathology by removing apoptotic hepatocytes in a manner largely dependent on scavenger receptors. Apoptotic hepatocytes not readily removed by KCs become secondarily necrotic and release high-mobility group box 1 (HMGB-1 protein, promoting organ infiltration by inflammatory cells, particularly neutrophils. Overall, these results indicate that KCs resolve rather than worsen liver immunopathology.

  19. Mesenchymal stem cell-conditioned medium prevents radiation-induced liver injury by inhibiting inflammation and protecting sinusoidal endothelial cells

    International Nuclear Information System (INIS)

    Chen Yixing; Zeng Zhaochong; Sun Jing; Huang Yan; Zhang Zhenyu; Zeng Haiying

    2015-01-01

    Current management of radiation-induced liver injury is limited. Sinusoidal endothelial cell (SEC) apoptosis and inflammation are considered to be initiating events in hepatic damage. We hypothesized that mesenchymal stem cells (MSCs) possess anti-apoptotic and anti-inflammatory actions during hepatic irradiation, acting via paracrine mechanisms. This study aims to examine whether MSC-derived bioactive components are protective against radiation-induced liver injury in rats. MSC-conditioned medium (MSC-CM) was generated from rat bone marrow–derived MSCs. The effect of MSC-CM on the viability of irradiated SECs was examined by flow cytometric analysis. Activation of the Akt and ERK pathways was analyzed by western blot. MSC-CM was also delivered to Sprague–Dawley rats immediately before receiving liver irradiation, followed by testing for pathological features, changes in serum hyaluronic acid, ALT, and inflammatory cytokine levels, and liver cell apoptosis. MSC-CM enhanced the viability of irradiated SECs in vitro and induced Akt and ERK phosphorylation in these cells. Infusion of MSC-CM immediately before liver irradiation provided a significant anti-apoptotic effect on SECs and improved the histopathological features of injury in the irradiated liver. MSC-CM also reduced the secretion and expression of inflammatory cytokines and increased the expression of anti-inflammatory cytokines. MSC-derived bioactive components could be a novel therapeutic approach for treating radiation-induced liver injury. (author)

  20. Phytosterols Promote Liver Injury and Kupffer Cell Activation in Parenteral Nutrition–Associated Liver Disease

    Science.gov (United States)

    El Kasmi, Karim C.; Anderson, Aimee L.; Devereaux, Michael W.; Vue, Padade M.; Zhang, Wujuan; Setchell, Kenneth D. R.; Karpen, Saul J.; Sokol, Ronald J.

    2014-01-01

    Parenteral nutrition–associated liver disease (PNALD) is a serious complication of PN in infants who do not tolerate enteral feedings, especially those with acquired or congenital intestinal diseases. Yet, the mechanisms underlying PNALD are poorly understood. It has been suggested that a component of soy oil (SO) lipid emulsions in PN solutions, such as plant sterols (phytosterols), may be responsible for PNALD, and that use of fish oil (FO)–based lipid emulsions may be protective. We used a mouse model of PNALD combining PN infusion with intestinal injury to demonstrate that SO-based PN solution causes liver damage and hepatic macrophage activation and that PN solutions that are FO-based or devoid of all lipids prevent these processes. We have furthermore demonstrated that a factor in the SO lipid emulsions, stigmasterol, promotes cholestasis, liver injury, and liver macrophage activation in this model and that this effect may be mediated through suppression of canalicular bile transporter expression (Abcb11/BSEP, Abcc2/MRP2) via antagonism of the nuclear receptors Fxr and Lxr, and failure of up-regulation of the hepatic sterol exporters (Abcg5/g8/ABCG5/8). This study provides experimental evidence that plant sterols in lipid emulsions are a major factor responsible for PNALD and that the absence or reduction of plant sterols is one of the mechanisms for hepatic protection in infants receiving FO-based PN or lipid minimization PN treatment. Modification of lipid constituents in PN solutions is thus a promising strategy to reduce incidence and severity of PNALD. PMID:24107776

  1. Liver fibrosis alleviation after co-transplantation of hematopoietic stem cells with mesenchymal stem cells in patients with thalassemia major.

    Science.gov (United States)

    Ghavamzadeh, Ardeshir; Sotoudeh, Masoud; Hashemi Taheri, Amir Pejman; Alimoghaddam, Kamran; Pashaiefar, Hossein; Jalili, Mahdi; Shahi, Farhad; Jahani, Mohammad; Yaghmaie, Marjan

    2018-02-01

    The aims of this study are to determine the replacement rate of damaged hepatocytes by donor-derived cells in sex-mismatched recipient patients with thalassemia major and to determine whether co-transplantation of mesenchymal stem cells and hematopoietic stem cells (HSCs) can alleviate liver fibrosis. Ten sex-mismatched donor-recipient pairs who received co-transplantation of HSCs with mesenchymal stem cells were included in our study. Liver biopsy was performed before transplantation. Two other liver biopsies were performed between 2 and 5 years after transplantation. The specimens were studied for the presence of donor-derived epithelial cells or hepatocytes using fluorescence in situ hybridization by X- and Y-centromeric probes and immunohistochemical staining for pancytokeratin, CD45, and a hepatocyte-specific antigen. All sex-mismatched tissue samples demonstrated donor-derived hepatocyte independent of donor gender. XY-positive epithelial cells or hepatocytes accounted for 11 to 25% of the cells in histologic sections of female recipients in the first follow-up. It rose to 47-95% in the second follow-up. Although not statistically significant, four out of ten patients showed signs of improvement in liver fibrosis. Our results showed that co-transplantation of HSC with mesenchymal stem cells increases the rate of replacement of recipient hepatocytes by donor-derived cells and may improve liver fibrosis.

  2. T cells infiltrate the liver and kill hepatocytes in HLA-B(∗)57:01-associated floxacillin-induced liver injury.

    Science.gov (United States)

    Wuillemin, Natascha; Terracciano, Luigi; Beltraminelli, Helmut; Schlapbach, Christoph; Fontana, Stefano; Krähenbühl, Stephan; Pichler, Werner J; Yerly, Daniel

    2014-06-01

    Drug-induced liver injury is a major safety issue. It can cause severe disease and is a common cause of the withdrawal of drugs from the pharmaceutical market. Recent studies have identified the HLA-B(∗)57:01 allele as a risk factor for floxacillin (FLUX)-induced liver injury and have suggested a role for cytotoxic CD8(+) T cells in the pathomechanism of liver injury caused by FLUX. This study aimed to confirm the importance of FLUX-reacting cytotoxic lymphocytes in the pathomechanism of liver injury and to dissect the involved mechanisms of cytotoxicity. IHC staining of a liver biopsy from a patient with FLUX-induced liver injury revealed periportal inflammation and the infiltration of cytotoxic CD3(+) CD8(+) lymphocytes into the liver. The infiltration of cytotoxic lymphocytes into the liver of a patient with FLUX-induced liver injury demonstrates the importance of FLUX-reacting T cells in the underlying pathomechanism. Cytotoxicity of FLUX-reacting T cells from 10 HLA-B(∗)57:01(+) healthy donors toward autologous target cells and HLA-B(∗)57:01-transduced hepatocytes was analyzed in vitro. Cytotoxicity of FLUX-reacting T cells was concentration dependent and required concentrations in the range of peak serum levels after FLUX administration. Killing of target cells was mediated by different cytotoxic mechanisms. Our findings emphasize the role of the adaptive immune system and especially of activated drug-reacting T cells in human leukocyte antigen-associated, drug-induced liver injury. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  3. Suppression of Human Liver Cancer Cell Migration and Invasion via the GABAA Receptor

    International Nuclear Information System (INIS)

    Chen, Zhi-ao; Bao, Mei-yan; Xu, Yong-fen; Zha, Ruo-peng; Shi, Hai-bing; Chen, Tao-yang; He, Xiang-huo

    2012-01-01

    To investigate the roles of the γ-aminobutyric acid (GABA) in the metastasis of hepatocellular carcinoma (HCC) and to explore the potential of a novel therapeutic approach for the treatment of HCC. The expression levels of GABA receptor subunit genes in various HCC cell lines and patients‘ tissues were detected by quantitative real-time polymerase chain reaction and Western blot analysis. Transwell cell migration and invasion assays were carried out for functional analysis. The effects of GABA on liver cancer cell cytoskeletal were determined by immunofluorescence staining. And the effects of GABA on HCC metastasis in nude mice were evaluated using an in vivo orthotopic model of liver cancer. The mRNA level of GABA receptor subunits varied between the primary hepatocellular carcinoma tissue and the adjacent non-tumor liver tissue. GABA inhibited human liver cancer cell migration and invasion via the ionotropic GABA A receptor as a result of the induction of liver cancer cell cytoskeletal reorganization. Pretreatment with GABA also significantly reduced intrahepatic liver metastasis and primary tumor formation in vivo. These findings introduce a potential and novel therapeutic approach for the treatment of cancer patients based on the modulation of the GABAergic system

  4. Cutting Edge: Eosinophils Undergo Caspase-1-Mediated Pyroptosis in Response to Necrotic Liver Cells.

    Science.gov (United States)

    Palacios-Macapagal, Daphne; Connor, Jane; Mustelin, Tomas; Ramalingam, Thirumalai R; Wynn, Thomas A; Davidson, Todd S

    2017-08-01

    Many chronic liver disorders are characterized by dysregulated immune responses and hepatocyte death. We used an in vivo model to study the immune response to necrotic liver injury and found that necrotic liver cells induced eosinophil recruitment. Necrotic liver induced eosinophil IL-1β and IL-18 secretion, degranulation, and cell death. Caspase-1 inhibitors blocked all of these responses. Caspase-1-mediated cell death with accompanying cytokine release is the hallmark of a novel form of cell death termed pyroptosis. To confirm this response in a disease model, we isolated eosinophils from the livers of Schistosoma mansoni -infected mice. S. mansoni eggs lodge in the hepatic sinusoids of infected mice, resulting in hepatocyte death, inflammation, and progressive liver fibrosis. This response is typified by massive eosinophilia, and we were able to confirm pyroptosis in the infiltrating eosinophils. This demonstrated that pyroptosis is a cellular pathway used by eosinophils in response to large-scale hepatic cell death. Copyright © 2017 by The American Association of Immunologists, Inc.

  5. The Use of Induced Pluripotent Stem Cells for the Study and Treatment of Liver Diseases.

    Science.gov (United States)

    Hansel, Marc C; Davila, Julio C; Vosough, Massoud; Gramignoli, Roberto; Skvorak, Kristen J; Dorko, Kenneth; Marongiu, Fabio; Blake, William; Strom, Stephen C

    2016-02-01

    Liver disease is a major global health concern. Liver cirrhosis is one of the leading causes of death in the world and currently the only therapeutic option for end-stage liver disease (e.g., acute liver failure, cirrhosis, chronic hepatitis, cholestatic diseases, metabolic diseases, and malignant neoplasms) is orthotropic liver transplantation. Transplantation of hepatocytes has been proposed and used as an alternative to whole organ transplant to stabilize and prolong the lives of patients in some clinical cases. Although these experimental therapies have demonstrated promising and beneficial results, their routine use remains a challenge due to the shortage of donor livers available for cell isolation, variable quality of those tissues, the potential need for lifelong immunosuppression in the transplant recipient, and high costs. Therefore, new therapeutic strategies and more reliable clinical treatments are urgently needed. Recent and continuous technological advances in the development of stem cells suggest they may be beneficial in this respect. In this review, we summarize the history of stem cell and induced pluripotent stem cell (iPSC) technology in the context of hepatic differentiation and discuss the potential applications the technology may offer for human liver disease modeling and treatment. This includes developing safer drugs and cell-based therapies to improve the outcomes of patients with currently incurable health illnesses. We also review promising advances in other disease areas to highlight how the stem cell technology could be applied to liver diseases in the future. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  6. Effect of adoptive transfer or depletion of regulatory T cells on triptolide-induced liver injury

    Directory of Open Access Journals (Sweden)

    Xinzhi eWang

    2016-04-01

    Full Text Available ObjectiveThe aim of this study is to clarify the role of regulatory T cell (Treg in triptolide (TP-induced hepatotoxicity. MethodsFemale C57BL/6 mice received either adoptive transfer of Tregs or depletion of Tregs, then underwent TP administration and were sacrificed 24 hours after TP administration. Liver injury was determined according to ALT and AST levels in serum and histopathological change in liver tissue. Hepatic frequencies of Treg cells and the mRNA expression levles of transcription factor FoxP3 and RORγt, IL-10, SOCS and Notch/Notch ligand were investigated.ResultsDuring TP-induced liver injury, hepatic Treg and IL-10 decreased, while Th17 cell transcription factor RORγt, SOCS signaling and Notch signaling increased, accompanied with liver inflammation. Adoptive transfer of Tregs ameliorated the severity of TP-induced liver injury, accompanied with increased levels of hepatic Treg and IL-10. Adoptive transfer of Tregs remarkably inhibited the expression of RORγt, SOCS3, Notch1 and Notch3. On the contrary, depletion of Treg cells in TP-administered mice resulted in a notable increase of RORγt, SOCS1, SOCS3 and Notch3, while the Treg and IL-10 of liver decreased. Consistent with the exacerbation of liver injury, higher serum levels of ALT and AST were detected in Treg-depleted mice. ConclusionsThese results showed that adoptive transfer or depletion of Tregs attenuated or aggravated TP-induced liver injury, suggesting that Tregs could play important roles in the progression of liver injury. SOCS proteins and Notch signaling affected Tregs, which may contribute to the pathogenesis of TP-induced hepatotoxicity.

  7. Age-related changes in the endocytic capacity of rat liver Kupffer and endothelial cells

    International Nuclear Information System (INIS)

    Brouwer, A.; Barelds, R.J.; Knook, D.L.

    1985-01-01

    There are many indications that the functional capacity of the reticuloendothelial system (RES) declines with age. The aim of this study was to investigate the cellular basis of age-related changes in the clearance function of the RES. The experiments were focused mainly on Kupffer and endothelial cells of the liver which represent a major part of the RES and are primarily responsible for clearance of colloidal material from the circulation. The clearance capacity of the RES was tested clinically and experimentally by intravenous injection of colloids, such as radiolabeled heat-aggregated colloidal albumin. Age-related changes in the endocytosis of 125 I-labeled colloidal albumin (CA) in rats were determined by clearance and organ distribution of different doses of intravenously injected CA, uptake of CA by Kupffer and endothelial liver cells in vivo as determined after isolation of the cells from injected rats and kinetic studies on CA uptake by Kupffer cells in culture. The results show that, at a low dose, the clearance of CA is primarily determined by liver blood flow. At a higher saturating dose, plasma clearance and uptake by the liver are not significantly decreased with age. Endocytosis by endothelial cells, which accounts for about 60% of that of the whole liver, is also unchanged with age. In contrast, a significant decrease in endocytic capacity was observed for Kupffer cells in vivo. This age-related functional decline was also observed in Kupffer cells which were isolated from rats of different ages and maintained in culture

  8. AMC-Bio-Artificial Liver culturing enhances mitochondrial biogenesis in human liver cell lines: The role of oxygen, medium perfusion and 3D configuration

    NARCIS (Netherlands)

    Adam, Aziza A. A.; van Wenum, Martien; van der Mark, Vincent A.; Jongejan, Aldo; Moerland, Perry D.; Houtkooper, Riekelt H.; Wanders, Ronald J. A.; Oude Elferink, Ronald P.; Chamuleau, Robert A. F. M.; Hoekstra, Ruurdtje

    2017-01-01

    Human liver cell lines, like HepaRG and C3A, acquire higher functionality when cultured in the AMC-Bio-Artificial Liver (AMC-BAL). The three main differences between BAL and monolayer culture are the oxygenation (40% vs 20%O2), dynamic vs absent medium perfusion and 3D vs 2D configuration. Here, we

  9. PPARα agonists up-regulate organic cation transporters in rat liver cells

    International Nuclear Information System (INIS)

    Luci, Sebastian; Geissler, Stefanie; Koenig, Bettina; Koch, Alexander; Stangl, Gabriele I.; Hirche, Frank; Eder, Klaus

    2006-01-01

    It has been shown that clofibrate treatment increases the carnitine concentration in the liver of rats. However, the molecular mechanism is still unknown. In this study, we observed for the first time that treatment of rats with the peroxisome proliferator activated receptor (PPAR)-α agonist clofibrate increases hepatic mRNA concentrations of organic cation transporters (OCTNs)-1 and -2 which act as transporters of carnitine into the cell. In rat hepatoma (Fao) cells, treatment with WY-14,643 also increased the mRNA concentration of OCTN-2. mRNA concentrations of enzymes involved in carnitine biosynthesis were not altered by treatment with the PPARα agonists in livers of rats and in Fao cells. We conclude that PPARα agonists increase carnitine concentrations in livers of rats and cells by an increased uptake of carnitine into the cell but not by an increased carnitine biosynthesis

  10. Liver-derived systemic factors drive β-cell hyperplasia in insulin resistant states

    Energy Technology Data Exchange (ETDEWEB)

    El Ouaamari, Abdelfattah; Kawamori, Dan; Dirice, Ercument; Liew, Chong Wee; Shadrach, Jennifer L.; Hu, Jiang; Katsuta, Hitoshi; Hollister-Lock, Jennifer; Qian, Weijun; Wagers, Amy J.; Kulkarni, Rohit N.

    2013-02-21

    Integrative organ cross-talk regulates key aspects of energy homeostasis and its dysregulation may underlie metabolic disorders such as obesity and diabetes. To test the hypothesis that cross-talk between the liver and pancreatic islets modulates β-cell growth in response to insulin resistance, we used the Liver-specific Insulin Receptor Knockout (LIRKO) mouse, a unique model that exhibits dramatic islet hyperplasia. Using complementary in vivo parabiosis and transplantation assays, and in vitro islet culture approaches, we demonstrate that humoral, non-neural, non-cell autonomous factor(s) induce β-cell proliferation in LIRKO mice. Furthermore, we report that a hepatocyte-derived factor(s) stimulates mouse and human β-cell proliferation in ex vivo assays, independent of ambient glucose and insulin levels. These data implicate the liver as a critical source of β-cell growth factors in insulin resistant states.

  11. NKT-cell subsets: promoters and protectors in inflammatory liver disease.

    Science.gov (United States)

    Kumar, Vipin

    2013-09-01

    Natural killer T cells (NKT) are innate-like cells which are abundant in liver sinusoids and express the cell surface receptors of NK cells (e.g., NK1.1 (mouse) or CD161+/CD56+(human)) as well as an antigen receptor (TCR) characteristic of conventional T cells. NKT cells recognize lipid antigens in the context of CD1d, a non-polymorphic MHC class I-like molecule. Activation of NKT cells has a profound influence on the immune response against tumors and infectious organisms and in autoimmune diseases. NKT cells can be categorized into at least two distinct subsets: iNKT or type I use a semi-invariant TCR, whereas type II NKT TCRs are more diverse. Recent evidence suggests that NKT-cell subsets can play opposing roles early in non-microbial liver inflammation in that type I NKT are proinflammatory whereas type II NKT cells inhibit type I NKT-mediated liver injury. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  12. Induction of insulin secretion in engineered liver cells by nitric oxide

    Directory of Open Access Journals (Sweden)

    Özcan Sabire

    2007-10-01

    Full Text Available Abstract Background Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs in a glucose-independent manner. In the present study we engineered liver cells to produce and secrete insulin and insulin secretion can be stimulated via the nitric oxide pathway. Results Expression of either human insulin or the beta cell specific transcription factors PDX-1, NeuroD1 and MafA in the Hepa1-6 cell line or primary liver cells via adenoviral gene transfer, results in production and secretion of insulin. Although, the secretion of insulin is not significantly increased in response to high glucose, treatment of these engineered liver cells with L-arginine stimulates insulin secretion up to three-fold. This L-arginine-mediated insulin release is dependent on the production of nitric oxide. Conclusion Liver cells can be engineered to produce insulin and insulin secretion can be induced by treatment with L-arginine via the production of nitric oxide.

  13. The heterologous expression strategies of antimicrobial peptides in microbial systems.

    Science.gov (United States)

    Deng, Ting; Ge, Haoran; He, Huahua; Liu, Yao; Zhai, Chao; Feng, Liang; Yi, Li

    2017-12-01

    Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Pathogenesis of Nonalcoholic Steatohepatitis: Interactions between Liver Parenchymal and Nonparenchymal Cells

    Directory of Open Access Journals (Sweden)

    Nancy Magee

    2016-01-01

    Full Text Available Nonalcoholic fatty liver disease (NAFLD is the most common type of chronic liver disease in the Western countries, affecting up to 25% of the general population and becoming a major health concern in both adults and children. NAFLD encompasses the entire spectrum of fatty liver disease in individuals without significant alcohol consumption, ranging from nonalcoholic fatty liver (NAFL to nonalcoholic steatohepatitis (NASH and cirrhosis. NASH is a manifestation of the metabolic syndrome and hepatic disorders with the presence of steatosis, hepatocyte injury (ballooning, inflammation, and, in some patients, progressive fibrosis leading to cirrhosis. The pathogenesis of NASH is a complex process and implicates cell interactions between liver parenchymal and nonparenchymal cells as well as crosstalk between various immune cell populations in liver. Lipotoxicity appears to be the central driver of hepatic cellular injury via oxidative stress and endoplasmic reticulum (ER stress. This review focuses on the contributions of hepatocytes and nonparenchymal cells to NASH, assessing their potential applications to the development of novel therapeutic agents. Currently, there are limited pharmacological treatments for NASH; therefore, an increased understanding of NASH pathogenesis is pertinent to improve disease interventions in the future.

  15. Transplantation of co-aggregates of Sertoli cells and islet cells into liver without immunosuppression.

    Science.gov (United States)

    Takemoto, Naohiro; Liu, Xibao; Takii, Kento; Teramura, Yuji; Iwata, Hiroo

    2014-02-15

    Transplantation of islets of Langerhans (islets) was used to treat insulin-dependent diabetes mellitus. However, islet grafts must be maintained by administration of immunosuppressive drugs, which can lead to complications in the long term. An approach that avoids immunosuppressive drug use is desirable. Co-aggregates of Sertoli cells and islet cells from BALB/c mice that were prepared by the hanging drop method were transplanted into C57BL/6 mouse liver through the portal vein as in human clinical islet transplantation. The core part of the aggregates contained mainly Sertoli cells, and these cells were surrounded by islet cells. The co-aggregates retained the functions of both Sertoli and islet cells. When 800 co-aggregates were transplanted into seven C57BL/6 mice via the portal vein, six of seven recipient mice demonstrated quasi-normoglycemia for more than 100 days. The hanging drop method is suitable for preparing aggregates of Sertoli and islet cells for transplantation. Notably, transplantation of these allogeneic co-aggregates into mice with chemically induced diabetes via the portal vein resulted in long-term graft survival without systemic immunosuppression.

  16. Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Takanori Takebe

    2017-12-01

    Full Text Available Summary: Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC. By conducting massive “reverse” screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108. Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations. : With the goal of clinical translation of liver bud transplant therapy, Takebe et al. established a massive organoid production platform from endoderm, endothelial, and mesenchymal progenitor populations specified entirely from human iPSCs, reproducibly demonstrating functionality both in vitro and in vivo. Keywords: iPSC, liver bud, organoid, transplantation, self-organization, endothelial, mesenchymal, liver failure, clinical grade

  17. Follicular helper T cells promote liver pathology in mice during Schistosoma japonicum infection.

    Directory of Open Access Journals (Sweden)

    Xiaojun Chen

    2014-05-01

    Full Text Available Following Schistosoma japonicum (S. japonicum infection, granulomatous responses are induced by parasite eggs trapped in host organs, particular in the liver, during the acute stage of disease. While excessive liver granulomatous responses can lead to more severe fibrosis and circulatory impairment in chronically infected host. However, the exact mechanism of hepatic granuloma formation has remained obscure. In this study, we for the first time showed that follicular helper T (Tfh cells are recruited to the liver to upregulate hepatic granuloma formation and liver injury in S. japonicum-infected mice, and identified a novel function of macrophages in Tfh cell induction. In addition, our results showed that the generation of Tfh cells driven by macrophages is dependent on cell-cell contact and the level of inducible costimulator ligand (ICOSL on macrophages which is regulated by CD40-CD40L signaling. Our findings uncovered a previously unappreciated role for Tfh cells in liver pathology caused by S. japonicum infection in mice.

  18. Cancer Stem Cells in Primary Liver Cancers: Pathological Concepts and Imaging Findings

    Energy Technology Data Exchange (ETDEWEB)

    Joo, Ijin [Department of Radiology, Seoul National University Hospital, Seoul 110-744 (Korea, Republic of); Kim, Haeryoung [Department of Pathology, Seoul National University Bundang Hospital, Seongnam 463-707 (Korea, Republic of); Lee, Jeong Min [Department of Radiology, Seoul National University Hospital, Seoul 110-744 (Korea, Republic of)

    2015-11-01

    There is accumulating evidence that cancer stem cells (CSCs) play an integral role in the initiation of hepatocarcinogenesis and the maintaining of tumor growth. Liver CSCs derived from hepatic stem/progenitor cells have the potential to differentiate into either hepatocytes or cholangiocytes. Primary liver cancers originating from CSCs constitute a heterogeneous histopathologic spectrum, including hepatocellular carcinoma, combined hepatocellular-cholangiocarcinoma, and intrahepatic cholangiocarcinoma with various radiologic manifestations. In this article, we reviewed the recent concepts of CSCs in the development of primary liver cancers, focusing on their pathological and radiological findings. Awareness of the pathological concepts and imaging findings of primary liver cancers with features of CSCs is critical for accurate diagnosis, prediction of outcome, and appropriate treatment options for patients.

  19. Cancer Stem Cells in Primary Liver Cancers: Pathological Concepts and Imaging Findings

    International Nuclear Information System (INIS)

    Joo, Ijin; Kim, Haeryoung; Lee, Jeong Min

    2015-01-01

    There is accumulating evidence that cancer stem cells (CSCs) play an integral role in the initiation of hepatocarcinogenesis and the maintaining of tumor growth. Liver CSCs derived from hepatic stem/progenitor cells have the potential to differentiate into either hepatocytes or cholangiocytes. Primary liver cancers originating from CSCs constitute a heterogeneous histopathologic spectrum, including hepatocellular carcinoma, combined hepatocellular-cholangiocarcinoma, and intrahepatic cholangiocarcinoma with various radiologic manifestations. In this article, we reviewed the recent concepts of CSCs in the development of primary liver cancers, focusing on their pathological and radiological findings. Awareness of the pathological concepts and imaging findings of primary liver cancers with features of CSCs is critical for accurate diagnosis, prediction of outcome, and appropriate treatment options for patients

  20. NKT cell subsets as key participants in liver physiology and pathology.

    Science.gov (United States)

    Bandyopadhyay, Keya; Marrero, Idania; Kumar, Vipin

    2016-05-01

    Natural killer T (NKT) cells are innate-like lymphocytes that generally recognize lipid antigens and are enriched in microvascular compartments of the liver. NKT cells can be activated by self- or microbial-lipid antigens and by signaling through toll-like receptors. Following activation, NKT cells rapidly secrete pro-inflammatory or anti-inflammatory cytokines and chemokines, and thereby determine the milieu for subsequent immunity or tolerance. It is becoming clear that two different subsets of NKT cells-type I and type II-have different modes of antigen recognition and have opposing roles in inflammatory liver diseases. Here we focus mainly on the roles of both NKT cell subsets in the maintenance of immune tolerance and inflammatory diseases in liver. Furthermore, how the differential activation of type I and type II NKT cells influences other innate cells and adaptive immune cells to result in important consequences for tissue integrity is discussed. It is crucial that better reagents, including CD1d tetramers, be used in clinical studies to define the roles of NKT cells in liver diseases in patients.

  1. Ultrasonic features and radionuclide correlation in liver cell adenoma and focal nodular hyperlasia.

    Science.gov (United States)

    Sandler, M A; Petrocelli, R D; Marks, D S; Lopez, R

    1980-05-01

    Ultrasonic features of three cases of liver cell adenoma (LCA) and two cases of focal nodular hyperplasia (FNH) are presented. These tumors have similar sonographic appearances presenting either as solid masses or containing sonolucent areas due to hemorrhage or necrosis. Although these ultrasonic features in patients with an area of decreased activity on 99mTc-sulfur colloid (Tc-SC) liver scans are not specific for LCA or FNH, such findings in the appropriate clinical setting are suggestive of these lesions. The combination of a solid mass on ultrasonography and a normal Tc-SC radioisotope liver study may be relatively specific for uncomplicated FNH.

  2. Ultrasonic features and radionuclide correlation in liver cell adenoma and focal nodular hyperplasia

    International Nuclear Information System (INIS)

    Sandler, M.A.; Petrocelli, R.D.; Marks, D.S.; Lopez, R.

    1980-01-01

    Ultrasonic features of three cases of liver cell adenoma (LCA) and two cases of focal nodular hyperplasia (FNH) are presented. These tumors have similar sonographic appearances presenting either as solid masses or containing sonolucent areas due to hemorrhage or necrosis. Although these ultrasonic features in patients wth an area of decreased activity on /sup 99m/Tc-sulfur colloid (Tc-SC) liver scans are not specific for LCA or FNH, such findings in the appropriate clinical setting are suggestive of these lesions. The combination of a solid mass on ultrasonography and a normal Tc-SC radioisotope liver study may be relatively specific for uncomplicated FNH

  3. Elevated Liver Enzymes

    Science.gov (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  4. Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells.

    OpenAIRE

    Felipe, A; Valdes, R; Santo, B; Lloberas, J; Casado, J; Pastor-Anglada, M

    1998-01-01

    Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent...

  5. Tumor induced hepatic myeloid derived suppressor cells can cause moderate liver damage.

    Directory of Open Access Journals (Sweden)

    Tobias Eggert

    Full Text Available Subcutaneous tumors induce the accumulation of myeloid derived suppressor cells (MDSC not only in blood and spleens, but also in livers of these animals. Unexpectedly, we observed a moderate increase in serum transaminases in mice with EL4 subcutaneous tumors, which prompted us to study the relationship of hepatic MDSC accumulation and liver injury. MDSC were the predominant immune cell population expanding in livers of all subcutaneous tumor models investigated (RIL175, B16, EL4, CT26 and BNL, while liver injury was only observed in EL4 and B16 tumor-bearing mice. Elimination of hepatic MDSC in EL4 tumor-bearing mice using low dose 5-fluorouracil (5-FU treatment reversed transaminase elevation and adoptive transfer of hepatic MDSC from B16 tumor-bearing mice caused transaminase elevation indicating a direct MDSC mediated effect. Surprisingly, hepatic MDSC from B16 tumor-bearing mice partially lost their damage-inducing potency when transferred into mice bearing non damage-inducing RIL175 tumors. Furthermore, MDSC expansion and MDSC-mediated liver injury further increased with growing tumor burden and was associated with different cytokines including GM-CSF, VEGF, interleukin-6, CCL2 and KC, depending on the tumor model used. In contrast to previous findings, which have implicated MDSC only in protection from T cell-mediated hepatitis, we show that tumor-induced hepatic MDSC themselves can cause moderate liver damage.

  6. Tumor induced hepatic myeloid derived suppressor cells can cause moderate liver damage.

    Science.gov (United States)

    Eggert, Tobias; Medina-Echeverz, José; Kapanadze, Tamar; Kruhlak, Michael J; Korangy, Firouzeh; Greten, Tim F

    2014-01-01

    Subcutaneous tumors induce the accumulation of myeloid derived suppressor cells (MDSC) not only in blood and spleens, but also in livers of these animals. Unexpectedly, we observed a moderate increase in serum transaminases in mice with EL4 subcutaneous tumors, which prompted us to study the relationship of hepatic MDSC accumulation and liver injury. MDSC were the predominant immune cell population expanding in livers of all subcutaneous tumor models investigated (RIL175, B16, EL4, CT26 and BNL), while liver injury was only observed in EL4 and B16 tumor-bearing mice. Elimination of hepatic MDSC in EL4 tumor-bearing mice using low dose 5-fluorouracil (5-FU) treatment reversed transaminase elevation and adoptive transfer of hepatic MDSC from B16 tumor-bearing mice caused transaminase elevation indicating a direct MDSC mediated effect. Surprisingly, hepatic MDSC from B16 tumor-bearing mice partially lost their damage-inducing potency when transferred into mice bearing non damage-inducing RIL175 tumors. Furthermore, MDSC expansion and MDSC-mediated liver injury further increased with growing tumor burden and was associated with different cytokines including GM-CSF, VEGF, interleukin-6, CCL2 and KC, depending on the tumor model used. In contrast to previous findings, which have implicated MDSC only in protection from T cell-mediated hepatitis, we show that tumor-induced hepatic MDSC themselves can cause moderate liver damage.

  7. A Complex Interplay between Wnt/β-Catenin Signalling and the Cell Cycle in the Adult Liver

    Directory of Open Access Journals (Sweden)

    Angélique Gougelet

    2012-01-01

    Full Text Available Canonical Wnt signalling, governed by its effector β-catenin, is known for a long time as playing an important role in development, tissue homeostasis, and cancer. In the liver, it was unravelled as both an oncogenic pathway involved in a subset of liver cancers and a physiological signalling identified as the “zonation-keeper” of the quiescent liver lobule. This duality has encouraged to explore the role of canonical Wnt in liver regeneration and liver-cell proliferation mainly using murine genetic models of β-catenin overactivation or inactivation. These studies definitely integrate Wnt signalling within the hepatic network driving regeneration and proliferation. We will review here the current knowledge concerning the mitogenic effect of Wnt, to switch on its specific role in the liver, which is quiescent but with a great capacity to regenerate. The duality of β-catenin signalling, associated both with liver quiescence and liver-cell proliferation, will be brought forward.

  8. A Complex Interplay between Wnt/β-Catenin Signalling and the Cell Cycle in the Adult Liver.

    Science.gov (United States)

    Gougelet, Angélique; Colnot, Sabine

    2012-01-01

    Canonical Wnt signalling, governed by its effector β-catenin, is known for a long time as playing an important role in development, tissue homeostasis, and cancer. In the liver, it was unravelled as both an oncogenic pathway involved in a subset of liver cancers and a physiological signalling identified as the "zonation-keeper" of the quiescent liver lobule. This duality has encouraged to explore the role of canonical Wnt in liver regeneration and liver-cell proliferation mainly using murine genetic models of β-catenin overactivation or inactivation. These studies definitely integrate Wnt signalling within the hepatic network driving regeneration and proliferation. We will review here the current knowledge concerning the mitogenic effect of Wnt, to switch on its specific role in the liver, which is quiescent but with a great capacity to regenerate. The duality of β-catenin signalling, associated both with liver quiescence and liver-cell proliferation, will be brought forward.

  9. NKT cell subsets as key participants in liver physiology and pathology

    Science.gov (United States)

    Bandyopadhyay, Keya; Marrero, Idania; Kumar, Vipin

    2016-01-01

    Natural killer T (NKT) cells are innate-like lymphocytes that generally recognize lipid antigens and are enriched in microvascular compartments of the liver. NKT cells can be activated by self- or microbial-lipid antigens and by signaling through toll-like receptors. Following activation, NKT cells rapidly secrete pro-inflammatory or anti-inflammatory cytokines and chemokines, and thereby determine the milieu for subsequent immunity or tolerance. It is becoming clear that two different subsets of NKT cells—type I and type II—have different modes of antigen recognition and have opposing roles in inflammatory liver diseases. Here we focus mainly on the roles of both NKT cell subsets in the maintenance of immune tolerance and inflammatory diseases in liver. Furthermore, how the differential activation of type I and type II NKT cells influences other innate cells and adaptive immune cells to result in important consequences for tissue integrity is discussed. It is crucial that better reagents, including CD1d tetramers, be used in clinical studies to define the roles of NKT cells in liver diseases in patients. PMID:26972772

  10. CALCIUM-DRIVEN TRANSCRIPTION OF CARDIAC SPECIFYING GENE PROGRAM IN LIVER STEM CELLS

    Science.gov (United States)

    We have previously shown that a cloned liver stem cell line (WB F344) acquires a cardiac phenotype when seeded in a cardiac microenvironment in vivo and ex vivo. Here we investigated the mechanisms of this transdifferentiation in early (cell, rat neonatal ventricu...

  11. Phosphatase of Regenerating Liver-3 Promotes Motility and Metastasis of Mouse Melanoma Cells

    Science.gov (United States)

    Wu, Xiaopeng; Zeng, Hu; Zhang, Xianming; Zhao, Ying; Sha, Haibo; Ge, Xiaomei; Zhang, Minyue; Gao, Xiang; Xu, Qiang

    2004-01-01

    Recent reports suggested that phosphatase of regenerating liver (PRL)-3 might be involved in colorectal carcinoma metastasis with an unknown mechanism. Here we demonstrated that PRL-3 expression was up-regulated in human liver carcinoma compared with normal liver. PRL-3 was also highly expressed in metastatic melanoma B16-BL6 cells but not in its lowly metastatic parental cell line, B16 cells. B16 cells transfected with PRL-3 cDNA displayed morphological transformation from epithelial-like shape to fibroblast-like shape. PRL-3-overexpressed cells showed much higher migratory ability, which could be reversed by specific anti-sense oligodeoxynucleotide and the phosphatase inhibitors sodium orthovanadate or potassium bisperoxo oxovanadate V. Meanwhile, the expression of the catalytically inactive PRL-3 mutations (D72A or C104S) significantly reduced the cell migratory capability. In addition, PRL-3 transfectants demonstrated altered extracellular matrix adhesive property and up-regulated integrin-mediated cell spreading efficiency. Furthermore, we confirmed that PRL-3 could facilitate lung and liver metastasis of B16 cells in an experimental metastasis model in mice, consistent with accelerated proliferation and growth rate both in vitro and in vivo. Together, these observations provide convincing evidence that PRL-3 truly plays a causal role in tumor metastasis. PMID:15161639

  12. Comparative retention of fission fragment 147Pm in regenerated and fetal liver on induction of chromosome aberrations in these cells

    International Nuclear Information System (INIS)

    Zhu Shoupeng; Zheng Siying; Wang Liuyi; Yang Shujin

    1989-01-01

    The purpose of the present study is to ascertain comparative retention of fission fragment 147 Pm in regenerated and fetal liver on induction of chromosome aberrations in these cells. The results indicated that retention of 147 Pm in regenerated liver was about 700 times than in fetal liver. The cumulative absorption dose in regenerated liver was about 2.87 Gy, while in fetal liver-only 0.004 Gy. Under the same conditions, the incidence rate of chromosome aberrations in regenerated liver cells induced by 147 Pm was 50.2%, and in fetal liver cells-about 28.3%. It should be concluded that the radiosensitivity to 147 Pm was not uniform among the regenerated and fetal liver cells. The study suggested that fetal liver cells show to be more radiosensitive to 147 Pm than regenerated liver cells. Among the type of aberrations in both cells induced by 147 Pm, chromatid breakages were predominant, accompanied with a few chromosome breakages

  13. PTP1B confers liver fibrosis by regulating the activation of hepatic stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Pei-Jie; Cai, Shuang-Peng; Yang, Yang; Li, Wan-Xia; Huang, Cheng; Meng, Xiao-Ming; Li, Jun, E-mail: lj@ahmu.edu.cn

    2016-02-01

    Liver fibrosis is a reversible wound-healing response to chronic hepatic injuries. Activation of hepatic stellate cells (HSCs) plays a pivotal role in the development of hepatic fibrosis. The currently accepted mechanism for the resolution of liver fibrosis is the apoptosis and inactivation of activated HSCs. Protein tyrosine phosphatase 1B (PTP1B), a prototype of non-receptor protein tyrosine phosphatase, is proved to be a vital modulator in cardiac fibrogenesis. However, the precise role of PTP1B on liver fibrosis and HSC activation is still unclear. Our study showed that the expression of PTP1B was elevated in fibrotic liver but reduced after spontaneous recovery. Moreover, stimulation of HSC-T6 cells with transforming growth factor-β1 (TGF-β1) resulted in a dose/time-dependent increase of PTP1B mRNA and protein. Co-incubation of HSC-T6 cells with PTP1B-siRNA inhibited the cell proliferation and activation induced by TGF-β1. Additionally, both mRNA and protein of PTP1B were dramatically decreased in inactivated HSCs after treated with adipogenic differentiation mixture (MDI). Over-expression of PTP1B hindered the inactivation of HSC-T6 cells induced by MDI. These observations revealed a regulatory role of PTP1B in liver fibrosis and implied PTP1B as a potential therapeutic target. - Highlights: • The expression of PTP1B in the fibrotic livers and recovery livers • The expression of PTP1B in activated and inactivated HSCs • Blockade of PTP1B inhibited the TGF-β1-induced proliferation and activation of HSCs. • Over-expression of PTP1B abolished the inactivation of HSCs induced by MDI.

  14. PTP1B confers liver fibrosis by regulating the activation of hepatic stellate cells

    International Nuclear Information System (INIS)

    Chen, Pei-Jie; Cai, Shuang-Peng; Yang, Yang; Li, Wan-Xia; Huang, Cheng; Meng, Xiao-Ming; Li, Jun

    2016-01-01

    Liver fibrosis is a reversible wound-healing response to chronic hepatic injuries. Activation of hepatic stellate cells (HSCs) plays a pivotal role in the development of hepatic fibrosis. The currently accepted mechanism for the resolution of liver fibrosis is the apoptosis and inactivation of activated HSCs. Protein tyrosine phosphatase 1B (PTP1B), a prototype of non-receptor protein tyrosine phosphatase, is proved to be a vital modulator in cardiac fibrogenesis. However, the precise role of PTP1B on liver fibrosis and HSC activation is still unclear. Our study showed that the expression of PTP1B was elevated in fibrotic liver but reduced after spontaneous recovery. Moreover, stimulation of HSC-T6 cells with transforming growth factor-β1 (TGF-β1) resulted in a dose/time-dependent increase of PTP1B mRNA and protein. Co-incubation of HSC-T6 cells with PTP1B-siRNA inhibited the cell proliferation and activation induced by TGF-β1. Additionally, both mRNA and protein of PTP1B were dramatically decreased in inactivated HSCs after treated with adipogenic differentiation mixture (MDI). Over-expression of PTP1B hindered the inactivation of HSC-T6 cells induced by MDI. These observations revealed a regulatory role of PTP1B in liver fibrosis and implied PTP1B as a potential therapeutic target. - Highlights: • The expression of PTP1B in the fibrotic livers and recovery livers • The expression of PTP1B in activated and inactivated HSCs • Blockade of PTP1B inhibited the TGF-β1-induced proliferation and activation of HSCs. • Over-expression of PTP1B abolished the inactivation of HSCs induced by MDI.

  15. Influence of matrix nature on the functional efficacy of biomedical cell product for the regeneration of damaged liver (experimental model of acute liver failure

    Directory of Open Access Journals (Sweden)

    S. V. Gautier

    2017-01-01

    Full Text Available Aim. A comparative analysis of the functional efficacy of biomedical cell products (BMCP for the regeneration of damaged liver based on biopolymer scaffolded porous and hydrogel matrices was performed on the experimental model of acute liver failure. Materials and methods. Matrices allowed for clinical use were employed for BMCP in the form of a sponge made from biopolymer nanostructured composite material (BNCM based on a highly purified bacterial copolymers of poly (β-hydroxybutyrate-co-β-oxyvalerate and polyethylene glycol and a hydrogel matrix from biopolymer microheterogeneous collagen-containing hydrogel (BMCH. Cellular component of BMCP was represented by liver cells and multipotent mesenchymal bone marrow stem cells. The functional efficacy of BMCP for the regeneration of damaged liver was evaluated on the experimental model of acute liver failure in Wistar rats (n = 40 via biochemical, morphological, and immunohistochemical methods. Results. When BMCP was implanted to regenerate the damaged liver on the basis of the scaffolded BNCM or hydrogel BMCH matrices, the lethality in rats with acute liver failure was absent; while in control it was 66.6%. Restoration of the activity of cytolytic enzyme levels and protein-synthetic liver function began on day 9 after modeling acute liver failure, in contrast to the control group, where recovery occurred only by days 18–21. Both matrices maintained the viability and functional activity of liver cells up to 90 days with the formation of blood vessels in BMCP. The obtained data confirm that scaffolded BNCM matrix and hydrogel BMCH matrix retain for a long time (up to 90 days the vital activity of the adherent cells in the BMCP composition, which allows using them to correct acute liver failure. At the same time, hydrogel matrix due to the presence of bioactive components contributes to the creation of the best conditions for adhesion and cell activity which accelerate the regeneration processes

  16. Method for determining heterologous biosynthesis pathways

    KAUST Repository

    Gao, Xin

    2017-08-10

    The present invention relates to a method and system for dynamically analyzing, determining, predicting and displaying ranked suitable heterologous biosynthesis pathways for a specified host. The present invention addresses the problem of finding suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived and developed to systematically and dynamically search for, determine, analyze, and display promising heterologous pathways while considering competing endogenous reactions in a given host organism.

  17. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3D Porous Polymer Scaffold for Liver Tissue Engineering

    DEFF Research Database (Denmark)

    Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan

    A huge shortage of liver organs for transplantation has motivated the research field of tissue engineering to develop bioartificial liver tissue and even a whole liver. The goal of NanoBio4Trans is to create a vascularized bioartificial liver tissue, initially as a liver-support system. Due...... to limitations of primary hepatocytes regarding availability and maintenance of functionality, stem cells and especially human induced pluripotent stem cells (hIPS cells) are an attractive cell source for liver tissue engineering. The aim of this part of NanoBio4Trans is to optimize culture and hepatic...... differentiation of hIPS-derived definitive endoderm (DE) cells in a 3D porous polymer scaffold built-in a perfusable bioreactor. The use of a microfluidic bioreactor array enables the culture of 16 independent tissues in one experimental run and thereby an optimization study to be performed....

  18. Lysosomal and endosomal heterogeneity in the liver: A comparison of the intracellular pathways of endocytosis in rat liver cells

    International Nuclear Information System (INIS)

    Kindberg, G.M.; Tolleshaug, H.; Gjoen, T.; Berg, T.

    1991-01-01

    Air-filled albumin microspheres, asialoorosomucoid and formaldehyde-treated serum albumin are selectively taken up by endocytosis in rat liver Kupffer cells, parenchymal cells and endothelial cells, respectively. Intracellular transport and degradation of endocytosed material were studied by subcellular fractionation in sucrose and Nycodenz gradients after intravenous injection of the ligand. By using ligands labeled with 125I-tyramine-cellobiose, the subcellular distribution of labeled degradation products can be studied because they are trapped at the site of formation. The results show that the kinetics of intracellular transport are different in hepatic parenchymal, endothelial and Kupffer cells. In endothelial cells, the ligand is associated with two types of endosomes during the first minutes after internalization and then is transferred rapidly to the lysosomes. In parenchymal cells, 125I-tyramine-cellobiose-asialoorosomucoid was located in a relatively slowly sedimenting vesicle during the first minute after internalization and subsequently in denser endosomes. Degradation of 125I-tyramine-cellobiose-asialoorosomucoid in parenchymal cells started later than that of 125I-tyramine-cellobiose-formaldehyde-treated serum albumin in endothelial cells. Furthermore, the ligand seemed to be transferred relatively slowly from endosomes to lysosomes, and most of the undegraded ligand was in the endosomes. The rate-limiting step of proteolysis in parenchymal cells is probably the transport from endosomes to lysosomes. In Kupffer cells, most 125I-tyramine-cellobiose-microspheres are found as undegraded material in very dense endosomes up to 3 hr after injection. After 20 hr, most of the ligand is degraded in lysosomes distributed at a lower density than the endosomes in Nycodenz and sucrose gradients

  19. Heterologous production of peptides in plants: fusion proteins and beyond.

    Science.gov (United States)

    Viana, Juliane Flávia Cançado; Dias, Simoni Campos; Franco, Octávio Luiz; Lacorte, Cristiano

    2013-11-01

    Recombinant DNA technology has allowed the ectopic production of proteins and peptides of different organisms leading to biopharmaceutical production in large cultures of bacterial, yeasts and mammalian cells. Otherwise, the expression of recombinant proteins and peptides in plants is an attractive alternative presenting several advantages over the commonly used expression systems including reduced production costs, easy scale-up and reduced risks of pathogen contamination. Different types of proteins and peptides have been expressed in plants, including antibodies, antigens, and proteins and peptides of medical, veterinary and industrial applications. However, apart from providing a proof of concept, the use of plants as platforms for heterologous protein and peptide production still depends on key steps towards optimization including the enhancement of expression levels, manipulation of post-transcriptional modifications and improvements in purification methods. In this review, strategies to increase heterologous protein and peptide stability and accumulation are discussed, focusing on the expression of peptides through the use of gene fusions.

  20. Prostaglandin E2 Regulates Liver versus Pancreas Cell Fate Decisions and Endodermal Outgrowth

    Science.gov (United States)

    Nissim, Sahar; Sherwood, Richard I.; Wucherpfennig, Julia; Saunders, Diane; Harris, James M.; Esain, Virginie; Carroll, Kelli J.; Frechette, Gregory M.; Kim, Andrew J.; Hwang, Katie L.; Cutting, Claire C.; Elledge, Susanna; North, Trista E.; Goessling, Wolfram

    2014-01-01

    SUMMARY The liver and pancreas arise from common endodermal progenitors. How these distinct cell fates are specified is poorly understood. Here, we describe prostaglandin E2 (PGE2) as a regulator of endodermal fate specification during development. Modulating PGE2 activity has opposing effects on liver-versus-pancreas specification in zebrafish embryos as well as mouse endodermal progenitors. The PGE2 synthetic enzyme cox2a and receptor ep2a are patterned such that cells closest to PGE2 synthesis acquire a liver fate whereas more distant cells acquire a pancreas fate. PGE2 interacts with the bmp2b pathway to regulate fate specification. At later stages of development, PGE2 acting via the ep4a receptor promotes outgrowth of both the liver and pancreas. PGE2 remains important for adult organ growth, as it modulates liver regeneration. This work provides in vivo evidence that PGE2 may act as a morphogen to regulate cell fate decisions and outgrowth of the embryonic endodermal anlagen. PMID:24530296

  1. Cross-activating invariant NKT cells and kupffer cells suppress cholestatic liver injury in a mouse model of biliary obstruction.

    Directory of Open Access Journals (Sweden)

    Caroline C Duwaerts

    Full Text Available Both Kupffer cells and invariant natural killer T (iNKT cells suppress neutrophil-dependent liver injury in a mouse model of biliary obstruction. We hypothesize that these roles are interdependent and require iNKT cell-Kupffer cell cross-activation. Female, wild-type and iNKT cell-deficient C57Bl/6 mice were injected with magnetic beads 3 days prior to bile duct ligation (BDL in order to facilitate subsequent Kupffer cell isolation. On day three post-BDL, the animals were euthanized and the livers dissected. Necrosis was scored; Kupffer cells were isolated and cell surface marker expression (flow cytometry, mRNA expression (qtPCR, nitric oxide (NO (. production (Griess reaction, and protein secretion (cytometric bead-array or ELISAs were determined. To address the potential role of NO (. in suppressing neutrophil accumulation, a group of WT mice received 1400W, a specific inducible nitric oxide synthase (iNOS inhibitor, prior to BDL. To clarify the mechanisms underlying Kupffer cell-iNKT cell cross-activation, WT animals were administered anti-IFN-γ or anti-lymphocyte function-associated antigen (LFA-1 antibody prior to BDL. Compared to their WT counterparts, Kupffer cells obtained from BDL iNKT cell-deficient mice expressed lower iNOS mRNA levels, produced less NO (. , and secreted more neutrophil chemoattractants. Both iNOS inhibition and IFN-γ neutralization increased neutrophil accumulation in the livers of BDL WT mice. Anti-LFA-1 pre-treatment reduced iNKT cell accumulation in these same animals. These data indicate that the LFA-1-dependent cross-activation of iNKT cells and Kupffer cells inhibits neutrophil accumulation and cholestatic liver injury.

  2. Estradiol inhibits hepatic stellate cell area and collagen synthesis in the chicken liver.

    Science.gov (United States)

    Nishimura, Shotaro; Teshima, Akifumi; Kawabata, Fuminori; Tabata, Shoji

    2017-11-01

    Hepatic stellate cells (HSCs) are the main collagen-producing cells in the liver. The HSC area and amount of collagen fibers are different between male and female chickens. This study was performed to confirm the effect of estradiol on collagen synthesis in the growing chicken liver. Blood estradiol levels in chicks were compared at 4 and 8 weeks of age, and the collagen fibril network in liver tissue was observed at 8 weeks by scanning electron microscopy. Intraperitoneal administrations of estradiol and tamoxifen to male and female chicks, respectively, were performed daily from 5 to 8 weeks of age. The areas of HSCs and collagen contents were measured in the liver tissue. The blood estradiol level was higher in females than in males, and the collagen fibril network was denser in males than in females at 8 weeks of age. Estradiol administration in males induced decreases in the HSC area and collagen content of the liver. Conversely, tamoxifen administration in females induced an increase in the HSC area but did not facilitate collagen synthesis. Based on these results, estradiol inhibits the area and collagen synthesis of HSCs in the growing chicken liver under normal physiological conditions. © 2017 Japanese Society of Animal Science.

  3. An in silico platform for the design of heterologous pathways in nonnative metabolite production

    Directory of Open Access Journals (Sweden)

    Chatsurachai Sunisa

    2012-05-01

    Full Text Available Abstract Background Microorganisms are used as cell factories to produce valuable compounds in pharmaceuticals, biofuels, and other industrial processes. Incorporating heterologous metabolic pathways into well-characterized hosts is a major strategy for obtaining these target metabolites and improving productivity. However, selecting appropriate heterologous metabolic pathways for a host microorganism remains difficult owing to the complexity of metabolic networks. Hence, metabolic network design could benefit greatly from the availability of an in silico platform for heterologous pathway searching. Results We developed an algorithm for finding feasible heterologous pathways by which nonnative target metabolites are produced by host microorganisms, using Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae as templates. Using this algorithm, we screened heterologous pathways for the production of all possible nonnative target metabolites contained within databases. We then assessed the feasibility of the target productions using flux balance analysis, by which we could identify target metabolites associated with maximum cellular growth rate. Conclusions This in silico platform, designed for targeted searching of heterologous metabolic reactions, provides essential information for cell factory improvement.

  4. Research advances in sorafenib-induced apoptotic signaling pathways in liver cancer cells

    Directory of Open Access Journals (Sweden)

    ZHANG Chaoya

    2016-04-01

    Full Text Available Currently, sorafenib is the multi-target inhibitor for the treatment of advanced primary liver cancer, and can effectively prolong the progression-free survival and overall survival in patients with advanced primary liver cancer. The application of sorafenib in the targeted therapy for liver cancer has become a hot topic. Major targets or signaling pathways include Raf/Mek/Erk, Jak/Stat, PI3K/Akt/mTOR, VEGFR and PDGFR, STAT, microRNA, Wnt/β-catenin, autolysosome, and tumor-related proteins, and sorafenib can regulate the proliferation, differentiation, metastasis, and apoptosis of liver cancer cells through these targets. This article reviews the current research on the action of sorafenib on these targets or signaling pathways to provide useful references for further clinical research on sorafenib.

  5. Impaired liver regeneration is associated with reduced cyclin B1 in natural killer T cell-deficient mice.

    Science.gov (United States)

    Ben Ya'acov, Ami; Meir, Hadar; Zolotaryova, Lydia; Ilan, Yaron; Shteyer, Eyal

    2017-03-23

    It has been shown that the proportion of natural killer T cells is markedly elevated during liver regeneration and their activation under different conditions can modulate this process. As natural killer T cells and liver injury are central in liver regeneration, elucidating their role is important. The aim of the current study is to explore the role of natural killer T cells in impaired liver regeneration. Concanvalin A was injected 4 days before partial hepatectomy to natural killer T cells- deficient mice or to anti CD1d1-treated mice. Ki-67 and proliferating cell nuclear antigen were used to measure hepatocytes proliferation. Expression of hepatic cyclin B1 and proliferating cell nuclear antigen were evaluated by Western Blot and liver injury was assessed by ALT and histology. Natural killer T cells- deficient or mice injected with anti CD1d antibodies exhibited reduced liver regeneration. These mice were considerably resistant to ConA-induced liver injury. In the absence of NKT cells hepatic proliferating cell nuclear antigen and cyclin B1 decreased in mice injected with Concanvalin A before partial hepatectomy. This was accompanied with reduced serum interleukin-6 levels. Natural killer T cells play an important role in liver regeneration, which is associated with cyclin B1 and interleukin-6.

  6. Method for determining heterologous biosynthesis pathways

    KAUST Repository

    Gao, Xin; Kuwahara, Hiroyuki; Alazmi, Meshari Saud; Cui, Xuefeng

    2017-01-01

    suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived

  7. Intra-Hepatic Depletion of Mucosal-Associated Invariant T Cells in Hepatitis C Virus-Induced Liver Inflammation.

    Science.gov (United States)

    Bolte, Fabian J; O'Keefe, Ashley C; Webb, Lauren M; Serti, Elisavet; Rivera, Elenita; Liang, T Jake; Ghany, Marc; Rehermann, Barbara

    2017-11-01

    Chronic hepatitis affects phenotypes of innate and adaptive immune cells. Mucosal-associated invariant T (MAIT) cells are enriched in the liver as compared with the blood, respond to intra-hepatic cytokines, and (via the semi-invariant T-cell receptor) to bacteria translocated from the gut. Little is known about the role of MAIT cells in livers of patients with chronic hepatitis C virus (HCV) infection and their fate after antiviral therapy. We collected blood samples from 42 patients with chronic HCV infection who achieved a sustained virologic response after 12 weeks of treatment with sofosbuvir and velpatasvir. Mononuclear cells were isolated from blood before treatment, at weeks 4 and 12 during treatment, and 24 weeks after the end of treatment. Liver biopsies were collected from 37 of the patients prior to and at week 4 of treatment. Mononuclear cells from 56 blood donors and 10 livers that were not suitable for transplantation were used as controls. Liver samples were assessed histologically for inflammation and fibrosis. Mononuclear cells from liver and blood were studied by flow cytometry and analyzed for responses to cytokine and bacterial stimulation. The frequency of MAIT cells among T cells was significantly lower in blood and liver samples of patients with HCV infection than of controls (median, 1.31% vs 2.32% for blood samples, P = .0048; and median, 4.34% vs 13.40% for liver samples, P = .001). There was an inverse correlation between the frequency of MAIT cells in the liver and histologically determined levels of liver inflammation (r = -.5437, P = .0006) and fibrosis (r = -.5829, P = .0002). MAIT cells from the liver had higher levels of activation and cytotoxicity than MAIT cells from blood (P liver inflammation and MAIT cell activation and cytotoxicity, and increased the MAIT cell frequency among intra-hepatic but not blood T cells. The MAIT cell response to T-cell receptor-mediated stimulation did not change during the 12 weeks of

  8. Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

    Science.gov (United States)

    Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

    2016-01-01

    Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models. PMID:27077489

  9. Nucleolar activity after 3-methylcholanthrene treatment of rat liver cells studied by silver staining procedure

    Energy Technology Data Exchange (ETDEWEB)

    Komaromy, L.; Tigyi, A.

    1986-01-01

    The influence of a single dose of 3-methylcholanthrene (3-MC) was studied in nucleoli of young rat liver cells by means of conventional and ultracytochemical methods. The nucleolar activity was stimulated in the authors experimental conditions: the appearance of the fibrillar centers in the liver cell nucleoli as well as the silver staining protein content of the fibrillar centers and the dense fibrillar component were increased by 3-MC. The results suggest that the activity of ribosomal genes was increased following 3-MC treatment.

  10. JNK1 induces hedgehog signaling from stellate cells to accelerate liver regeneration in mice.

    Science.gov (United States)

    Langiewicz, Magda; Graf, Rolf; Humar, Bostjan; Clavien, Pierre A

    2018-04-27

    To improve outcomes of two-staged hepatectomies for large/multiple liver tumors, portal vein ligation (PVL) has been combined with parenchymal transection (coined ALPPS; Associated Liver Partition and Portal vein ligation for Staged hepatectomy) to greatly accelerate liver regeneration. In a novel ALPPS mouse model, we have reported paracrine Indian hedgehog (IHH) signaling from stellate cells as an early contributor to augmented regeneration. Here, we sought to identify upstream regulators of IHH. ALPPS in mice was compared against PVL and additional control surgeries. Potential IHH regulators were identified through in silico mining of transcriptomic data. JNK1 activity was reduced through SP600125 to evaluate its effects on IHH signaling. Recombinant IHH was injected after JNK diminution to substantiate their relationship during accelerated liver regeneration. Mining linked Ihh to Mapk8. JNK1 upregulation after ALPPS was validated and preceded the IHH peak. On immunofluorescence, JNK1 and IHH co-localized in ASMA-positive non-parenchymal cells. Inhibition of JNK1 prior to ALPPS surgery reduced liver weight gain to PVL levels and was accompanied by downregulation of hepatocellular proliferation and the IHH-GLI1-CCND1 axis. In JNK1-inhibited mice, recombinant IHH restored ALPPS-like acceleration of regeneration and re-elevated JNK1 activity, suggesting the presence of a positive IHH-JNK1 feedback loop. JNK1-mediated induction of IHH paracrine signaling from HSCs is essential for accelerated regeneration of parenchymal mass. The JNK1-IHH axis is a mechanism unique to ALPPS surgery and may point to therapeutic alternatives for patients with insufficient regenerative capacity. ALPPS, a novel two-staged hepatectomy, induces an unprecedented acceleration of liver regeneration to enable treatment of unresectable liver tumors. Here, we demonstrate JNK1-IHH signaling as a mechanism underlying the regenerative acceleration induced by ALPPS. Copyright © 2018 European

  11. Hyper-IL-15 suppresses metastatic and autochthonous liver cancer by promoting tumour-specific CD8+ T cell responses.

    Science.gov (United States)

    Cheng, Liang; Du, Xuexiang; Wang, Zheng; Ju, Jianqi; Jia, Mingming; Huang, Qibin; Xing, Qiao; Xu, Meng; Tan, Yi; Liu, Mingyue; Du, Peishuang; Su, Lishan; Wang, Shengdian

    2014-12-01

    Liver cancer has a very dismal prognosis due to lack of effective therapy. Here, we studied the therapeutic effects of hyper-interleukin15 (hyper-IL-15), which is composed of IL-15 and the sushi domain of the IL-15 receptor α chain, on metastatic and autochthonous liver cancers. Liver metastatic tumour models were established by intraportally injecting syngeneic mice with murine CT26 colon carcinoma cells or B16-OVA melanoma cells. Primary hepatocellular carcinoma (HCC) was induced by diethylnitrosamine (DEN). A hydrodynamics-based gene delivery method was used to achieve sustained hyper-IL-15 expression in the liver. Liver gene delivery of hyper-IL-15 robustly expanded CD8(+) T and NK cells, leading to a long-term (more than 40 days) accumulation of CD8(+) T cells in vivo, especially in the liver. Hyper-IL-15 treatment exerted remarkable therapeutic effects on well-established liver metastatic tumours and even on DEN-induced autochthonous HCC, and these effects were abolished by depletion of CD8(+) T cells but not NK cells. Hyper-IL-15 triggered IL-12 and interferon-γ production and reduced the expression of co-inhibitory molecules on dendritic cells in the liver. Adoptive transfer of T cell receptor (TCR) transgenic OT-1 cells showed that hyper-IL-15 preferentially expanded tumour-specific CD8(+) T cells and promoted their interferon-γ synthesis and cytotoxicity. Liver delivery of hyper-IL-15 provides an effective therapy against well-established metastatic and autochthonous liver cancers in mouse models by preferentially expanding tumour-specific CD8(+) T cells and promoting their anti-tumour effects. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  12. Hydrogen peroxide stimulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells

    Energy Technology Data Exchange (ETDEWEB)

    Shibata, Ayano; Tanabe, Eriko; Inoue, Serina; Kitayoshi, Misaho; Okimoto, Souta; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2013-04-12

    Highlights: •Hydrogen peroxide stimulates cell motility of WB-F344 cells. •LPA{sub 3} is induced by hydrogen peroxide in WB-F344 cells. •Cell motility by hydrogen peroxide is inhibited in LPA{sub 3} knockdown cells. •LPA signaling is involved in cell migration by hydrogen peroxide. -- Abstract: Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1 μM for 48 h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA{sub 3} on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA{sub 3} may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide.

  13. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

    Science.gov (United States)

    Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

    2017-01-01

    Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. © FASEB.

  14. Identification of Pathways in Liver Repair Potentially Targeted by Secretory Proteins from Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Sandra Winkler

    2016-07-01

    Full Text Available Background: The beneficial impact of mesenchymal stem cells (MSC on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1 increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor signalling pathways as relevant networks. Relationships to transforming growth factor β (TGF-β and hypoxia-inducible factor 1-α (HIF1-α signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

  15. Etanercept blocks inflammatory responses orchestrated by TNF-α to promote transplanted cell engraftment and proliferation in rat liver

    Science.gov (United States)

    Viswanathan, Preeti; Kapoor, Sorabh; Kumaran, Vinay; Joseph, Brigid; Gupta, Sanjeev

    2014-01-01

    Engraftment of transplanted cells is critical for liver-directed cell therapy but most transplanted cells are rapidly cleared from liver sinusoids by proinflammatory cytokines/chemokines/receptors after activation of neutrophils or Kupffer cells. To define whether TNF-α served roles in cell-transplantation-induced hepatic inflammation, we used TNF-α antagonist, etanercept, for studies in syngeneic rat hepatocyte transplantation systems. After cell transplantation, multiple cytokines/chemokines/receptors were overexpressed, whereas etanercept prior to cell transplantation essentially normalized these responses. Moreover, ETN downregulated cell transplantation-induced intrahepatic release of secretory cytokines, such as high mobility group box 1. These effects of etanercept decreased cell transplantation-induced activation of neutrophils but not of Kupffer cells. Transplanted cell engraftment improved by several-fold in etanercept-treated animals. These gains in cell engraftment were repeatedly realized after pretreatment of animals with etanercept before multiple cell transplantation sessions. Transplanted cell numbers did not change over time indicating absence of cell proliferation after etanercept alone. By contrast, in animals preconditioned with retrorsine and partial hepatectomy, cell transplantation after etanercept pretreatment significantly accelerated liver repopulation compared with control rats. We concluded that TNF-α played a major role in orchestrating cell transplantation-induced inflammation through regulation of multiple cytokines/chemokines/receptor expression. As TNF-α antagonism by etanercept decreased transplanted cell clearance, improved cell engraftment and accelerated liver repopulation, this pharmacological approach to control hepatic inflammation will help optimize clinical strategies for liver cell therapy. PMID:24844924

  16. Betatrophin: A liver-derived hormone for the pancreatic β-cell proliferation.

    Science.gov (United States)

    Raghow, Rajendra

    2013-12-15

    The pancreatic β-cell failure which invariably accompanies insulin resistance in the liver and skeletal muscle is a hallmark of type-2 diabetes mellitus (T2DM). The persistent hyperglycemia of T2DM is often treated with anti-diabetic drugs with or without subcutaneous insulin injections, neither of which mimic the physiological glycemic control seen in individuals with fully functional pancreas. A sought after goal for the treatment of T2DM has been to harness the regenerative potential of pancreatic β-cells that might obviate a need for exogenous insulin injections. A new study towards attaining this aim was reported by Yi et al, who have characterized a liver-derived protein, named betatrophin, capable of inducing pancreatic β-cell proliferation in mice. Using a variety of in vitro and in vivo methods, Yi et al, have shown that betatrophin was expressed mainly in the liver and adipose tissue of mice. Exogenous expression of betatrophin in the liver led to dramatic increase in the pancreatic β-cell mass and higher output of insulin in mice that also concomitantly elicited improved glucose tolerance. The authors discovered that betatrophin was also present in the human plasma. Surprisingly, betatrophin has been previously described by three other names, i.e., re-feeding-induced fat and liver protein, lipasin and atypical angiopoeitin-like 8, by three independent laboratories, as nutritionally regulated liver-enriched factors that control serum triglyceride levels and lipid metabolism. Yi et al demonstration of betatrophin, as a circulating hormone that regulates β-cell proliferation, if successfully translated in the clinic, holds the potential to change the course of current therapies for diabetes.

  17. Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

    International Nuclear Information System (INIS)

    Hirose, Yoshikazu; Itoh, Tohru; Miyajima, Atsushi

    2009-01-01

    Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog (Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk + hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk + hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk + hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.

  18. Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

    Science.gov (United States)

    Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

    2018-01-01

    Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

  19. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

    Science.gov (United States)

    Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-03-10

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

  20. Stellate-cell lipidosis in liver biopsy specimens. Recognition and significance.

    Science.gov (United States)

    Levine, Pascale Hummel; Delgado, Yara; Theise, Neil D; West, A Brian

    2003-02-01

    Hepatic stellate-cell lipidosis due to hypervitaminosis A can lead to cirrhosis, which can be averted by restricting vitamin A intake. Other causes, including the use of synthetic retinoids, have been postulated. We studied the frequency and etiology of stellate-cell lipidosis in patients undergoing liver biopsy for reasons other than vitamin A abuse. Fourteen cases (1.1%) were identified retrospectively among 1,235 nontransplant liver biopsy specimens examined from January 1995 through December 1999. Diagnostic criteria included the following: lipid-laden cells in the space of Disse; small, dark, crescent-shaped nuclei with inconspicuous nucleoli; and wispy cytoplasmic strands separating fat droplets. Patient details, reason for biopsy, and medication use were studied. Reasons for biopsy included hepatitis C (10 cases), abnormal liver enzyme levels (2 cases), methotrexate use (1 case), and alcohol abuse (1 case). Hypervitaminosis A was not suspected clinically in the 5 patients who used oral vitamin A or 3 who used topical tretinoin (Retin-A). In 6 patients, no cause of stellate-cell lipidosis was discerned. Stellate-cell lipidosis should be reported to alert clinicians to a potentially preventable form of liver injury.

  1. Cocktail of chemical compounds robustly promoting cell reprogramming protects liver against acute injury

    Directory of Open Access Journals (Sweden)

    Yuewen Tang

    2017-02-01

    Full Text Available Abstract Tissue damage induces cells into reprogramming-like cellular state, which contributes to tissue regeneration. However, whether factors promoting the cell reprogramming favor tissue regeneration remains elusive. Here we identified combination of small chemical compounds including drug cocktails robustly promoting in vitro cell reprogramming. We then administrated the drug cocktails to mice with acute liver injuries induced by partial hepatectomy or toxic treatment. Our results demonstrated that the drug cocktails which promoted cell reprogramming in vitro improved liver regeneration and hepatic function in vivo after acute injuries. The underlying mechanism could be that expression of pluripotent genes activated after injury is further upregulated by drug cocktails. Thus our study offers proof-of-concept evidence that cocktail of clinical compounds improving cell reprogramming favors tissue recovery after acute damages, which is an attractive strategy for regenerative purpose.

  2. The spleen as an extramedullary source of inflammatory cells responding to acetaminophen-induced liver injury

    International Nuclear Information System (INIS)

    Mandal, Mili; Gardner, Carol R.; Sun, Richard; Choi, Hyejeong; Lad, Sonali; Mishin, Vladimir; Laskin, Jeffrey D.; Laskin, Debra L.

    2016-01-01

    Macrophages have been shown to play a role in acetaminophen (APAP)-induced hepatotoxicity, contributing to both pro- and anti-inflammatory processes. In these studies, we analyzed the role of the spleen as an extramedullary source of hepatic macrophages. APAP administration (300 mg/kg, i.p.) to control mice resulted in an increase in CD11b + infiltrating Ly6G + granulocytic and Ly6G − monocytic cells in the spleen and the liver. The majority of the Ly6G + cells were also positive for the monocyte/macrophage activation marker, Ly6C, suggesting a myeloid derived suppressor cell (MDSC) phenotype. By comparison, Ly6G − cells consisted of 3 subpopulations expressing high, intermediate, and low levels of Ly6C. Splenectomy was associated with increases in mature (F4/80 + ) and immature (F4/80 − ) pro-inflammatory Ly6C hi macrophages and mature anti-inflammatory (Ly6C lo ) macrophages in the liver after APAP; increases in MDSCs were also noted in the livers of splenectomized (SPX) mice after APAP. This was associated with increases in APAP-induced expression of chemokine receptors regulating pro-inflammatory (CCR2) and anti-inflammatory (CX3CR1) macrophage trafficking. In contrast, APAP-induced increases in pro-inflammatory galectin-3 + macrophages were blunted in livers of SPX mice relative to control mice, along with hepatic expression of TNF-α, as well as the anti-inflammatory macrophage markers, FIZZ-1 and YM-1. These data demonstrate that multiple subpopulations of pro- and anti-inflammatory cells respond to APAP-induced injury, and that these cells originate from distinct hematopoietic reservoirs. - Highlights: • Multiple inflammatory cell subpopulations accumulate in the spleen and liver following acetaminophen (APAP) intoxication. • Splenectomy alters liver inflammatory cell populations responding to APAP. • Inflammatory cells accumulating in the liver in response to APAP originate from the spleen and the bone marrow. • Hepatotoxicity is reduced in

  3. The spleen as an extramedullary source of inflammatory cells responding to acetaminophen-induced liver injury

    Energy Technology Data Exchange (ETDEWEB)

    Mandal, Mili, E-mail: milimandal@gmail.com [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Gardner, Carol R., E-mail: cgardner@pharmacy.rutgers.edu [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Sun, Richard, E-mail: fishpower52@gmail.com [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Choi, Hyejeong, E-mail: choi@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Lad, Sonali, E-mail: sonurose92@gmail.com [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Mishin, Vladimir, E-mail: mishinv@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Jeffrey D., E-mail: jlaskin@eohsi.rutgers.edu [Department of Environmental and Occupational Health, School of Public Health, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States)

    2016-08-01

    Macrophages have been shown to play a role in acetaminophen (APAP)-induced hepatotoxicity, contributing to both pro- and anti-inflammatory processes. In these studies, we analyzed the role of the spleen as an extramedullary source of hepatic macrophages. APAP administration (300 mg/kg, i.p.) to control mice resulted in an increase in CD11b{sup +} infiltrating Ly6G{sup +} granulocytic and Ly6G{sup −} monocytic cells in the spleen and the liver. The majority of the Ly6G{sup +} cells were also positive for the monocyte/macrophage activation marker, Ly6C, suggesting a myeloid derived suppressor cell (MDSC) phenotype. By comparison, Ly6G{sup −} cells consisted of 3 subpopulations expressing high, intermediate, and low levels of Ly6C. Splenectomy was associated with increases in mature (F4/80{sup +}) and immature (F4/80{sup −}) pro-inflammatory Ly6C{sup hi} macrophages and mature anti-inflammatory (Ly6C{sup lo}) macrophages in the liver after APAP; increases in MDSCs were also noted in the livers of splenectomized (SPX) mice after APAP. This was associated with increases in APAP-induced expression of chemokine receptors regulating pro-inflammatory (CCR2) and anti-inflammatory (CX3CR1) macrophage trafficking. In contrast, APAP-induced increases in pro-inflammatory galectin-3{sup +} macrophages were blunted in livers of SPX mice relative to control mice, along with hepatic expression of TNF-α, as well as the anti-inflammatory macrophage markers, FIZZ-1 and YM-1. These data demonstrate that multiple subpopulations of pro- and anti-inflammatory cells respond to APAP-induced injury, and that these cells originate from distinct hematopoietic reservoirs. - Highlights: • Multiple inflammatory cell subpopulations accumulate in the spleen and liver following acetaminophen (APAP) intoxication. • Splenectomy alters liver inflammatory cell populations responding to APAP. • Inflammatory cells accumulating in the liver in response to APAP originate from the spleen and the

  4. Repair effect of transplantation of bone marrow mesenchymal stem cells on liver injury in severe burned rats and its mechanism

    International Nuclear Information System (INIS)

    Chen Hao; Zhou Yubo; Zhang Ying; Qin Yonggang; Guo Li; Yin Fei; Meng Chunyang; Yang Xiaoyu

    2014-01-01

    Objective: To investigate the repair effect of transplantation of bone marrow mesenchymal stem cells (BMSCs) on liver injury in severe burned rats, and to clarify its mechanism. Methods: The BMSCs of rats were isolated, cultured, amplified, identified, and labeled in vitro. 30 Wistar rats were randomly divided into normal control group (n=10), model group (n=10) and cell therapy group (n=10). The burned rat model was established. The BMSCs labeled by chlormethyl-benzamidodialkylcarbocyanine (CM-Dil) were transplanted into the rats in cell therapy group by retro-orbital intravenous injection and the saline was injected into the rats in model group. The general status of all rats were observed. The liver tissues of rats were obtained 2 weeks after transplantation, and the pathohistological changes were observed and the pathohistological scores were detected; the apoptotic rate of liver cells was detected by TUNEL method; the engraftment of BMSCs in liver tissues of the rats was observed under laser scanning confocal microscope. Results: 2 weeks after transplantation, the rats in model group were obviously malaise dispirited and the rats in cell therapy group showed obviously better, and the body weight of the rats in cell therapy group was higher than that in model group (P<0.05). The pathohistological results showed the normal liver lobules of the rats in model group disappeared, and the liver cords disordered, and some liver sinusoids dilated and congested, lymphocytes infiltrated with occasional focal aggregating, and cell edema was found, cytoplasm loose and steatosis were seen in liver tissue. However, the pathohistological changes of liver tissue of the rats in cell therapy group were significantly better than those in model group. The pathohistological score of the rats in cell therapy group was significantly lower than that in model group (P<0.05). The TUNEL staining results showed that there were lots of apoptotic liver cells in liver tissue of the rats in

  5. Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation.

    Science.gov (United States)

    Grandvalet, Cosette; Assad-García, Juan Simón; Chu-Ky, Son; Tollot, Marie; Guzzo, Jean; Gresti, Joseph; Tourdot-Maréchal, Raphaëlle

    2008-09-01

    Cyclopropane fatty acid (CFA) synthesis was investigated in Oenococcus oeni. The data obtained demonstrated that acid-grown cells or cells harvested in the stationary growth phase showed changes in fatty acid composition similar to those of ethanol-grown cells. An increase of the CFA content and a decrease of the oleic acid content were observed. The biosynthesis of CFAs from unsaturated fatty acid phospholipids is catalysed by CFA synthases. Quantitative real-time-PCR experiments were performed on the cfa gene of O. oeni, which encodes a putative CFA synthase. The level of cfa transcripts increased when cells were harvested in stationary phase and when cells were grown in the presence of ethanol or at low pH, suggesting transcriptional regulation of the cfa gene under different stress conditions. In contrast to Escherichia coli, only one functional promoter was identified upstream of the cfa gene of O. oeni. The function of the cfa gene was confirmed by complementation of a cfa-deficient E. coli strain. Nevertheless, the complementation remained partial because the conversion percentage of unsaturated fatty acids into CFA of the complemented strain was much lower than that of the wild-type strain. Moreover, a prevalence of cycC19 : 0 was observed in the membrane of the complemented strain. This could be due to a specific affinity of the CFA synthase from O. oeni. In spite of this partial complementation, the complemented strain of E. coli totally recovered its viability after ethanol shock (10 %, v/v) whereas its viability was only partly recovered after an acid shock at pH 3.0.

  6. Methylation of Septin9 mediated by DNMT3a enhances hepatic stellate cells activation and liver fibrogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yuting, E-mail: wuyuting1302@sina.com; Bu, Fangtian; Yu, Haixia; Li, Wanxia; Huang, Cheng; Meng, Xiaoming; Zhang, Lei; Ma, Taotao; Li, Jun, E-mail: lj@ahmu.edu.cn

    2017-01-15

    Liver fibrosis, resulting from chronic and persistent injury to the liver, is a worldwide health problem. Advanced liver fibrosis results in cirrhosis, liver failure and even hepatocellular cancer (HCC), often eventually requiring liver transplantation, poses a huge health burden on the global community. However, the specific pathogenesis of liver fibrosis remains not fully understood. Numerous basic and clinical studies have provided evidence that epigenetic modifications, especially DNA methylation, might contribute to the activation of hepatic stellate cells (HSCs), the pivotal cell type responsible for the fibrous scar in liver. Here, reduced representation bisulfite sequencing (RRBS) and bisulfite pyrosequencing PCR (BSP) analysis identified hypermethylation status of Septin9 (Sept9) gene in liver fibrogenesis. Sept9 protein was dramatically decreased in livers of CCl4-treated mice and immortalized HSC-T6 cells exposed to TGF-β1. Nevertheless, the suppression of Sept9 could be blocked by DNMT3a-siRNA and DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-azadC). Overexpressed Sept9 attenuated TGF-β1-induced expression of myofibroblast markers α-SMA and Col1a1, accompanied by up-regulation of cell apoptosis-related proteins. Conversely, RNAi-mediated silencing of Sept9 enhanced accumulation of extracellular matrix. These observations suggested that Sept9 contributed to alleviate liver fibrosis might partially through promoting activated HSCs apoptosis and this anti-fibrogenesis effect might be blocked by DNMT-3a mediated methylation of Sept9. Therefore, pharmacological agents that inhibit Sept9 methylation and increase its expression could be considered as valuable treatments for liver fibrosis. - Highlights: • This is the first report of Sept9 methylation and function in liver fibrosis. • Ectopic expression of Sept9 could block the liver fibrogenesis. • DNMT3a might be responsible for the suppression of Sept9 in liver fibrosis.

  7. Methylation of Septin9 mediated by DNMT3a enhances hepatic stellate cells activation and liver fibrogenesis

    International Nuclear Information System (INIS)

    Wu, Yuting; Bu, Fangtian; Yu, Haixia; Li, Wanxia; Huang, Cheng; Meng, Xiaoming; Zhang, Lei; Ma, Taotao; Li, Jun

    2017-01-01

    Liver fibrosis, resulting from chronic and persistent injury to the liver, is a worldwide health problem. Advanced liver fibrosis results in cirrhosis, liver failure and even hepatocellular cancer (HCC), often eventually requiring liver transplantation, poses a huge health burden on the global community. However, the specific pathogenesis of liver fibrosis remains not fully understood. Numerous basic and clinical studies have provided evidence that epigenetic modifications, especially DNA methylation, might contribute to the activation of hepatic stellate cells (HSCs), the pivotal cell type responsible for the fibrous scar in liver. Here, reduced representation bisulfite sequencing (RRBS) and bisulfite pyrosequencing PCR (BSP) analysis identified hypermethylation status of Septin9 (Sept9) gene in liver fibrogenesis. Sept9 protein was dramatically decreased in livers of CCl4-treated mice and immortalized HSC-T6 cells exposed to TGF-β1. Nevertheless, the suppression of Sept9 could be blocked by DNMT3a-siRNA and DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-azadC). Overexpressed Sept9 attenuated TGF-β1-induced expression of myofibroblast markers α-SMA and Col1a1, accompanied by up-regulation of cell apoptosis-related proteins. Conversely, RNAi-mediated silencing of Sept9 enhanced accumulation of extracellular matrix. These observations suggested that Sept9 contributed to alleviate liver fibrosis might partially through promoting activated HSCs apoptosis and this anti-fibrogenesis effect might be blocked by DNMT-3a mediated methylation of Sept9. Therefore, pharmacological agents that inhibit Sept9 methylation and increase its expression could be considered as valuable treatments for liver fibrosis. - Highlights: • This is the first report of Sept9 methylation and function in liver fibrosis. • Ectopic expression of Sept9 could block the liver fibrogenesis. • DNMT3a might be responsible for the suppression of Sept9 in liver fibrosis.

  8. Advanced glycation end products promote ChREBP expression and cell proliferation in liver cancer cells by increasing reactive oxygen species.

    Science.gov (United States)

    Chen, Hanbei; Li, Yakui; Zhu, Yemin; Wu, Lifang; Meng, Jian; Lin, Ning; Yang, Dianqiang; Li, Minle; Ding, WenJin; Tong, Xuemei; Su, Qing

    2017-08-01

    The aim of the study was to elucidate the mechanism by which advanced glycation end products (AGEs) promote cell proliferation in liver cancer cells.We treated liver cancer HepG2 cells with 200 mg/L AGEs or bovine serum albumin (BSA) and assayed for cell viability, cell cycle, and apoptosis. We performed real-time PCR and Western blot analysis for RNA and protein levels of carbohydrate responsive element-binding protein (ChREBP) in AGEs- or BSA-treated HepG2 cells. We analyzed the level of reactive oxygen species (ROS) in HepG2 cells treated with AGEs or BSA.We found that increased S-phase cell percentage and decreased apoptosis contributed to AGEs-induced liver cancer cell proliferation. Real-time PCR and Western blot analysis showed that AGEs stimulated RNA and protein levels of ChREBP, a transcription factor promoting glycolysis and maintaining cell proliferation in liver cancer cells. Intriguingly, the level of ROS was higher in AGEs-treated liver cancer cells. Treating liver cancer cells with antioxidant N-acetyl cystein (NAC) partly blocked AGEs-induced ChREBP expression and cell proliferation.Our results suggest that the AGEs-ROS-ChREBP pathway plays a critical role in promoting ChREBP expression and liver cancer cell proliferation.

  9. Potential genotoxic and cytotoxicity of emamectin benzoate in human normal liver cells.

    Science.gov (United States)

    Zhang, Zhijie; Zhao, Xinyu; Qin, Xiaosong

    2017-10-10

    Pesticide residue inducing cancer-related health problems draw people more attention recently. Emamectin benzoate (EMB) has been widely used in agriculture around the world based on its specificity targets. Although potential risk and the molecular mechanism of EMB toxicity to human liver has not been well-characterized. Unlike well-reported toxicity upon central nervous system, potential genotoxic and cytotoxicity of EMB in human liver cell was ignored and very limited. In this study, we identify genotoxicity and cytotoxicity of EMB to human normal liver cells (QSG7701 cell line) in vitro . We demonstrate that EMB inhibited the viability of QSG7701 cells and induced the DNA damage. Established assays of cytotoxicity were performed to characterize the mechanism of EMB toxicity on QSG7701 cells. Typical chromatin condensation and DNA fragmentation indicated the apoptosis of QSG7701 cells induced by EMB. And the intracellular biochemical results demonstrated that EMB-enhanced apoptosis of QSG7701 cells concurrent with generated ROS, a loss of mitochondrial membrane potential, the cytochrome-c release, up regulate the Bax/Bcl-2 and the activation of caspase-9/-3. Our results of EMB induces the death of QSG7701 cells maybe via mitochondrial-mediated intrinsic apoptotic pathways would contribute to promote the awareness of EMB as an extensive used pesticide to human being effects and reveal the underlying mechanisms of potential genotoxic.

  10. Differential genomic effects of six different TiO2 nanomaterials on human liver HepG2 cells

    Science.gov (United States)

    Engineered nanoparticles are reported to cause liver toxicity in vivo. To better assess the mechanism of the in vivo liver toxicity, we used the human hepatocarcinoma cells (HepG2) as a model system. Human HepG2 cells were exposed to 6 TiO2 nanomaterials (with dry primary partic...

  11. Development of intraepithelial T lymphocytes in the intestine of irradiated SCID mice by adult liver hematopoietic stem cells from normal mice

    International Nuclear Information System (INIS)

    Yamagiwa, Satoshi; Seki, Shuhji; Shirai, Katsuaki; Yoshida, Yuhei; Miyaji, Chikako; Watanabe, Hisami; Abo, Toru

    1999-01-01

    Background/Aims: We recently reported the adult mouse liver to contain c-kit + stem cells that can give rise to multilineage leukocytes. This study was designed to determine whether or not adult mouse liver stem cells can generate intraepithelial T cells in the intestine as well as to examine the possibility that adult liver c-kit + stem cells originate from the fetal liver. Methods: Adult liver mononuclear cells, bone marrow (BM) cells, liver c-kit + cells or bone BM c-kit + cells of BALB/c mice were i.v. transferred into 4 Gy irradiated CB17/-SCID mice. In other experiments, fetal liver cells from Ly5.1 C57BL/6 mice and T cell depleted adult BM cells from Ly5.2 C57BL/6 mice were simultaneously transferred into irradiated C57BL/6 SCID mice (Ly5.2). At 1 to 8 weeks after cell transfer, the SCID mice were examined. Results: Not only BM cells and BM c-kit + cells but also liver mononuclear cells and liver c-kit + cells reconstituted γδT cells, CD4 + CD8 + double-positive T cells and CDiα + β - T cells of intestinal intraepithelial lymphocytes of SCID mice. Injection of a mixture of fetal liver cells from Ly5.1 C57BL/6 mice and adult BM cells from Ly5.2 C57BL/6 mice into Ly5.2 C57BL/6 SCID mice induced both Ly5.1 and Ly5.2 T cells, while also generating c-kit + cells of both Ly5.1 and Ly5.2 origins in the liver. Conclusions: Adult mouse liver stem cells were able to generate intestinal intraepithelial T cells of the SCID mice, and it is thus suggested that some adult liver stem cells may indeed be derived from the fetal liver. (au)

  12. Kluyveromyces marxianus as a host for heterologous protein synthesis.

    Science.gov (United States)

    Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

    2016-07-01

    The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

  13. Liver Immunology

    Science.gov (United States)

    Bogdanos, Dimitrios P.; Gao, Bin; Gershwin, M. Eric

    2014-01-01

    The liver is the largest organ in the body and is generally regarded by non-immunologists as not having lymphoid function. However, such is far from accurate. This review highlights the importance of the liver as a lymphoid organ. Firstly, we discuss experimental data surrounding the role of liver as a lymphoid organ. The liver facilitates a tolerance rather than immunoreactivity, which protects the host from antigenic overload of dietary components and drugs derived from the gut and is also instrumental to fetal immune tolerance. Loss of liver tolerance leads to autoaggressive phenomena which if are not controlled by regulatory lymphoid populations may lead to the induction of autoimmune liver diseases. Liver-related lymphoid subpopulations also act as critical antigen-presenting cells. The study of the immunological properties of liver and delineation of the microenvironment of the intrahepatic milieu in normal and diseased livers provides a platform to understand the hierarchy of a series of detrimental events which lead to immune-mediated destruction of the liver and the rejection of liver allografts. The majority of emphasis within this review will be on the normal mononuclear cell composition of the liver. However, within this context, we will discus select, but not all, immune mediated liver disease and attempt to place these data in the context of human autoimmunity. PMID:23720323

  14. Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids.

    Directory of Open Access Journals (Sweden)

    Cyrill Géraud

    Full Text Available Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ, i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.

  15. Clonogenic cell line survival of a human liver cancer cell line SMMC-7721 after carbon ion irradiation with different LET

    International Nuclear Information System (INIS)

    Lei Suwen; Su Xu; Wang Jifang; Li Wenjian

    2003-01-01

    Objective: To investigate the survival fraction of a human liver cancer cell line SMMC-7721 following irradiation with carbon ions with different LET. Methods: cells of the human liver cancer cell line SMMC-7721 were irradiated with carbon ions (LET=30 and 70 keV/μm). The survival fraction was determined with clonogenic assay after 9 days incubation in a 5% CO 2 incubator at 37 degree C. Results: When the survival fractions of 70 keV/μm were D s = 0.1 and D s=0.01 absorption dose were 2.94 and 5.88 Gy respectively, and those of 30 keV/μm were 4.00 and 8.00 Gy respectively. Conclusion: For the SMMC-7721 cell line, 70 keV/μm is more effective for cell killing than 30 keV/μm

  16. Cell-specific delivery of biologicals : Problems, pitfalls and possibilities of antifibrotic compounds in the liver

    NARCIS (Netherlands)

    Poelstra, Klaas; Beljaars, Leonie; Melgert, Barbro N

    2013-01-01

    Liver fibrosis is a complex disease affecting millions of people world-wide. It involves the activation of several cell types whose activities are tightly controlled by endogenous mediators. No pharmacotherapy is available for this disease, despite the fact that many experimental drugs are very

  17. Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging

    NARCIS (Netherlands)

    Pfeifer, A.; Kessler, T.; Yang, M.; Baranov, E.; Kootstra, N.; Cheresh, D. A.; Hoffman, R. M.; Verma, I. M.

    2001-01-01

    Viral vectors based on lentiviruses, such as the human immunodeficiency virus, are able to transduce a broad spectrum of nondividing cells in vivo. This ability of lentiviral vectors makes them an attractive vehicle for gene transfer into the liver. In order to determine the requirements for

  18. Effects of gamma-irradiation on the glycogen and lipid contents of the rat liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Nahed, R H.A.; Al-Zahaby, Al-Ahmmady, S.; Sanad, S M.K.; Roushdy, H M

    1986-01-01

    Histochemical changes in the glycogen and lipid contents of the rat liver cells were studied at different intervals following whole body gamma-irradiation at the exposure dose level of 600 rads. The glycogen and lipid contents were significantly altered, the changes were time-dependent.

  19. Short-chain flavor ester synthesis in organic media by an E. coli whole-cell biocatalyst expressing a newly characterized heterologous lipase.

    Directory of Open Access Journals (Sweden)

    Guillaume Brault

    Full Text Available Short-chain aliphatic esters are small volatile molecules that produce fruity and pleasant aromas and flavors. Most of these esters are artificially produced or extracted from natural sources at high cost. It is, however, possible to 'naturally' produce these molecules using biocatalysts such as lipases and esterases. A gene coding for a newly uncovered lipase was isolated from a previous metagenomic study and cloned into E. coli BL21 (DE3 for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media. The recombinant lipase (LipIAF5-2 showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. Using a simple cetyl-trimethylammonium bromide pretreatment increased the synthetic activity by a six-fold factor and the whole-cell biocatalyst showed the highest activity at 40°C with a relatively high water content of 10% (w/w. The whole-cell biocatalyst showed excellent tolerance to alcohol and short-chain fatty acid denaturation. Substrate affinity was equally effective with all primary alcohols tested as acyl acceptors, with a slight preference for methanol. The best transesterification conversion of 50 mmol glyceryl triacetate into isoamyl acetate (banana fragrance provided near 100% yield after 24 hours using 10% biocatalyst loading (w/w in a fluidized bed reactor, allowing recycling of the biocatalyst up to five times. These results show promising potential for an industrial approach aimed at the biosynthesis of short-chain esters, namely for natural flavor and fragrance production in micro-aqueous media.

  20. Nuclear receptor CAR (NR1I3) is essential for DDC-induced liver injury and oval cell proliferation in mouse liver.

    Science.gov (United States)

    Yamazaki, Yuichi; Moore, Rick; Negishi, Masahiko

    2011-11-01

    The liver is endowed with the ability to regenerate hepatocytes in response to injury. When this regeneration ability is impaired during liver injury, oval cells, which are considered to be postnatal hepatic progenitors, proliferate and differentiate into hepatocytes. Here we have demonstrated that 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) activates the nuclear receptor constitutive active/androstane receptor (CAR), resulting in proliferation of oval cells in mouse liver. Activation of CAR by DDC was shown by hepatic nuclear CAR accumulation and cytochrome P450 (CYP)2B10 mRNA induction after feeding a 0.1% DDC-containing diet to Car(+/+) mice. After being fed the DDC diet, Car(+/+), but not Car(-/-) mice, developed severe liver injury and an A6 antibody-stained ductular reaction in an area around the portal tract. Oval cell proliferation was confirmed by laser capture microdissection and real-time PCR; mRNAs for the two oval cell markers epithelial cell adhesion molecule and TROP2 were specifically induced in the periportal region of DDC diet-fed Car(+/+), but not Car(-/-) mice. Although rates of both hepatocyte growth and death were initially enhanced only in DDC diet-fed Car(+/+) mice, growth was attenuated when oval cells proliferated, whereas death continued unabated. DDC-induced liver injury, which differs from other CAR activators such as phenobarbital, occurred in the periportal region where cells developed hypertrophy, accumulated porphyrin crystals and inflammation developed, all in association with the proliferation of oval cells. Thus, CAR provides an excellent experimental model for further investigations into its roles in liver regeneration, as well as the development of diseases such as hepatocellular carcinoma.

  1. Drug-Induced Liver Injury: Cascade of Events Leading to Cell Death, Apoptosis or Necrosis

    Directory of Open Access Journals (Sweden)

    Andrea Iorga

    2017-05-01

    Full Text Available Drug-induced liver injury (DILI can broadly be divided into predictable and dose dependent such as acetaminophen (APAP and unpredictable or idiosyncratic DILI (IDILI. Liver injury from drug hepatotoxicity (whether idiosyncratic or predictable results in hepatocyte cell death and inflammation. The cascade of events leading to DILI and the cell death subroutine (apoptosis or necrosis of the cell depend largely on the culprit drug. Direct toxins to hepatocytes likely induce oxidative organelle stress (such as endoplasmic reticulum (ER and mitochondrial stress leading to necrosis or apoptosis, while cell death in idiosyncratic DILI (IDILI is usually the result of engagement of the innate and adaptive immune system (likely apoptotic, involving death receptors (DR. Here, we review the hepatocyte cell death pathways both in direct hepatotoxicity such as in APAP DILI as well as in IDILI. We examine the known signaling pathways in APAP toxicity, a model of necrotic liver cell death. We also explore what is known about the genetic basis of IDILI and the molecular pathways leading to immune activation and how these events can trigger hepatotoxicity and cell death.

  2. Impact of recombinant adenovirus serotype 35 priming versus boosting of a Plasmodium falciparum protein: Characterization of T- and B-Cell responses to liver-stage antigen 1

    NARCIS (Netherlands)

    Rodriguez, Ariane; Goudsmit, Jaap; Companjen, Arjen; Mintardjo, Ratna; Gillissen, Gert; Tax, Dennis; Sijtsma, Jeroen; Weverling, Gerrit Jan; Holterman, Lennart; Lanar, David E.; Havenga, Menzo J. E.; Radosevic, Katarina

    2008-01-01

    Prime-boost vaccination regimens with heterologous antigen delivery systems have indicated that redirection of the immune response is feasible. We showed earlier that T-cell responses to circumsporozoite (CS) protein improved significantly when the protein is primed with recombinant adenovirus

  3. Study on bone marrow mesenchymal stem cells in repairing of radiation induced acute liver injury of rats

    International Nuclear Information System (INIS)

    Bao Yongxing; Lou Fan; Zhao Huarong; Zhu Huhu; Ma Yan; Wen Hao

    2010-01-01

    Objective: To investigate the role of mesenchymal stem cells in the repair of radiation induced liver injury. Methods: 12 female SD rats were irradiated with 20 Gy 6 MV X-rays on the right lobe of the liver, to establish the model of radiation induced liver injury. The rats were divided randomly into two groups as invention group and control group, and transplanted with 1 ml male mesenchymal suspension or 1 ml normal saline in 4 hours after radiotherapy. The morphological changes of liver were observed. The existence of sex determining gene Y(SRY) and the level of alpha-smooth muscle actin (a-SMA) were detected. Results: Some injury of right lobe liver in two groups were observed, and the injury degree of right lobe liver in intervention group were lower than that of control group. The amount of SRY positive cells in the right lobe liver of intervention group was higher than that in the left lobe liver (t = 3.77, P <0.05). The positive expression rate of a-SMA in right lobe liver of intervention group was lower than that of control group. Conclusions: Acute radiation induced liver injury could lead BMSCs' homing in order to decrease the degree of liver fibrosis. (authors)

  4. Administration of multipotent mesenchymal stromal cells restores liver regeneration and improves liver function in obese mice with hepatic steatosis after partial hepatectomy.

    Science.gov (United States)

    Ezquer, Fernando; Bahamonde, Javiera; Huang, Ya-Lin; Ezquer, Marcelo

    2017-01-28

    The liver has the remarkable capacity to regenerate in order to compensate for lost or damaged hepatic tissue. However, pre-existing pathological abnormalities, such as hepatic steatosis (HS), inhibits the endogenous regenerative process, becoming an obstacle for liver surgery and living donor transplantation. Recent evidence indicates that multipotent mesenchymal stromal cells (MSCs) administration can improve hepatic function and increase the potential for liver regeneration in patients with liver damage. Since HS is the most common form of chronic hepatic illness, in this study we evaluated the role of MSCs in liver regeneration in an animal model of severe HS with impaired liver regeneration. C57BL/6 mice were fed with a regular diet (normal mice) or with a high-fat diet (obese mice) to induce HS. After 30 weeks of diet exposure, 70% hepatectomy (Hpx) was performed and normal and obese mice were divided into two groups that received 5 × 10 5 MSCs or vehicle via the tail vein immediately after Hpx. We confirmed a significant inhibition of hepatic regeneration when liver steatosis was present, while the hepatic regenerative response was promoted by infusion of MSCs. Specifically, MSC administration improved the hepatocyte proliferative response, PCNA-labeling index, DNA synthesis, liver function, and also reduced the number of apoptotic hepatocytes. These effects may be associated to the paracrine secretion of trophic factors by MSCs and the hepatic upregulation of key cytokines and growth factors relevant for cell proliferation, which ultimately improves the survival rate of the mice. MSCs represent a promising therapeutic strategy to improve liver regeneration in patients with HS as well as for increasing the number of donor organs available for transplantation.

  5. In Vitro and In Vivo Hepatic Differentiation of Adult Somatic Stem Cells and Extraembryonic Stem Cells for Treating End Stage Liver Diseases

    Directory of Open Access Journals (Sweden)

    Chenxia Hu

    2015-01-01

    Full Text Available The shortage of liver donors is a major handicap that prevents most patients from receiving liver transplantation and places them on a waiting list for donated liver tissue. Then, primary hepatocyte transplantation and bioartificial livers have emerged as two alternative treatments for these often fatal diseases. However, another problem has emerged. Functional hepatocytes for liver regeneration are in short supply, and they will dedifferentiate immediately in vitro after they are isolated from liver tissue. Alternative stem-cell-based therapeutic strategies, including hepatic stem cells (HSCs, embryonic stem cells (ESCs, induced pluripotent stem cells (iPSCs, and mesenchymal stem cells (MSCs, are more promising, and more attention has been devoted to these approaches because of the high potency and proliferation ability of the cells. This review will focus on the general characteristics and the progress in hepatic differentiation of adult somatic stem cells and extraembryonic stem cells in vitro and in vivo for the treatment of end stage liver diseases. The hepatic differentiation of stem cells would offer an ideal and promising source for cell therapy and tissue engineering for treating liver diseases.

  6. Thinking outside the liver: induced pluripotent stem cells for hepatic applications.

    Science.gov (United States)

    Subba Rao, Mekala; Sasikala, Mitnala; Nageshwar Reddy, D

    2013-06-14

    The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications.

  7. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Roskams, Tania [Department of Morphology and Molecular Pathology, University of Leuven (Belgium); Oben, Jude A., E-mail: j.oben@ucl.ac.uk [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Department of Gastroenterology and Hepatology, Guy' s and St Thomas' Hospital, London SE1 7EH (United Kingdom)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  8. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    International Nuclear Information System (INIS)

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

    2012-01-01

    Highlights: ► Cigarette smoke may induce liver fibrosis via nicotine receptors. ► Nicotine induces proliferation of hepatic stellate cells (HSCs). ► Nicotine activates hepatic fibrogenic pathways. ► Nicotine receptor antagonists attenuate HSC proliferation. ► Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine – which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed – RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-α2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-β1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type (α1, β1, delta and epsilon) and neuronal type (α3, α6, α7, β2 and β4) nAChR subunits at the mRNA level. Among these subunits, α3, α7, β1 and ε were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-α2 and TGF-β1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. α1 and α3-nAChR mRNA expression was significantly upregulated in NASH fibrosis compared to normal livers. Conclusion: Nicotine at levels in smokers’ blood is pro-fibrogenic, through

  9. Analysis of the apoptosis of brain and liver cells induced by ionizing radiation

    International Nuclear Information System (INIS)

    Konoplya, E.F.; Litvinchuk, A.V.; Zajtseva, O.A.; Stashkevich, D.G.

    2010-01-01

    The PCR-analysis of the expression of two oncogenes (bax/bcl-2) in the liver and brain tissues of the animals after being exposed to gamma-rays at doses of 1,0 and 4,0 Gy at remote terms after the action of (30, 60, 90 days) was made. The proofs of start-up of the genetic program of apoptosis of brain and liver cells were obtained. This can be a reason for functional dysfunction of a particular organ and the whole organism. (authors)

  10. Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.

    Directory of Open Access Journals (Sweden)

    Anne Frentzen

    2011-04-01

    Full Text Available Hepatitis C virus (HCV is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

  11. Transcriptome atlas of eight liver cell types uncovers effects of ...

    Indian Academy of Sciences (India)

    2010-12-06

    Dec 6, 2010 ... by autocrine or paracrine systems can reduce antigen present- ing capacity of immune cells, ... polypeptide (GNAQ), glycosylphosphatidylinositol specific phosphorlipase C ...... profiling of prostate cancer. BMC Mol. Biol. 8, 25.

  12. Computed tomography of the spleen and liver in sickle cell disease

    International Nuclear Information System (INIS)

    Magid, D.; Fishman, E.K.; Siegelman, S.S.

    1984-01-01

    The spleen was assessed in 10 patients with sickle cell disease studied with computed tomography (CT) for abdominal pain and/or unexplained fever. Patients with homozygous sickle cell anemia were found to have small, densely calcified spleens with occasional low-density infarcts. Five of six had hepatomegaly, and there was one case each of hepatic abscess, infarcts, and hemochromatosis. All patients with heterozygous sickle cell disease were found to have splenomegaly, with a variety of findings including acute hemorrhage, acute and chronic infarcts, rupture, and possible sequestration. It was concluded that CT is useful for evaluating the status of the spleen and liver in symptomatic patients with sickle cell disease

  13. Progenitor cells in liver regeneration: molecular responses controlling their activation and expansion

    DEFF Research Database (Denmark)

    Santoni-Rugiu, Eric; Jelnes, Peter; Thorgeirsson, Snorri S

    2005-01-01

    created in the liver by a certain insult. This review will focus on molecular responses controlling activation and expansion of the hepatic progenitor cell niche, emphasizing similarities and differences in the microenvironments orchestrating regeneration by recruitment of progenitor cell populations...... cells, and recruited inflammatory cells as well as the variety of growth-modulating molecules produced and/or harboured by these elements. The cellular and molecular responses to different regenerative stimuli seem to depend on the injury inflicted and consequently on the molecular microenvironment...

  14. Manipulation of the Host Cell Membrane during Plasmodium Liver Stage Egress

    Directory of Open Access Journals (Sweden)

    Paul-Christian Burda

    2017-04-01

    Full Text Available A crucial step in the life cycle of Plasmodium parasites is the transition from the liver stage to the blood stage. Hepatocyte-derived merozoites reach the blood vessels of the liver inside host cell-derived vesicles called merosomes. The molecular basis of merosome formation is only partially understood. Here we show that Plasmodium berghei liver stage merozoites, upon rupture of the parasitophorous vacuole membrane, destabilize the host cell membrane (HCM and induce separation of the host cell actin cytoskeleton from the HCM. At the same time, the phospholipid and protein composition of the HCM appears to be substantially altered. This includes the loss of a phosphatidylinositol 4,5-bisphosphate (PIP2 reporter and the PIP2-dependent actin-plasma membrane linker ezrin from the HCM. Furthermore, transmembrane domain-containing proteins and palmitoylated and myristoylated proteins, as well as glycosylphosphatidylinositol-anchored proteins, lose their HCM localization. Collectively, these findings provide an explanation of HCM destabilization during Plasmodium liver stage egress and thereby contribute to our understanding of the molecular mechanisms that lead to merosome formation.

  15. Autoserum: An Optimal Supplement for Bone Marrow Mesenchymal Stem Cells of Liver-Injured Rats

    Directory of Open Access Journals (Sweden)

    Qinglin Zhang

    2015-01-01

    Full Text Available Mesenchymal stem cells (MSCs are an attractive source for the clinical cell therapy of liver injury. Although the use of adult serum, platelet lysate, or cord blood serum solves some of the problems caused by fetal bovine serum (FBS, the allogeneic immune response, contamination, and donor-to-donor and donor-to-receptor differences still obstruct the application of MSCs. In this study, the influences of autoserum from liver-injured rats (LIRs and allogeneic serum from healthy rats on the isolation and culture of bone marrow MSCs (BMSCs were examined and compared to FBS. The results showed that BMSCs cultured with autoserum or allogeneic serum exhibited better MSC-specific morphology, lower rate of cell senescent, and higher proliferation kinetics than those with FBS. In addition, autoserum promoted the osteogenic differentiation potential of BMSCs as allogeneic serum did. Although there were no significant differences in proliferation activity, immunophenotypic characterization, and differentiation potential between BMSCs cultured with autoserum and those with allogeneic serum, the potential adverse immunological reactions in patients with allogeneic material transplantation must be considered. We therefore believe that the autoserum from liver-injured patients may be a better choice for MSC expansion to meet the needs of liver injury therapy.

  16. Comparison Of Liver Cell Models Using The Basel Phenotyping Cocktail

    Directory of Open Access Journals (Sweden)

    Benjamin Berger

    2016-11-01

    Full Text Available Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH cultures. We investigated the suit-ability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4] in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6 and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least 4-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20µM rifampicin, mRNA increased 3 to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6 and 17-fold (CYP3A4 for 2D-PHH and 4-fold (CYP3A4 for HepG2. In 3D-PHH at least a 2-fold in-crease in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model.Our studies show that 3D-PHH and (with some limitations HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro. HepG2 cells are less suited to as-sess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro.

  17. Th-17 cells infiltrate the liver in human biliary atresia and are related to surgical outcome.

    Science.gov (United States)

    Hill, Richard; Quaglia, Alberto; Hussain, Munther; Hadzic, Nedim; Mieli-Vergani, Giorgina; Vergani, Diego; Davenport, Mark

    2015-08-01

    Biliary atresia (BA), a cholangiopathy of unknown etiology is associated with intrahepatic mononuclear cell infiltrate. An abnormal reaction to viral exposure has been hypothesized in some cases. We aimed to investigate the nature of the CD4+ hepatic infiltrate in defined clinical variants of BA by quantification of inflammatory cell components. Liver biopsies of infants obtained at Kasai portoenterostomy (KPE) were stained immunohistochemically using monoclonal antibodies to Tbet, GATA-3, FOXP3 and interleukin (IL) 17, identifying Th-1, Th-2, Tregs and Th-17 cells respectively. T cells were counted with the aid of a graticule. Data are reported as median (range) of cells per high-power-field (×400) and compared using nonparametric statistical tests with P≤0.05 regarded as significant. Liver biopsies from BA (n=37) and age-matched cholestatic controls (e.g. alpha-1-anti trypsin deficiency, Alagilles syndrome, n=12) were investigated. BA infants were divided into three groups: cytomegalovirus IgM +ve (CMV; n=9); BA splenic malformation (BASM; n=9) and isolated BA (IBA; n=19). All T-cell subsets were present in the portal tracts, with an overrepresentation of Th-1 (PTh-17 (PTh-2 (P=0.94) or Tregs (P=0.15), compared to controls. Th-1 cells predominated in the CMV group; (18 [7-37] vs. 3 [0-14] [BASM] and vs. 5 [3-23] [IBA]; PTh-17 cells. The degree of Th-1 cell infiltrate inversely correlated with platelet count (rS=-0.49; PTh-17 cells were fewer (6 [2-11] vs. 11 [8-20]; P=0.02) in infants who cleared their jaundice (n=15, Th-17 cells infiltrate the liver in BA and are associated with a worse surgical outcome; a Th-1 profile predominates in CMV-associated BA. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Cloning and heterologous expression of a hydrophobin gene Ltr.hyd from the tiger milk mushroom Lentinus tuber-regium in yeast-like cells of Tremella fuciformis

    Directory of Open Access Journals (Sweden)

    Dongmei Liu

    2018-03-01

    Full Text Available Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier. Keywords: Agrobacterium tumefaciens, Emulsifier, Expression vector, Filamentous fungi, Gel electrophoresis, Glyceraldehyde-3-phosphate dehydrogenase, Heterogenous expression, Hydrophobin, Quantitative real-time PCR, Southern blot, Surface activity

  19. Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines

    OpenAIRE

    Gasnier, C?line; Benachour, Nora; Clair, Emilie; Travert, Carine; Langlois, Fr?d?ric; Laurant, Claire; Decroix-Laporte, C?cile; S?ralini, Gilles-Eric

    2010-01-01

    Abstract Background Worldwide used pesticides containing different adjuvants like Roundup formulations, which are glyphosate-based herbicides, can provoke some in vivo toxicity and in human cells. These pesticides are commonly found in the environment, surface waters and as food residues of Roundup tolerant genetically modified plants. In order to know their effects on cells from liver, a major detoxification organ, we have studied their mechanism of action and possible protection by precise ...

  20. Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

    Science.gov (United States)

    Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

    2018-03-05

    Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  1. Substance P increases liver fibrosis by differential changes in senescence of cholangiocytes and hepatic stellate cells.

    Science.gov (United States)

    Wan, Ying; Meng, Fanyin; Wu, Nan; Zhou, Tianhao; Venter, Julie; Francis, Heather; Kennedy, Lindsey; Glaser, Trenton; Bernuzzi, Francesca; Invernizzi, Pietro; Glaser, Shannon; Huang, Qiaobing; Alpini, Gianfranco

    2017-08-01

    Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct-ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin-1 receptor (NK-1R). To identify whether SP regulates liver fibrosis during cholestasis, wild-type or NK-1R knockout (NK-1R -/- ) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2 -/- ) mice treated with either an NK-1R antagonist (L-733,060) or saline were used. Additionally, wild-type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK-1R in both BDL and Mdr2 -/- mice compared to wild-type mice. Expression of tachykinin precursor 1 and NK-1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK-1R decreased BDL-induced liver fibrosis, and treatment with L-733,060 resulted in decreased liver fibrosis in Mdr2 -/- mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor-β1 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK-1R -/- mice with BDL surgery or Mdr2 -/- mice treated with L-733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L-733,060 inhibited SP-induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L-733,060 partially reversed the SP-induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP-induced increase of senescence-related gene expression in cultured cholangiocytes. Collectively, our results demonstrate the regulatory effects of the SP/NK-1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017;66:528-541). © 2017 by the American

  2. Suppression of immune-mediated liver injury after vaccination with attenuated pathogenic cells.

    Science.gov (United States)

    Mei, Yunhua; Wang, Ying; Xu, Lingyun

    2007-05-15

    Cell vaccination via immunization with attenuated pathogenic cells is an effective preventive method that has been successfully applied in several animal models of inflammatory or autoimmune diseases. Concanavalin A (Con A)-induced hepatitis (CIH) is a commonly used experimental model to study immune-mediated liver injury. Multiple cell types including T lymphocytes, macrophages and neutrophils have been found to be involved in the pathogenesis of CIH. In this study, we used attenuated spleen lymphocytes or peripheral blood lymphocytes as vaccines to investigate whether they could induce protective immune responses to prevent mice from developing CIH. We found that mice receiving such vaccination before CIH induction developed much milder diseases, exhibited a lower level of alanine aminotransferase (ALT) released into their plasma and had less inflammatory lesions in their livers. Such CIH-suppression is dose- and frequency-dependent. The suppressive effect was associated with inhibition of several major inflammatory mediators, pro-inflammatory cytokines and chemokines.

  3. SET mediates TCE-induced liver cell apoptosis through dephosphorylation and upregulation of nucleolin.

    Science.gov (United States)

    Ren, Xiaohu; Huang, Xinfeng; Yang, Xifei; Liu, Yungang; Liu, Wei; Huang, Haiyan; Wu, Desheng; Zou, Fei; Liu, Jianjun

    2017-06-20

    Trichloroethylene (TCE) is an occupational and environmental chemical that can cause severe hepatotoxicity. While our previous studies showed that the phosphatase inhibitor SET is a key mediator of TCE-induced liver cell apoptosis, the molecular mechanisms remain elusive. Using quantitative phosphoproteomic analysis, we report here that nucleolin is a SET-regulated phosphoprotein in human liver HL-7702 cells. Functional analysis suggested that SET promoted dephosphorylation of nucleolin, decreased its binding to its transcriptional activator, c-myc, and upregulated nucleolin expression in TCE-treated cells. Importantly, TCE-induced hepatocyte apoptosis was significantly attenuated when nucleolin was downregulated with specific siRNAs. These findings indicate that TCE may induce hepatocyte apoptosis via SET-mediated dephosphorylation and overexpression of nucleolin.

  4. Aging-associated oxidative stress inhibits liver progenitor cell activation in mice.

    Science.gov (United States)

    Cheng, Yiji; Wang, Xue; Wang, Bei; Zhou, Hong; Dang, Shipeng; Shi, Yufang; Hao, Li; Luo, Qingquan; Jin, Min; Zhou, Qianjun; Zhang, Yanyun

    2017-04-29

    Recent studies have discovered aging-associated changes of adult stem cells in various tissues and organs, which potentially contribute to the organismal aging. However, aging-associated changes of liver progenitor cells (LPCs) remain elusive. Employing young (2-month-old) and old (24-month-old) mice, we found diverse novel alterations in LPC activation during aging. LPCs in young mice could be activated and proliferate upon liver injury, whereas the counterparts in old mice failed to respond and proliferate, leading to the impaired liver regeneration. Surprisingly, isolated LPCs from young and old mice did not exhibit significant difference in their clonogenic and proliferative capacity. Later, we uncovered that the decreased activation and proliferation of LPCs were due to excessive reactive oxygen species produced by neutrophils infiltrated into niche, which was resulted from chemokine production from activated hepatic stellate cells during aging. This study demonstrates aging-associated changes in LPC activation and reveals critical roles for the stem cell niche, including neutrophils and hepatic stellate cells, in the negative regulation of LPCs during aging.

  5. Effects of quantum dots on the ROS amount of liver cancer stem cells.

    Science.gov (United States)

    Li, Kunmeng; Xia, Chunhui; Wang, Baiqi; Chen, Hetao; Wang, Tong; He, Qian; Cao, Hailong; Wang, Yu

    2017-07-01

    Liver cancer (LC) is a serious disease that threatens human lives. LC has a high recurrence rate and poor prognosis. LC stem cells (LCSCs) play critical roles in these processes. However, the mechanism remains unclear. Reactive oxygen species (ROS) can be used to determine cell apoptosis and proliferation. However, studies of the effects of exogenous nanomaterials on LCSC ROS changes are rarely reported. In this work, quantum dots (QDs) were prepared using a hydrothermal method, and QDs were further modified with polyethylene glycol (PEG) and bovine serum albumin (BSA) using a chemical approach. The effects of QDs, PEG-modified QDs (PEG@QDs) and BSA-modified QDs (BSA@QDs) on the amounts of ROS in liver cancer PLC/PRF/5 (PLC) cells and liver cancer stem cells (LCSCs) were principally investigated. The results showed that when the concentration of QDs, PEG@QDs, and BSA@QDs were 10nM and 90nM, the ROS amount in PLC cells increased by approximately 2- to 5-fold. However, when the concentrations of these nanomaterials were 10nM and 90nM, ROS levels in LCSCs were reduced by approximately 50%. This critical path potentially leads to drug resistance and recurrence of LC. This work provides an important indication for further study of LC drug resistance and recurrence. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Human endometrial regenerative cells alleviate carbon tetrachloride-induced acute liver injury in mice

    Directory of Open Access Journals (Sweden)

    Shanzheng Lu

    2016-10-01

    Full Text Available Abstract Background The endometrial regenerative cell (ERC is a novel type of adult mesenchymal stem cell isolated from menstrual blood. Previous studies demonstrated that ERCs possess unique immunoregulatory properties in vitro and in vivo, as well as the ability to differentiate into functional hepatocyte-like cells. For these reasons, the present study was undertaken to explore the effects of ERCs on carbon tetrachloride (CCl4–induced acute liver injury (ALI. Methods An ALI model in C57BL/6 mice was induced by administration of intraperitoneal injection of CCl4. Transplanted ERCs were intravenously injected (1 million/mouse into mice 30 min after ALI induction. Liver function, pathological and immunohistological changes, cell tracking, immune cell populations and cytokine profiles were assessed 24 h after the CCl4 induction. Results ERC treatment effectively decreased the CCl4-induced elevation of serum alanine aminotransferase (ALT and aspartate aminotransferase (AST activities and improved hepatic histopathological abnormalities compared to the untreated ALI group. Immunohistochemical staining showed that over-expression of lymphocyte antigen 6 complex, locus G (Ly6G was markedly inhibited, whereas expression of proliferating cell nuclear antigen (PCNA was increased after ERC treatment. Furthermore, the frequency of CD4+ and CD8+ T cell populations in the spleen was significantly down-regulated, while the percentage of splenic CD4+CD25+FOXP3+ regulatory T cells (Tregs was obviously up-regulated after ERC treatment. Moreover, splenic dendritic cells in ERC-treated mice exhibited dramatically decreased MHC-II expression. Cell tracking studies showed that transplanted PKH26-labeled ERCs engrafted to lung, spleen and injured liver. Compared to untreated controls, mice treated with ERCs had lower levels of IL-1β, IL-6, and TNF-α but higher level of IL-10 in both serum and liver. Conclusions Human ERCs protect the liver from acute injury

  7. The effect of apple (Malus Domestica juice on the damage of mice liver cells due to paracetamol treatment

    Directory of Open Access Journals (Sweden)

    Anthony Hartanto

    2009-07-01

    Full Text Available The liver is an important organ for body metabolism process. Liver disease is one of serious health problems in developing countries including Indonesia. Liver damage is caused by viral infection, toxic agent exposure (medications, alcohol, hormonal disturbance, neoplasm and autoimmune diseases. The use of high dose paracetamol to reduce pain also leads to liver damage. Apple (Malus domestica juice is a natural anti oxidant agent. This laboratory experimental study was performed to discover the effect of giving apple juice on damaged cell regeneration due to the use of paracetamol. The study was performed in 21 male mice from Swiss-Webster strain that were divided into group I, II, and III. Group, I served as control while group II received 1 mg/ml paracetamol dose for 5 days and Group III received 1 mg/ml paracetamol for 5 days and 1 ml of apple juice on the 5th to 10th day. The observation of the mice liver cells was conducted using a light microscope with 400x magnification to get the number of necrotic liver cells per view field. The results of this study showed a difference in the number of necrotic liver cells between Group II and III. ANOVA statistical test ( = 0.05 concluded that apple juice significantly helps regeneration process in damaged liver cells caused by paracetamol.

  8. The interaction of bacterial magnetosomes and human liver cancer cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Pingping, E-mail: wangpp@mail.iee.ac.cn [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); Chen, Chuanfang [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); Chen, Changyou [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yue; Pan, Weidong; Song, Tao [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China)

    2017-04-01

    As the biogenic magnetic nanomaterial, bacterial magnetic nanoparticles, namely magnetosomes, provide many advantages for potential biomedical applications. As such, interactions among magnetosomes and target cells should be elucidated to develop their bioapplications and evaluate their biocompatibilities. In this study, the interaction of magnetosomes and human liver cancer HepG2 cells was examined. Prussian blue staining revealed numerous stained particles in or on the cells. Intracellular iron concentrations, measured through inductively coupled plasma optical emission spectroscopy, increased with the increasing concentration of the magnetosomes. Transmission electron microscopy images showed that magnetosomes could be internalized in cells, mainly encapsulated in membrane vesicles, such as endosomes and lysosomes, and partly found as free particles in the cytosol. Some of the magnetosomes on cellular surfaces were encapsulated through cell membrane ruffling, which is the initiating process of endocytosis. Applying low temperature treatment and using specific endocytic inhibitors, we validated that macropinocytosis and clathrin-mediated endocytosis were involved in magnetosome uptake by HepG2 cells. Consequently, we revealed the interaction and intrinsic endocytic mechanisms of magnetosomes and HepG2 cells. This study provides a basis for the further research on bacterial magnetosome applications in liver diseases. - Highlights: • Bacterial magnetosomes interact with HepG2 cells in a dose-dependent manner. • Magnetosomes are wrapped by membrane ruffling on cell surface. • Internalized magnetosomes mainly localize in endosomes and lysosomes. • Macropinocytosis and CME are involved in the cellular uptake of magnetosomes.

  9. Cell Spheroids with Enhanced Aggressiveness to Mimic Human Liver Cancer In Vitro and In Vivo.

    Science.gov (United States)

    Jung, Hong-Ryul; Kang, Hyun Mi; Ryu, Jea-Woon; Kim, Dae-Soo; Noh, Kyung Hee; Kim, Eun-Su; Lee, Ho-Joon; Chung, Kyung-Sook; Cho, Hyun-Soo; Kim, Nam-Soon; Im, Dong-Soo; Lim, Jung Hwa; Jung, Cho-Rok

    2017-09-05

    We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial-mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.

  10. Action of DTPA on hepatic plutonium. II. DTPA-induced removal of monomeric plutonium from mouse liver parenchymal cells

    International Nuclear Information System (INIS)

    Bhattacharyya, M.H.; Peterson, D.P.; Lindenbaum, A.

    1978-01-01

    Liver parenchymal cells were isolated 6 and 24 hr following the administration of diethylenetriaminepentaacetic acid (DTPA, 0.25 mmole/kg) to mice previously injected with 239 Pu-citrate (4.4 μCi/kg). Isolated parenchymal cells contained 440 dpm Pu/10 6 cells at 24 hr after Pu injection, just prior to DTPA administration. The PU content decreased to 330 dpm/10 6 cells at 6 hr and 140 dpm/10 6 cells at 24 hr after DTPA administration. Thus DTPA induced a striking decrease in the Pu content of isolated liver parenchymal cells. Parenchymal cells isolated from control mice not treated with DTPA changed little in Pu content from 24 to 48 hr after Pu injection. By 24 hr after DTPA treatment, the decrease in the Pu content of isolated liver parenchymal cells could account for the DTPA-induced release of Pu from the intact liver. Thus in the liver DTPA appears to act preferentially on the Pu associated with parenchymal cells. Liver parenchymal cells isolated 6 hr after DTPA administration and containing 330 dpm Pu/10 6 cells were incubated in vitro in the absence of added DTPA. After 18 hr of incubation the cells contained 130 dpm Pu/10 6 cells. This level corresponds to the level observed in cells isolated 24 hr after DTPA administration. Cells isolated from untreated mice lost only 15% of their Pu content during a similar in vitro incubation. Thus, by 6 hr after DTPA administration to the mouse, isolated liver parenchymal cells appeared to retain their ability to release Pu in vitro with no need for additional exposure to DTPA. The physiological significance of this finding is discussed

  11. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Yeonhee Kim

    Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

  12. Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency.

    Science.gov (United States)

    Herrera Sanchez, Maria Beatriz; Previdi, Sara; Bruno, Stefania; Fonsato, Valentina; Deregibus, Maria Chiara; Kholia, Sharad; Petrillo, Sara; Tolosano, Emanuela; Critelli, Rossana; Spada, Marco; Romagnoli, Renato; Salizzoni, Mauro; Tetta, Ciro; Camussi, Giovanni

    2017-07-27

    Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia. HLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity. EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells. Treating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme

  13. A tryptophan derivative, ITE, enhances liver cell metabolic functions in vitro.

    Science.gov (United States)

    Zhang, Xiaoqian; Lu, Juan; He, Bin; Tang, Lingling; Liu, Xiaoli; Zhu, Danhua; Cao, Hongcui; Wang, Yingjie; Li, Lanjuan

    2017-01-01

    Cell encapsulation provides a three-dimensional support by incorporating isolated cells into microcapsules with the goal of simultaneously maintaining cell survival and function, as well as providing active transport for a bioreactor in vitro similarly to that observed in vivo. However, the biotra-nsformation and metabolic functions of the encapsulated cells are not satisfactory for clinical applications. For this purpose, in this study, hepatoma-derived Huh7 cells/C3A cells were treated with 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous non-toxic ligand for aryl hydrocarbon receptor, in monolayer cultures and on microspheres. The mRNA and protein levels, as well as the metabolic activities of drug metabolizing enzymes, albumin secretion and urea synthesis were determined. When the Huh7 and C3A cells cultured in a monolayer on two‑dimensional surfaces, ITE enhanced the protein levels and the metabolic activities of the major cytochrome P450 (CYP450) enzymes, CYP1A1, CYP1A2, CYP3A4 and CYP1B1, and slightly increased albumin secretion and urea synthesis. Moreover, when cultured on microspheres, ITE also substantially increased the protein levels and metabolic activities of CYP1A1, CYP1A2, CYP3A4 and CYP1B1 in both liver cell lines. On the whole, our findings indicate that ITE enhances the enzymatic activities of major CYP450 enzymes and the metabolic functions of liver cells cultured in monolayer or on microspheres, indicating that it may be utilized to improve the functions of hepatocytes. Thus, it may be used in the future for the treatment of liver diseases.

  14. A tryptophan derivative, ITE, enhances liver cell metabolic functions in vitro

    Science.gov (United States)

    Zhang, Xiaoqian; Lu, Juan; He, Bin; Tang, Lingling; Liu, Xiaoli; Zhu, Danhua; Cao, Hongcui; Wang, Yingjie; Li, Lanjuan

    2017-01-01

    Cell encapsulation provides a three-dimensional support by incorporating isolated cells into microcapsules with the goal of simultaneously maintaining cell survival and function, as well as providing active transport for a bioreactor in vitro similarly to that observed in vivo. However, the biotransformation and metabolic functions of the encapsulated cells are not satisfactory for clinical applications. For this purpose, in this study, hepatoma-derived Huh7 cells/C3A cells were treated with 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous non-toxic ligand for aryl hydrocarbon receptor, in monolayer cultures and on microspheres. The mRNA and protein levels, as well as the metabolic activities of drug metabolizing enzymes, albumin secretion and urea synthesis were determined. When the Huh7 and C3A cells cultured in a monolayer on two-dimensional surfaces, ITE enhanced the protein levels and the metabolic activities of the major cytochrome P450 (CYP450) enzymes, CYP1A1, CYP1A2, CYP3A4 and CYP1B1, and slightly increased albumin secretion and urea synthesis. Moreover, when cultured on microspheres, ITE also substantially increased the protein levels and metabolic activities of CYP1A1, CYP1A2, CYP3A4 and CYP1B1 in both liver cell lines. On the whole, our findings indicate that ITE enhances the enzymatic activities of major CYP450 enzymes and the metabolic functions of liver cells cultured in monolayer or on microspheres, indicating that it may be utilized to improve the functions of hepatocytes. Thus, it may be used in the future for the treatment of liver diseases. PMID:27959388

  15. Primary Diffuse Large B-Cell Lymphoma of the Liver in a Patient with Sjogren Syndrome

    Directory of Open Access Journals (Sweden)

    Vadim Gorodetskiy

    2016-01-01

    Full Text Available Sjögren’s syndrome (SS has the highest incidence of malignant lymphoproliferative disorders transformation among autoimmune diseases. We present a case of extranodal high grade lymphoma of the liver in a 52-year-old patient with long history of SS. Lymphoma manifested with sharp significant pain in the right hypochondrium, weakness, and profuse night sweats. Contrast-enhanced computed tomography scan (CT-scan of the abdomen revealed multiple low density foci with homogeneous structure and clear contours in both lobes of the liver. Histologically, proliferation of medium sized lymphoma cells with round-oval and slightly irregular nuclei with fine chromatin was shown. Immunohistochemical and molecular features of the tumors allowed diagnosis of diffuse large B-cell lymphoma (DLBCL. To exclude secondary liver lesion by non-Hodgkin lymphoma, chest and small pelvis CT-scan, endoscopy of upper and lower gastrointestinal tract and study of bone marrow were performed. After 8 cycles of R-CHOP chemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, the complete remission was achieved, which persists after 45 months of follow-up. Primary hepatic lymphomas are extremely rare, and previously only low-grade hepatic lymphomas have been described in SS. To our knowledge, the patient described here represents the first reported case of DLBCL with primary liver involvement in SS.

  16. Clinical utility of red cell distribution width in alcoholic and non-alcoholic liver cirrhosis.

    Science.gov (United States)

    Milić, Sandra; Mikolasević, Ivana; Radić, Mladen; Hauser, Goran; Stimac, Davor

    2011-09-01

    Red blood cell distribution width (RDW) is a measure of the variation of red blood cell width that is reported as apart of standard complete blood count. Red blood cell distribution width results are often used together with mean corpuscular volume (MCV) results to figure out mixed anemia. The aim of our study was to compare the values of RDW in alcoholic and non-alcoholic liver cirrhosis and to determine if RDW follows the severity of disease according to Child-Pugh score. We retrospectively analyzed 241 patients (176 men and 65 women) with liver cirrhosis and anemia, defined as a hemoglobin value reference range is 11-15%. Alcoholic liver cirrhosis had 204 patients (85%) while non-alcoholic cirrhosis had 37 patients (15%). In group of alcoholic cirrhosis the average RDW was 16.8%. In relation to severity of disease the average RDW for Child-Pugh A was 16.80%, for Child-Pugh B was 16.92%, for Child-Pugh C was 17.10%. In the group of non-alcoholic cirrhosis the average RDW was 16.73% and in relation to severity of disease for Child-Pugh A was 16.25%, for Child-Pugh B 17.01% and for Child-Pugh C was 16.87%. We didn't find statistically significant difference of RDW between alcoholic and non alcoholic cirrhosis (p > 0.05) and we didn't proved any statistically significant increase of RDW in relation to severity of disease in group of alcoholic cirrhosis (p = 0.915) nor in group of patients with non-alcoholic cirrhosis (p = 0.697). Our study showed that RDW had not any clinical value in differentiation of anemia neither in alcoholic and non-alcoholic liver cirrhosis nor in severity of liver disease.

  17. Histones activate the NLRP3 Inflammasome in Kupffer Cells during Sterile Inflammatory Liver Injury

    Science.gov (United States)

    Huang, Hai; Chen, Hui-Wei; Evankovich, John; Yan, Wei; Rosborough, Brian R.; Nace, Gary W.; Ding, Qing; Loughran, Patricia; Beer-Stolz, Donna; Billiar, Timothy R.; Esmon, Charles T.; Tsung, Allan

    2013-01-01

    Cellular processes that drive sterile inflammatory injury after hepatic ischemia/reperfusion (I/R) injury are not completely understood. Activation of the inflammasome plays a key role in response to invading intracellular pathogens, but mounting evidence suggests it also plays a role in inflammation driven by endogenous danger-associate molecular pattern (DAMP) molecules released after ischemic injury. The nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3) inflammasome is one such process, and the mechanism by which its activation results in damage and inflammatory responses following liver I/R is unknown. Here we report that both NLRP3 and its downstream target Caspase-1 are activated I/R and are essential for hepatic I/R injury as both NLRP3 and Caspase-1 KO mice are protected from injury. Furthermore, inflammasome-mediated injury is dependent on Caspase-1 expression in liver non-parenchymal cells. While upstream signals that activate the inflammasome during ischemic injury are not well characterized, we show that endogenous extracellular histones activate the NLRP3 inflammasome during liver I/R through Toll-like Receptor-9 (TLR9). This occurs through TLR9-dependent generation of reactive oxygen species. This mechanism is operant in resident liver Kupffer cells, which drive innate immune responses after I/R injury by recruiting additional cell types, including neutrophils and inflammatory monocytes. These novel findings illustrate a new mechanism by which extracellular histones and activation of NLRP3 inflammasome contribute to liver damage and activation of innate immunity during sterile inflammation. PMID:23904166

  18. Clinical Utility of Red Cell Distribution Width in Alcoholic and Non-alcoholic Liver Cirrhosis

    OpenAIRE

    Milić, Sandra; Mikolašević, Ivana; Radić, Mladen; Hauser, Goran; Štimac, Davor

    2011-01-01

    Red blood cell distribution width (RDW) is a measure of the variation of red blood cell width that is reported as a part of standard complete blood count. Red blood cell distribution width results are often used together with mean corpuscular volume (MCV) results to figure out mixed anemia. The aim of our study was to compare the values of RDW in alcoholic and non-alcoholic liver cirrhosis and to determine if RDW follows the severity of disease according to Child-Pugh score. We re...

  19. Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold.

    Science.gov (United States)

    Hassanein, Wessam; Uluer, Mehmet C; Langford, John; Woodall, Jhade D; Cimeno, Arielle; Dhru, Urmil; Werdesheim, Avraham; Harrison, Joshua; Rivera-Pratt, Carlos; Klepfer, Stephen; Khalifeh, Ali; Buckingham, Bryan; Brazio, Philip S; Parsell, Dawn; Klassen, Charlie; Drachenberg, Cinthia; Barth, Rolf N; LaMattina, John C

    2017-01-02

    Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.

  20. Hydrogen peroxide impairs autophagic flux in a cell model of nonalcoholic fatty liver disease

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Pengtao [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China); University of Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan District, Beijing 100049 (China); Huang, Zhen [Department of Abdominal Surgical Oncology, Cancer Hospital, Chinese Academy of Medical Sciences, 17 Panjiayuan Nanli, Chaoyang District, Beijing 100021 (China); Zhao, Hong, E-mail: zhaohong9@sina.com [Department of Abdominal Surgical Oncology, Cancer Hospital, Chinese Academy of Medical Sciences, 17 Panjiayuan Nanli, Chaoyang District, Beijing 100021 (China); Wei, Taotao, E-mail: weitt@moon.ibp.ac.cn [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China)

    2013-04-19

    Highlights: •Free fatty acids exposure induces elevated autophagy. •H{sub 2}O{sub 2} inhibits autophagic flux through impairing the fusion between autophagosomes and lysosomes. •Inhibition of autophagy potentiates H{sub 2}O{sub 2}-induced cell death. -- Abstract: Nonalcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease, but the pathogenesis of NAFLD is not fully clear. The aim of this study was to determine whether autophagy plays a role in the pathogenesis of NAFLD. We found that the levels of autophagy were elevated in hepatoma cells upon exposure to free fatty acids, as confirmed by the increase in the number of autophagosomes. However, exposure of hepatoma cells to H{sub 2}O{sub 2} and TNF-α, two typical “second hit” factors, increased the initiation of autophagy but inhibited the autophagic flux. The inhibition of autophagy sensitized cells to pro-apoptotic stimuli. Taken together, our results suggest that autophagy acts as a protective mechanism in the pathogenesis of NAFLD and that impairment of autophagy might induce more severe lesions of the liver. These findings will be a benefit to the understanding of the pathogenesis of NAFLD and might suggest a strategy for the prevention and cure of NAFLD.

  1. OPD4-positive T-cell lymphoma of the liver in systemic lupus erythematosus.

    Science.gov (United States)

    Tsutsumi, Y; Deng, Y L; Uchiyama, M; Kawano, K; Ikeda, Y

    1991-11-01

    Primary malignant lymphoma of the liver occupying the right lobe, 14 x 9 x 7 cm in size, developed in a 30-year-old man with a 4-year history of autoimmune hemolytic anemia. The diagnosis of systemic lupus erythematosus (SLE) accompanying thrombocytopenia had been made clinically 10 months earlier. The liver biopsy specimen revealed diffuse proliferation of large lymphoma cells expressing the activated helper/inducer T-cell phenotype (LCA+, UCHL1+, OPD4+, LN3+, MT1-, L26-, MB1-, Leu M1-, Ki-1-, KP1-). The lymphoma was successfully treated by chemotherapy and irradiation. Intractable thrombocytopenia provoked fatal esophageal hemorrhage. At autopsy, no lymphomatous lesion was identified, and the hepatic right lobe contained an encapsulated necrotic lesion without any viable tumor cells. The bone marrow revealed marked hyperplasia of erythroid and megakaryocytic series. Extramedullary hematopoiesis was demonstrated in the liver, spleen and lymph nodes. This is the second case of primary hepatic T-cell lymphoma associated with SLE.

  2. Correlation of serum GP73, SOD and GPC3 contents with cell proliferation and angiogenesis in liver cancer lesion

    Directory of Open Access Journals (Sweden)

    Hua Xin

    2017-11-01

    Full Text Available Objective: To study the correlation of serum GP73, SOD and GPC3 contents with cell proliferation and angiogenesis in liver cancer lesion. Methods: Patients who were diagnosed with primary liver cancer in Jianghan Oilfield General Hospital between June 2014 and February 2017 were selected as liver cancer group, and healthy subjects who received physical examination in Jianghan Oilfield General Hospital during the same period were selected as control group. Serum was collected from two groups of subjects to determine the contents of GP73, SOD and GPC3; liver cancer lesion and adjacent lesion were collected from liver cancer group to determine the expression of cell proliferation molecules and angiogenesis molecules. Results: Serum GP73 and GPC3 levels of liver cancer group were obviously higher than those of control group while SOD content was obviously lower than that of control group; DNMT3B, STC2, SIRT6, LETM1, EphB4, SULT2B1, HIF-1α, VEGF, Ang-2, HGF and TGF-β1 protein expression levels in liver cancer lesion of liver cancer group were significantly higher than those in adjacent lesion; DNMT3B, STC2, SIRT6, LETM1, EphB4, SULT2B1, HIF-1α, VEGF, Ang-2, HGF and TGF-β1 protein expression levels in liver cancer lesion of liver cancer group were positively correlated with serum GP73 and GPC3 levels, and negatively correlated with serum SOD level. Conclusion: The changes of GP73, SOD and GPC3 levels in the serum of patients with liver cancer are closely related to the cell proliferation and angiogenesis in liver cancer lesion.

  3. Long-chain PUFA in Granulocytes, Mononuclear Cells, and RBC in Patients With Cystic Fibrosis: Relation to Liver Disease

    DEFF Research Database (Denmark)

    Jorgensen, Marianne H.; Ott, Peter; Michaelsen, Kim F.

    2012-01-01

    -related liver disease were matched with 20 CF patients without. Blood samples were analysed for liver biochemistry and haematology. Granulocytes, mononuclear cells, and RBC were separated by density gradient centrifugation, and fatty acid composition was measured by gas chromatography. Hepatic ultrasound...

  4. Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line

    DEFF Research Database (Denmark)

    Jacobsen, Kari Stougaard; Nielsen, Kirstine Overgaard; Nordmann Winther, Thilde

    2016-01-01

    expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection...

  5. In vivo migration of labeled autologous natural killer cells to liver metastases in patients with colon carcinoma

    Directory of Open Access Journals (Sweden)

    Satolli Maria A

    2006-11-01

    Full Text Available Abstract Background Besides being the effectors of native anti-tumor cytotoxicity, NK cells participate in T-lymphocyte responses by promoting the maturation of dendritic cells (DC. Adherent NK (A-NK cells constitute a subset of IL-2-stimulated NK cells which show increased expression of integrins and the ability to adhere to solid surface and to migrate, infiltrate, and destroy cancer. A critical issue in therapy of metastatic disease is the optimization of NK cell migration to tumor tissues and their persistence therein. This study compares localization to liver metastases of autologous A-NK cells administered via the systemic (intravenous, i.v. versus locoregional (intraarterial, i.a. routes. Patients and methods A-NK cells expanded ex-vivo with IL-2 and labeled with 111In-oxine were injected i.a. in the liver of three colon carcinoma patients. After 30 days, each patient had a new preparation of 111In-A-NK cells injected i.v. Migration of these cells to various organs was evaluated by SPET and their differential localization to normal and neoplastic liver was demonstrated after i.v. injection of 99mTc-phytate. Results A-NK cells expressed a donor-dependent CD56+CD16+CD3- (NK or CD56+CD16+CD3+ (NKT phenotype. When injected i.v., these cells localized to the lung before being visible in the spleen and liver. By contrast, localization of i.a. injected A-NK cells was virtually confined to the spleen and liver. Binding of A-NK cells to liver neoplastic tissues was observed only after i.a. injections. Conclusion This unique study design demonstrates that A-NK cells adoptively transferred to the liver via the intraarterial route have preferential access and substantial accumulation to the tumor site.

  6. Successful orthotopic liver transplantation in an adult patient with sickle cell disease and review of the literature

    Directory of Open Access Journals (Sweden)

    Morey Blinder

    2013-05-01

    Full Text Available Sickle cell disease can lead to hepatic complications ranging from acute hepatic crises to chronic liver disease including intrahepatic cholestasis, and iron overload. Although uncommon, intrahepatic cholestasis may be severe and medical treatment of this complication is often ineffective. We report a case of a 37 year-old male patient with sickle cell anemia, who developed liver failure and underwent successful orthotopic liver transplantation. Both pre and post-operatively, he was maintained on red cell transfusions. He remains stable with improved liver function 42 months post transplant. The role for orthotopic liver transplantation is not well defined in patients with sickle cell disease, and the experience remains limited. Although considerable challenges of post-transplant graft complications remain, orthotopic liver transplantation should be considered as a treatment option for sickle cell disease patients with end-stage liver disease who have progressed despite conventional medical therapy. An extended period of red cell transfusion support may lessen the post-operative complications.

  7. Effect of Co-Culturing of Mice Liver Cells and Embryonic Carcinomatous Stem Cells on the Rate of Differentiation to Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    AA Pourfatollah

    2005-10-01

    Full Text Available Introduction: Considering the importance of co-culture in differentiation of embryonic stem cells, the aim of this study was evaluation of the effect of co-culturing fetal liver stroma cells with P19 cells on the line of differentiation. Materials and Methods: For this purpose, P19 cells were cultured directly in semisolid medium. These cells proliferated and primarily differentiated to colonies know as embryoid bodies (EBs after 8-12 days. The Ebs cells were trypsinized and dissociated to single or double cells. Then these cells were co-cultured on the mouse fetal liver feeder layer in the absence of exogenous factors. After 14-18 days, the colonies were studied morphologically by benzidine and giemsa staining and also counted under invert microscope. Results: The percentages of benzidine positive (or erythroid and negative colonies were 94% and 6% respectively and also the cells of colonies were studied by Giemsa staining. Results showed that they were myeloid or lymphoid type cells. Thus, the results show that in the presence of mouse fetal liver feeder layer, the number of erythroid colonies was increased. Conclusions: Therefore, this technique may be effective for differentiation of stem cells from different sources into hematopoietic cells and can be used in future for human cell therapy.

  8. LP-THAE induced tumor cell apoptosis of rabbit VX2 liver carcinoma

    International Nuclear Information System (INIS)

    Chen Shengli; Quan Yi; Huang Zicheng; Chen Guodong; Zhu Dongliang

    2007-01-01

    Objective: To research tumor cell apoptosis induced by Lp-THAE of rabbit VX2 liver implanted tumor. Methods: 27 New Zealand white rabbits implanted with VX2 tumor at left middle lobe of the liver divided into three groups: Group A(n= 9) Lp-THAE: treated through transhepatic artery catheterization; Group B(n=9) THAI and Group C(n=9) as control. The rabbits were executed at second to fifth day after treatment. HE dye microscopy was taken for counting the typical apoptosis cells and calculating apoptosis index (ApI). FITC-AnnexinV/PI assay was used for measuring apoptosis by flow cytometry. Results: The ApI of tumor central area and marginal area were (17.769±2.417)%, (4.129±1.172)%, P<0.01. The percentages of tumor cell apoptosis and tumor cell necrosis were (16.483±1.404)%, (9.478±0.964)%, P<0.01 and (43.559±5.053)%, (33.460±1.840)%, P=0.093. The total percentages of tumor cell apoptosis and necrosis were (60.042±13.979)%, (42.938±8.979)%, P< 0.01, at tumor center and marginal area in THAE group respectively. The ApI, percentages of tumor cell apoptosis and necrosis in THAE group were significantly higher than those of THAI group (P<0.01). The percentages of tumor cell apoptosis at tumor center area in THAE group were significantly higher than those of tumor marginal area(P<0.01). Conclusion: Induced tumor cell apoptosis and necrosis are two mechanisms of action for Lp-THAE treatment of liver carcinoma. (authors)

  9. Long-lasting inhibitory effects of fetal liver mesenchymal stem cells on T-lymphocyte proliferation.

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    Massimo Giuliani

    Full Text Available Human bone marrow mesenchymal stem cells (BM-MSC are multipotent progenitor cells that have transient immunomodulatory properties on Natural Killer (NK cells, Dendritic Cells (DC, and T cells. This study compared the use of MSC isolated from bone marrow and fetal liver (FL-MSC to determine which displayed the most efficient immunosuppressive effects on T cell activation. Although both types of MSC exhibit similar phenotype profile, FL-MSC displays a much more extended in vitro life-span and immunomodulatory properties. When co-cultured with CD3/CD28-stimulated T cells, both BM-MSC and FL-MSC affected T cell proliferation by inhibiting their entry into the cell cycle, by inducing the down-regulation of phospho-retinoblastoma (pRb, cyclins A and D1, as well as up-regulating p27(kip1 expression. The T cell inhibition by MSC was not due to the soluble HLA-G5 isoform, but to the surface expression of HLA-G1, as shown by the need of cell-cell contact and by the use of neutralizing anti-HLA-G antibodies. To note, in a HLA-G-mediated fashion, MSC facilitated the expansion of a CD4(low/CD8(low T subset that had decreased secretion of IFN-γ, and an induced secretion of the immunomodulatory cytokine IL-10. Because of their longer lasting in vitro immunosuppressive properties, mainly mediated by HLA-G, and their more efficient induction of IL-10 production and T cell apoptosis, fetal liver MSC could be considered a new tool for MSC therapy to prevent allograft rejection.

  10. Nonparenchymal cells cultivated from explants of fibrotic liver resemble endothelial and smooth muscle cells from blood vessel walls

    International Nuclear Information System (INIS)

    Voss, B.; Rauterberg, J.; Pott, G.; Brehmer, U.; Allam, S.; Lehmann, R.; von Bassewitz, D.B.

    1982-01-01

    Tissue specimens from human fibrotic liver obtained by needle biopsy were cultured. Two cell types emerged from the tissue explants. From their morphology and biosynthetic products they resembled smooth muscle cells and endothelial cells from blood vessel walls. In the endothelial cells, factor VIII-associated protein was demonstrated by indirect immunofluorescence. Synthesis of collagen types I and III, basement membrane collagen types IV and V, and fibronectin by both cell types was observed by immunofluorescence microscopy. Homogeneous cultures of smooth muscle cells were observed in subcultures. After incubation with [ 14 C]glycine, collagen was isolated and characterized by CM cellulose chromatography, and consisted mainly of types I and III. These data suggest involvement of mesenchymal cells in hepatic fibrosis; they presumably originate from blood vessel or sinusoidal walls

  11. Total intermittent Pringle maneuver during liver resection can induce intestinal epithelial cell damage and endotoxemia.

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    Simon A W G Dello

    Full Text Available OBJECTIVES: The intermittent Pringle maneuver (IPM is frequently applied to minimize blood loss during liver transection. Clamping the hepatoduodenal ligament blocks the hepatic inflow, which leads to a non circulating (hepatosplanchnic outflow. Also, IPM blocks the mesenteric venous drainage (as well as the splenic drainage with raising pressure in the microvascular network of the intestinal structures. It is unknown whether the IPM is harmful to the gut. The aim was to investigate intestinal epithelial cell damage reflected by circulating intestinal fatty acid binding protein levels (I-FABP in patients undergoing liver resection with IPM. METHODS: Patients who underwent liver surgery received total IPM (total-IPM or selective IPM (sel-IPM. A selective IPM was performed by selectively clamping the right portal pedicle. Patients without IPM served as controls (no-IPM. Arterial blood samples were taken immediately after incision, ischemia and reperfusion of the liver, transection, 8 hours after start of surgery and on the first post-operative day. RESULTS: 24 patients (13 males were included. 7 patients received cycles of 15 minutes and 5 patients received cycles of 30 minutes of hepatic inflow occlusion. 6 patients received cycles of 15 minutes selective hepatic occlusion and 6 patients underwent surgery without inflow occlusion. Application of total-IPM resulted in a significant increase in I-FABP 8 hours after start of surgery compared to baseline (p<0.005. In the no-IPM group and sel-IPM group no significant increase in I-FABP at any time point compared to baseline was observed. CONCLUSION: Total-IPM in patients undergoing liver resection is associated with a substantial increase in arterial I-FABP, pointing to intestinal epithelial injury during liver surgery. TRIAL REGISTRATION: ClinicalTrials.gov NCT01099475.

  12. Physiology and pathophysiology of apoptosis in epithelial cells of the liver, pancreas, and intestine.

    Science.gov (United States)

    Jones, B A; Gores, G J

    1997-12-01

    Cell death of gastrointestinal epithelial cells occurs by a process referred to as apoptosis. In this review, we succinctly define apoptosis and summarize the role of apoptosis in the physiology and pathophysiology of epithelial cells in the liver, pancreas, and small and large intestine. The physiological mediators regulating apoptosis in gastrointestinal epithelial cells, when known, are discussed. Selected pathophysiological consequences of excessive apoptosis and inhibition of apoptosis are used to illustrate the significance of apoptosis in disease processes. These examples demonstrate that excessive apoptosis may result in epithelial cell atrophy, injury, and dysfunction, whereas inhibition of apoptosis results in hyperplasia and promotes malignant transformation. The specific cellular mechanisms responsible for dysregulation of epithelial cell apoptosis during pathophysiological disturbances are emphasized. Potential future areas of physiological research regarding apoptosis in gastrointestinal epithelia are highlighted when appropriate.

  13. Ectopic expression of the calcium-binding protein parvalbumin in mouse liver endothelial cells

    DEFF Research Database (Denmark)

    Castillo, M B; Berchtold, M W; Rülicke, T

    1997-01-01

    To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical...... methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent...... vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased...

  14. Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

    Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

  15. The effect of experimental diabetes on phenylalanine metabolism in isolated liver cells.

    OpenAIRE

    Santana, M A; Fisher, M J; Bate, A J; Pogson, C I

    1985-01-01

    Chronic (10-day) diabetes was associated with increased metabolic flux through phenylalanine hydroxylase in isolated liver cells. This flux was stimulated by 0.1 microM-glucagon, but not by 10 microM-noradrenaline; 0.1 microM-insulin affected neither basal nor glucagon-stimulated flux. The increased rate of phenylalanine hydroxylation in diabetes was accompanied by parallel increases in enzyme activity (as measured with artificial cofactor) and immunoreactive-enzyme-protein content. In contra...

  16. Phenylalanine metabolism in isolated rat liver cells. Effects of glucagon and diabetes.

    OpenAIRE

    Carr, F P; Pogson, C I

    1981-01-01

    1. Methods are described for monitoring the metabolic flux through phenylalanine hydroxylase, the tyrosine catabolic pathway and phenylalanine: pyruvate transaminase in isolated liver cell incubations. 2. The relationship between hydroxylase flux and phenylalanine concentration is sigmoidal. 3. Glucagon increases hydroxylase activity at low, near-physiological, substrate concentrations only. The hormone does not affect the rate of formation of phenylpyruvate. 4. Experimental diabetes (for 10 ...

  17. Radioprotective role of H2S/CSE pathway in Chang liver cells

    International Nuclear Information System (INIS)

    Pan Yan; Ye Shuang; Yuan Dexiao; Zhang Jianghong; Bai Yang; Shao Chunlin

    2012-01-01

    Radiation-induced liver cell damage may be life-threatening. Here, we investigated whether hydrogen sulfide (H 2 S)/cystathionine γ-lyase (CSE) pathway could serve the protective role toward radiation in normal human liver cells. Our data showed that pretreatment of cells with H 2 S donor, sodium hydrosulfide (NaHS) significantly attenuated radiation induced micronuclei formation and improved cell viability. However, the use of DL-propargylglycine (PPG), a potent inhibitor of CSE, markedly enhanced the cell-killing effect induced by radiation. Exposure of cells to 2 Gy γ-radiation led to significant increases of the endogenous H 2 S content. The mRNA and protein expressions of CSE also increased after radiation in a time-dependent manner, while the expression of cystathionine β-synthase (CBS), another endogenous H 2 S synthetase, did not change significantly. Notably, radiation induced production of reactive oxygen species (ROS) was significantly reversed by the pretreatment of NaHS, while blockage of CSE activity resulted in an enhanced ROS production in irradiated cells. Moreover, NaHS markedly suppressed radiation-induced phosphorylation of P53, decrease of Bcl-2/Bax, and activity of nuclear factor kappaB (NF-κB). In conclusion, our finding demonstrates that H 2 S/CSE pathway plays a radioprotection role by inhibiting radiation-induced ROS production, P53 phosphorylation, NF-κB activation and decrease of Bcl-2/Bax, indicating that modulation of H 2 S may be a novel protection strategy for liver radiation injury in radiotherapy.

  18. Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

    Science.gov (United States)

    Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

    2015-01-01

    Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

  19. Ambiguous roles of innate lymphoid cells in chronic development of liver diseases.

    Science.gov (United States)

    Shen, Yue; Li, Jing; Wang, Si-Qi; Jiang, Wei

    2018-05-14

    Innate lymphoid cells (ILCs) are defined as a distinct arm of innate immunity. According to their profile of secreted cytokines and lineage-specific transcriptional factors, ILCs can be categorized into the following three groups: group 1 ILCs (including natural killer (NK) cells and ILC1s) are dependent on T-bet and can produce interferon-γ; group 2 ILCs (ILC2s) are dependent on GATA3 and can produce type 2 cytokines, including interleukin (IL)-5 and IL-13; and, group 3 ILCs (including lymphoid tissue-like cells and ILC3s) are dependent on RORγt and can produce IL-22 and IL-17. Collaborative with adaptive immunity, ILCs are highly reactive innate effectors that promptly orchestrate immunity, inflammation and tissue repair. Dysregulation of ILCs might result in inflammatory disorders. Evidence regarding the function of intrahepatic ILCs is emerging from longitudinal studies of inflammatory liver diseases wherein they exert both physiological and pathological functions, including immune homeostasis, defenses and surveillance. Their overall effect on the liver depends on the balance of their proinflammatory and antiinflammatory populations, specific microenvironment and stages of immune responses. Here, we review the current data about ILCs in chronic liver disease progression, to reveal their roles in different stages as well as to discuss their therapeutic potency as intervention targets.

  20. Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

    Science.gov (United States)

    Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

    2011-01-01

    Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

  1. Stromal Derived Factor-1/CXCR4 Axis Involved in Bone Marrow Mesenchymal Stem Cells Recruitment to Injured Liver

    Directory of Open Access Journals (Sweden)

    Kuai Xiao Ling

    2016-01-01

    Full Text Available The molecular mechanism of bone marrow mesenchymal stromal stem cells (BMSCs mobilization and migration to the liver was poorly understood. Stromal cell-derived factor-1 (SDF-1 participates in BMSCs homing and migration into injury organs. We try to investigate the role of SDF-1 signaling in BMSCs migration towards injured liver. The expression of CXCR4 in BMSCs at mRNA level and protein level was confirmed by RT-PCR, flow cytometry, and immunocytochemistry. The SDF-1 or liver lysates induced BMSCs migration was detected by transwell inserts. CXCR4 antagonist, AMD3100, and anti-CXCR4 antibody were used to inhibit the migration. The Sprague-Dawley rat liver injury model was established by intraperitoneal injection of thioacetamide. The concentration of SDF-1 increased as modeling time extended, which was determined by ELISA method. The Dir-labeled BMSCs were injected into the liver of the rats through portal vein. The cell migration in the liver was tracked by in vivo imaging system and the fluorescent intensity was measured. In vivo, BMSCs migrated into injured liver which was partially blocked by AMD3100 or anti-CXCR4 antibody. Taken together, the results demonstrated that the migration of BMSCs was regulated by SDF-1/CXCR4 signaling which involved in BMSCs recruitment to injured liver.

  2. Retinol and retinyl esters in parenchymal and nonparenchymal rat liver cell fractions after long-term administration of ethanol

    International Nuclear Information System (INIS)

    Rasmussen, M.; Blomhoff, R.; Helgerud, P.; Solberg, L.A.; Berg, T.; Norum, K.R.

    1985-01-01

    Chronic ethanol consumption reduces the liver retinoid store in man and rat. We have studied the effect of ethanol on some aspects of retinoid metabolism in parenchymal and nonparenchymal liver cells. Rats fed 36% of total energy intake as ethanol for 5-6 weeks had the liver retinoid concentration reduced to about one-third, as compared to pair-fed controls. The reduction in liver retinoid affected both the parenchymal and the nonparenchymal cell fractions. Plasma retinol level was normal. Liver uptake of injected chylomicron [3H]retinyl ester was similar in the experimental and control group. The transport of retinoid from the parenchymal to the nonparenchymal cells was not found to be significantly retarded in the ethanol-fed rats. Despite the reduction in total retinoid level in liver, the concentrations of unesterified retinol and retinyl oleate were increased in the ethanol fed rats. Hepatic retinol esterification was not significantly affected in the ethanol-fed rats. Since our study has demonstrated that liver uptake of chylomicron retinyl ester is not impaired in the ethanol-fed rat, we suggest that liver retinoid metabolism may be increased

  3. Genomic instability induced by 60Co γ ray radiation in normal human liver cells

    International Nuclear Information System (INIS)

    Gen Xiaohua; Guo Xianhua; Zuo Yahui; Wang Xiaoli; Wang Zhongwen

    2007-01-01

    Objective: To explore the genomic instability induced by 60 Co γ rays. Methods: The cloning efficiency and micronucleus efficiency of normal human liver cell irradiated by 60 Co γ rays were detected, and the method of single cell gel electrophoresis (SCGE) was carried out to measure DNA chains damage. The fast-growing cells were divided into different dose-groups and then irradiated by 60 Co γ rays. After 40 populations doubling, the progenies were secondly irradiated with 2 Gy 60 Co γ rays. Results: The cloning efficiency decreased with the increase of doses after the initial irradiation. After the survival cells were given second irradiation, both results of SCGE and micronucleus frequency showed that the second damage was correlated with the original irradiation doses. Conclusions: 60 Co γ rays can not only induce the immediate biological effects in liver cells, but also lead to the genomic instability in the descendants that leads to an enhanced frequency of genetic changes occurring among the progeny of the original irradiated cell. The expanding effect of second event helps to study the genomic instability. (authors)

  4. Gene targeting and cloning in pigs using fetal liver derived cells.

    Science.gov (United States)

    Waghmare, Sanjeev K; Estrada, Jose; Reyes, Luz; Li, Ping; Ivary, Bess; Sidner, Richard A; Burlak, Chris; Tector, A Joseph

    2011-12-01

    Since there are no pig embryonic stem cells, pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 wk. Nuclear donor cells that remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs. The goal of this study was to test the feasibility of using fetal liver-derived cells (FLDC) to perform gene targeting, and create a genetic knockout pig. FLDC were isolated and processed using a human liver stem cell protocol. Single copy α-1,3-galactosyl transferase knockout (GTKO) FLDCs were created using electroporation and neomycin resistant colonies were screened using PCR. Homozygous GTKO cells were created through loss of heterozygosity mutations in single GTKO FLDCs. Double GTKO FLDCs were used in somatic cell nuclear transfer (SCNT) to create GTKO pigs. FLDCs grew for more than 80 population doublings, maintaining normal karyotype. Gene targeting and loss of heterozygosity mutations produced homozygous GTKO FLDCs. FLDCs used in SCNT gave rise to homozygous GTKO pigs. FDLCs can be used in gene targeting and SCNT to produce genetically modified pigs. The increased life span in culture compared to fetal fibroblasts may facilitate genetic engineering in the pig. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells.

    Directory of Open Access Journals (Sweden)

    Silvia Berardis

    Full Text Available Adult-derived human liver stem/progenitor cells (ADHLSC are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity. ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays. Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10. Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.

  6. The fatty acid profile of rainbow trout liver cells modulates their tolerance to methylmercury and cadmium

    International Nuclear Information System (INIS)

    Ferain, Aline; Bonnineau, Chloé; Neefs, Ineke; Rees, Jean François; Larondelle, Yvan; Schamphelaere, Karel A.C.De; Debier, Cathy

    2016-01-01

    Highlights: • The phospholipid composition of rainbow trout liver cells was successfully changed. • Cell phospholipids influenced methylmercury (MeHg) and cadmium (Cd) toxicity. • Cells enriched in 18:3n-3, 20:5n-3 or 22:5n-6 were more resistant to MeHg and Cd. • Cell enrichment in 22:6n-3 increased resistance to Cd but not MeHg. - Abstract: The polyunsaturated fatty acid (PUFA) composition of fish tissues, which generally reflects that of the diet, affects various cellular properties such as membrane structure and fluidity, energy metabolism and susceptibility to oxidative stress. Since these cellular parameters can play an important role in the cellular response to organic and inorganic pollutants, a variation of the PUFA supply might modify the toxicity induced by such xenobiotics. In this work, we investigated whether the cellular fatty acid profile has an impact on the in vitro cell sensitivity to two environmental pollutants: methylmercury and cadmium. Firstly, the fatty acid composition of the rainbow trout liver cell line RTL-W1 was modified by enriching the growth medium with either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6) or docosapentaenoic acid (DPA, 22:5n-6). These modified cells and their control (no PUFA enrichment) were then challenged for 24 h with increasing concentrations of methylmercury or cadmium. We observed that (i) the phospholipid composition of the RTL-W1 cells was profoundly modulated by changing the PUFA content of the growth medium: major modifications were a high incorporation of the supplemented PUFA in the cellular phospholipids, the appearance of direct elongation and desaturation metabolites in the cellular phospholipids as well as a change in the gross phospholipid composition (PUFA and monounsaturated fatty acid (MUFA) levels and n-3/n-6 ratio); (ii) ALA, EPA and DPA enrichment significantly

  7. The fatty acid profile of rainbow trout liver cells modulates their tolerance to methylmercury and cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Ferain, Aline, E-mail: aline.ferain@uclouvain.be [Institute of Life Sciences, Université catholique de Louvain, Place Croix du Sud 2/L7.05.08, B-1348 Louvain-la-Neuve (Belgium); Bonnineau, Chloé [Institute of Life Sciences, Université catholique de Louvain, Place Croix du Sud 2/L7.05.08, B-1348 Louvain-la-Neuve (Belgium); Irstea, UR MALY, Centre de Lyon-Villeurbanne, rue de la Doua 5/32108, F-69616 Villeurbanne (France); Neefs, Ineke; Rees, Jean François; Larondelle, Yvan [Institute of Life Sciences, Université catholique de Louvain, Place Croix du Sud 2/L7.05.08, B-1348 Louvain-la-Neuve (Belgium); Schamphelaere, Karel A.C.De [Laboratory of Environmental Toxicology and Aquatic Ecology, Environmental Toxicology Unit, Ghent University, J. Plateaustraat 22, B-9000 Ghent (Belgium); Debier, Cathy [Institute of Life Sciences, Université catholique de Louvain, Place Croix du Sud 2/L7.05.08, B-1348 Louvain-la-Neuve (Belgium)

    2016-08-15

    Highlights: • The phospholipid composition of rainbow trout liver cells was successfully changed. • Cell phospholipids influenced methylmercury (MeHg) and cadmium (Cd) toxicity. • Cells enriched in 18:3n-3, 20:5n-3 or 22:5n-6 were more resistant to MeHg and Cd. • Cell enrichment in 22:6n-3 increased resistance to Cd but not MeHg. - Abstract: The polyunsaturated fatty acid (PUFA) composition of fish tissues, which generally reflects that of the diet, affects various cellular properties such as membrane structure and fluidity, energy metabolism and susceptibility to oxidative stress. Since these cellular parameters can play an important role in the cellular response to organic and inorganic pollutants, a variation of the PUFA supply might modify the toxicity induced by such xenobiotics. In this work, we investigated whether the cellular fatty acid profile has an impact on the in vitro cell sensitivity to two environmental pollutants: methylmercury and cadmium. Firstly, the fatty acid composition of the rainbow trout liver cell line RTL-W1 was modified by enriching the growth medium with either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6) or docosapentaenoic acid (DPA, 22:5n-6). These modified cells and their control (no PUFA enrichment) were then challenged for 24 h with increasing concentrations of methylmercury or cadmium. We observed that (i) the phospholipid composition of the RTL-W1 cells was profoundly modulated by changing the PUFA content of the growth medium: major modifications were a high incorporation of the supplemented PUFA in the cellular phospholipids, the appearance of direct elongation and desaturation metabolites in the cellular phospholipids as well as a change in the gross phospholipid composition (PUFA and monounsaturated fatty acid (MUFA) levels and n-3/n-6 ratio); (ii) ALA, EPA and DPA enrichment significantly

  8. Identification and Characterization of Mesenchymal-Epithelial Progenitor-Like Cells in Normal and Injured Rat Liver

    Science.gov (United States)

    Liu, Daqing; Yovchev, Mladen I.; Zhang, Jinghang; Alfieri, Alan A.; Tchaikovskaya, Tatyana; Laconi, Ezio; Dabeva, Mariana D.

    2016-01-01

    In normal rat liver, thymocyte antigen 1 (Thy1) is expressed in fibroblasts/myofibroblasts and in some blood progenitor cells. Thy1-expressing cells also accumulate in the liver during impaired liver regeneration. The origin and nature of these cells are not well understood. By using RT-PCR analysis and immunofluorescence microscopy, we describe the presence of rare Thy1+ cells in the liver lobule of normal animals, occasionally forming small collections of up to 20 cells. These cells constitute a small portion (1.7% to 1.8%) of nonparenchymal cells and reveal a mixed mesenchymal-epithelial phenotype, expressing E-cadherin, cytokeratin 18, and desmin. The most potent mitogens for mesenchymal-epithelial Thy1+ cells in vitro are the inflammatory cytokines interferon γ, IL-1, and platelet-derived growth factor-BB, which are not produced by Thy1+ cells. Thy1+ cells express all typical mesenchymal stem cell and hepatic progenitor cell markers and produce growth factor and cytokine mRNA (Hgf, Il6, Tgfa, and Tweak) for proteins that maintain oval cell growth and differentiation. Under appropriate conditions, mesenchymal-epithelial cells differentiate in vitro into hepatocyte-like cells. In this study, we show that the adult rat liver harbors a small pool of endogenous mesenchymal-epithelial cells not recognized previously. In the quiescent state, these cells express both mesenchymal and epithelial cell markers. They behave like hepatic stem cells/progenitors with dual phenotype, exhibiting high plasticity and long-lasting proliferative activity. PMID:25447047

  9. Oval cell response is attenuated by depletion of liver resident macrophages in the 2-AAF/partial hepatectomy rat.

    Directory of Open Access Journals (Sweden)

    Shuai Xiang

    Full Text Available BACKGROUND/AIMS: Macrophages are known to play an important role in hepatocyte mediated liver regeneration by secreting inflammatory mediators. However, there is little information available on the role of resident macrophages in oval cell mediated liver regeneration. In the present study we aimed to investigate the role of macrophages in oval cell expansion induced by 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH in rats. METHODOLOGY/PRINCIPAL FINDINGS: We depleted macrophages in the liver of 2-AAF/PH treated rats by injecting liposome encapsulated clodronate 48 hours before PH. Regeneration of remnant liver mass, as well as proliferation and differentiation of oval cells were measured. We found that macrophage-depleted rats suffered higher mortality and liver transaminase levels. We also showed that depletion of macrophages yielded a significant decrease of EPCAM and PCK positive oval cells in immunohistochemical stained liver sections 9 days after PH. Meanwhile, oval cell differentiation was also attenuated as a result of macrophage depletion, as large foci of small basophilic hepatocytes were observed by day 9 following hepatectomy in control rats whereas they were almost absent in macrophage depleted rats. Accordingly, real-time polymerase chain reaction analysis showed lower expression of albumin mRNA in macrophage depleted livers. Then we assessed whether macrophage depletion may affect hepatic production of stimulating cytokines for liver regeneration. We showed that macrophage-depletion significantly inhibited hepatic expression of tumor necrosis factor-α and interleukin-6, along with a lack of signal transducer and activator of transcription 3 phosphorylation during the early period following hepatectomy. CONCLUSIONS: These data indicate that macrophages play an important role in oval cell mediated liver regeneration in the 2-AAF/PH model.

  10. Impaired methylation as a novel mechanism for proteasome suppression in liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Osna, Natalia A., E-mail: nosna@UNMC.edu [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States); White, Ronda L.; Donohue, Terrence M. [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States); Beard, Michael R. [Department of Molecular Biosciences, University of Adelaide (Australia); Tuma, Dean J.; Kharbanda, Kusum K. [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States)

    2010-01-08

    The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.

  11. Is Osteopontin a Friend or Foe of Cell Apoptosis in Inflammatory Gastrointestinal and Liver Diseases?

    Science.gov (United States)

    Iida, Tomoya; Wagatsuma, Kohei; Hirayama, Daisuke; Nakase, Hiroshi

    2017-12-21

    Osteopontin (OPN) is involved in a variety of biological processes, including bone remodeling, innate immunity, acute and chronic inflammation, and cancer. The expression of OPN occurs in various tissues and cells, including intestinal epithelial cells and immune cells such as macrophages, dendritic cells, and T lymphocytes. OPN plays an important role in the efficient development of T helper 1 immune responses and cell survival by inhibiting apoptosis. The association of OPN with apoptosis has been investigated. In this review, we described the role of OPN in inflammatory gastrointestinal and liver diseases, focusing on the association of OPN with apoptosis. OPN changes its association with apoptosis depending on the type of disease and the phase of disease activity, acting as a promoter or a suppressor of inflammation and inflammatory carcinogenesis. It is essential that the roles of OPN in those diseases are elucidated, and treatments based on its mechanism are developed.

  12. Acute liver failure in a patient with sickle cell/β+ thalassaemia

    International Nuclear Information System (INIS)

    Wigg, A.J.; Mounkley, A.D.; Cochlan, D.; Somers, S.

    2001-01-01

    We describe a rare, severe, vaso-occlusive presentation of sickle cell disease, named sickle cell intrahepatic cholestasis (SCIC). Patients with sickle cell/β + thalassaemia frequently have mild vaso-occlusive symptoms and only one case of SCIC developing in a patient with sickle cell/β + thalassaemia has been previously described in the world literature. The present report represents only the second described case of SCIC in a patient with sickle cell/β + thalassaemia. An abdominal computed tomography scan and Doppler ultrasound studies demonstrated massive hepatomegaly (25 cm span). Liver biopsy was performed and demonstrated dilatation and congestion of erythrocytes, severe cholestasis and fibrosis. The case demonstrates the importance of early recognition and institution of adequate therapy. Initial and correct diagnosis does not require biopsy or surgery which carry substantial risks of bleeding and mortality

  13. The Impact of Liver Cell Injury on Health-Related Quality of Life in Patients with Chronic Liver Disease.

    Directory of Open Access Journals (Sweden)

    Yvonne Alt

    Full Text Available Patients with chronic liver disease often suffer from unspecific symptoms and report severe impairment in the quality of life. The underlying mechanisms are multifactorial and include disease-specific but also liver related causes. The current analysis evaluated the association of hepatocellular apoptosis in non-viral chronic liver disease and health-related quality of life (HRQL. Furthermore we examined factors, which influence patient's physical and mental well-being.A total of 150 patients with non-infectious chronic liver disease were included between January 2014 and June 2015. The German version of the Chronic Liver Disease Questionnaire (CLDQ-D, a liver disease specific instrument to assess HRQL, was employed. Hepatocellular apoptosis was determined by measuring Cytokeratin 18 (CK18, M30 Apoptosense ELISA.Female gender (5.24 vs. 5.54, p = 0.04, diabetes mellitus type II (4.75 vs. 5.46, p<0.001 and daily drug intake (5.24 vs. 6.01, p = 0.003 were associated with a significant impairment in HRQL. HRQL was not significantly different between the examined liver diseases. Levels of CK18 were the highest in patients with NASH compared to all other disease entities (p<0.001. Interestingly, CK18 exhibited significant correlations with obesity (p<0.001 and hyperlipidemia (p<0.001. In patients with cirrhosis levels of CK18 correlated with the MELD score (r = 0.18, p = 0.03 and were significantly higher compared to patients without existing cirrhosis (265.5 U/l vs. 186.9U/l, p = 0.047. Additionally, CK18 showed a significant correlation with the presence and the degree of hepatic fibrosis (p = 0.003 and inflammation (p<0.001 in liver histology. Finally, there was a small negative association between CLDQ and CK18 (r = -0.16, p = 0.048.Different parameters are influencing HRQL and CK18 levels in chronic non-viral liver disease and the amount of hepatocellular apoptosis correlates with the impairment in HRQL in chronic non-viral liver diseases. These

  14. The let-7/Lin28 axis regulates activation of hepatic stellate cells in alcoholic liver injury.

    Science.gov (United States)

    McDaniel, Kelly; Huang, Li; Sato, Keisaku; Wu, Nan; Annable, Tami; Zhou, Tianhao; Ramos-Lorenzo, Sugeily; Wan, Ying; Huang, Qiaobing; Francis, Heather; Glaser, Shannon; Tsukamoto, Hidekazu; Alpini, Gianfranco; Meng, Fanyin

    2017-07-07

    The let-7/Lin28 axis is associated with the regulation of key cellular regulatory genes known as microRNAs in various human disorders and cancer development. This study evaluated the role of the let-7/Lin28 axis in regulating a mesenchymal phenotype of hepatic stellate cells in alcoholic liver injury. We identified that ethanol feeding significantly down-regulated several members of the let-7 family in mouse liver, including let-7a and let-7b. Similarly, the treatment of human hepatic stellate cells (HSCs) with lipopolysaccharide (LPS) and transforming growth factor-β (TGF-β) significantly decreased the expressions of let-7a and let-7b. Conversely, overexpression of let-7a and let-7b suppressed the myofibroblastic activation of cultured human HSCs induced by LPS and TGF-β, as evidenced by repressed ACTA2 (α-actin 2), COL1A1 (collagen 1A1), TIMP1 (TIMP metallopeptidase inhibitor 1), and FN1 (fibronectin 1); this supports the notion that HSC activation is controlled by let-7. A combination of bioinformatics, dual-luciferase reporter assay, and Western blot analysis revealed that Lin28B and high-mobility group AT-hook (HMGA2) were the direct targets of let-7a and let-7b. Furthermore, Lin28B deficiency increased the expression of let-7a/let-7b as well as reduced HSC activation and liver fibrosis in mice with alcoholic liver injury. This feedback regulation of let-7 by Lin28B is verified in hepatic stellate cells isolated by laser capture microdissection from the model. The identification of the let-7/Lin28 axis as an important regulator of HSC activation as well as its upstream modulators and down-stream targets will provide insights into the involvement of altered microRNA expression in contributing to the pathogenesis of alcoholic liver fibrosis and novel therapeutic approaches for human alcoholic liver diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Treatment of diffuse large B-cell lymphoma of the liver with yttrium-90 microsphere embolization.

    Science.gov (United States)

    Fenske, Timothy S; Benjamin, Heather; Kroft, Steven H; Hohenwalter, Eric J; Rilling, William S

    2008-11-01

    A 41-year-old male with a 4-year history of chronic hepatitis C presented with a 1-month history of abdominal pain, fatigue, weight loss, and night sweats. Laboratory examinations, chest, abdomen, and pelvic CT scans, PET-CT scans, ultrasound-guided needle biopsies of liver lesions, bone-marrow biopsy, flow cytometry, and immunohistochemical staining for B-cell markers including CD20. Chemoresistant diffuse large B-cell lymphoma, with gradual loss of CD20 antigen expression. Embolization of hepatic tumors using yttrium-90 microspheres (Therasphere, Theragenics Corporation, Buford, GA).

  16. TRAIL enhances paracetamol-induced liver sinusoidal endothelial cell death in a Bim- and Bid-dependent manner

    Science.gov (United States)

    Badmann, A; Langsch, S; Keogh, A; Brunner, T; Kaufmann, T; Corazza, N

    2012-01-01

    Paracetamol (acetaminophen, APAP) is a universally used analgesic and antipyretic agent. Considered safe at therapeutic doses, overdoses cause acute liver damage characterized by centrilobular hepatic necrosis. One of the major clinical problems of paracetamol-induced liver disease is the development of hemorrhagic alterations. Although hepatocytes represent the main target of the cytotoxic effect of paracetamol overdose, perturbations within the endothelium involving morphological changes of liver sinusoidal endothelial cells (LSECs) have also been described in paracetamol-induced liver disease. Recently, we have shown that paracetamol-induced liver damage is synergistically enhanced by the TRAIL signaling pathway. As LSECs are constantly exposed to activated immune cells expressing death ligands, including TRAIL, we investigated the effect of TRAIL on paracetamol-induced LSEC death. We here demonstrate for the first time that TRAIL strongly enhances paracetamol-mediated LSEC death with typical features of apoptosis. Inhibition of caspases using specific inhibitors resulted in a strong reduction of cell death. TRAIL appears to enhance paracetamol-induced LSEC death via the activation of the pro-apoptotic BH3-only proteins Bid and Bim, which initiate the mitochondrial apoptotic pathway. Taken together this study shows that the liver endothelial layer, mainly LSECs, represent a direct target of the cytotoxic effect of paracetamol and that activation of TRAIL receptor synergistically enhances paracetamol-induced LSEC death via the mitochondrial apoptotic pathway. TRAIL-mediated acceleration of paracetamol-induced cell death may thus contribute to the pathogenesis of paracetamol-induced liver damage. PMID:23254290

  17. Heterologous transporter expression for improved fatty alcohol secretion in yeast

    DEFF Research Database (Denmark)

    Hu, Yating; Zhu, Zhiwei; Nielsen, Jens

    2017-01-01

    The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production...... transporters tested, human FATP1 was shown to mediate fatty alcohol export in a high fatty alcohol production yeast strain. An approximately five-fold increase of fatty alcohol secretion was achieved. The results indicate that the overall cell fitness benefited from fatty alcohol secretion and that the acyl......-CoA synthase activity of FATP1 contributed to increased cell growth as well. This is the first study that enabled an increased cell fitness for fatty alcohol production by heterologous transporter expression in yeast, and this investigation indicates a new potential function of FATP1, which has been known...

  18. Disruption of contact inhibition in rat liver epithelial cells by various types of AhR ligands

    Energy Technology Data Exchange (ETDEWEB)

    Vondracek, J.; Chramostova, K.; Kozubik, A. [Institute of Biophysics, Brno (Czech Republic); Krcmar, P.; Machala, M. [Veterinary Research Institute, Brno (Czech Republic)

    2004-09-15

    The maintenance of a balance between cell gain and cell loss is essential for proper liver function. The exact role of aryl hydrocarbon receptor (AhR) in regulating cell proliferation and apoptosis of liver cells remains unclear, since ligand-dependent activation of AhR has been shown to induce cell cycle arrest, proliferation, differentiation or apoptosis, depending on the cellular model used. AhR can directly interact with retinoblastoma protein in hepatic cells, forming protein complexes that can efficiently block cell cycle progression by inducing G1 arrest, or to induce the expression of inhibitors of cyclin-dependent kinases, such as p271. On the other hand, it has been suggested that AhR could play a stimulatory role in cell proliferation, either directly or by mediating a release from contact inhibition. It is now generally accepted that progenitor cells exist in the liver, are activated in various liver diseases and can form a potential target cell population for both tumor initiating and tumor promoting chemicals4. 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) has been found to release rat liver epithelial cells from contact inhibition by upregulating cyclin A expression and cyclin A/cdk2 activity. Our previous studies have shown that a number of AhR ligands5,6 can stimulate proliferation of confluent of rat liver epithelial ''stem-like'' WB-F344 cells. Such mechanism could play a role in liver tumor promotion. In the present study, we used flavonoid compounds that have been reported to act either as pure agonists, such as beta-naphthoflavone (BNF), or as partial/complete antagonists of AhR - alpha-naphthoflavone (ANF) and 3'-methoxy-4'-nitroflavone (3'M4'NF), in order to investigate effects of AhR agonists/antagonists on confluent rat liver epithelial cells. The present study aimed to investigate the effects of model flavonoids on the release of rat liver epithelial cells from contact inhibition, and on inducibility of

  19. Induction of Morphological Changes in Human Embryo Liver Cells by the Pyrrolizidine Alkaloid Lasiocarpine

    Science.gov (United States)

    Armstrong, Sylvia J.; Zuckerman, A. J.; Bird, R. G.

    1972-01-01

    The pyrrolizidine alkaloids have been implicated in the aetiology of liver disease in man and in animals. Studies of the effects of lasiocarpine indicate that they have several and perhaps independent effects on human liver cells in culture. These may be summarized as follows: 1. Nuclear and nucleolar changes which are probably related to the alkylation of DNA and ensuing inhibition of nucleic acid and protein synthesis. 2. The induction of possible chromosomal damage and mutation. 3. A generalized reduction of the metabolic activities of the cells due to membrane and mitochondrial damage, and to alkylation and inactivation of cell enzymes and proteins. 4. A long-term inhibition of mitosis leading to the formation of giant cells (“megalocytes”). The morphological effects induced by a number of the pyrrolizidine alkaloids were very similar but the pattern of metabolic changes varied somewhat. It is believed that the hepatotoxic effects are not due to the pyrrolizidine alkaloids themselves but to metabolic derivatives formed by the cell. ImagesFigs. 3-5Figs. 1-2 PMID:5032090

  20. Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells

    Science.gov (United States)

    Zalzman, Michal; Gupta, Sanjeev; Giri, Ranjit K.; Berkovich, Irina; Sappal, Baljit S.; Karnieli, Ohad; Zern, Mark A.; Fleischer, Norman; Efrat, Shimon

    2003-06-01

    Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

  1. Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

    Science.gov (United States)

    El Baz, Hanan; Demerdash, Zeinab; Kamel, Manal; Atta, Shimaa; Salah, Faten; Hassan, Salwa; Hammam, Olfat; Khalil, Heba; Meshaal, Safa; Raafat, Inas

    2018-02-01

    Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

  2. Liver scintigraphy

    International Nuclear Information System (INIS)

    Tateno, Yukio

    1996-01-01

    Liver scintigraphy can be classified into 3 major categories according to the properties of the radiopharmaceuticals used, i.e., methods using radiopharmaceuticals which are (1) incorporated by hepatocytes, (2) taken up by reticulo endothelial cells, and (3) distributed in the blood pool of the liver. Of these three categories, the liver scintigraphy of the present research falls into category 2. Radiopharmaceuticals which are taken up by endothelial cells include 198 Au colloids and 99m Tc-labelled colloids. Liver scintigraphy takes advantage of the property by which colloidal microparticles are phagocytosed by Kupffer cells, and reflect the distribution of endothelial cells and the intensity of their phagocytic capacity. This examination is indicated in the following situations: (i) when you suspect a localized intrahepatic lesion (tumour, abscess, cyst, etc.), (ii) when you want to follow the course of therapy of a localized lesion, (iii) when you suspect liver cirrhosis, (iv) when you want to know the severity of liver cirrhosis or hepatitis, (v) when there is hepatomegaly and you want to determine the morphology of the liver, (vi) differential diagnosis of upper abdominal masses, and (vii) when there are abnormalities of the right diaphragm and you want to know their relation to the liver

  3. Ultrasound-guided cytology of spleen and liver: a prognostic tool in canine cutaneous mast cell tumor.

    Science.gov (United States)

    Stefanello, D; Valenti, P; Faverzani, S; Bronzo, V; Fiorbianco, V; Pinto da Cunha, N; Romussi, S; Cantatore, M; Caniatti, M

    2009-01-01

    In the clinical staging of cutaneous mast cell tumors (cMCT), the diagnosis of metastasis is controversial based on cytological examination of lymph nodes, spleen, liver, bone marrow, and blood. To define the prognostic role of ultrasound-guided cytology of spleen and liver in cMCT. The results of cytological evaluation were compared in relation with survival time. Fifty-two client-owned dogs with a diagnosis of cMCT. Selection of cases was based on cytological evaluation of liver and spleen to detect infiltration at distant sites. The Kaplan Meier method was used to compare survival in dogs with and without infiltration of spleen and liver (log-rank test P dogs with cMCT had mast cell infiltration of spleen, liver, or both and 4 of these dogs had involvement of the regional lymph nodes. The majority of dogs had 2 or more ultrasonographically abnormal findings simultaneously in spleen and liver. Nine dogs had grade II cMCT, and 1 had grade III cMCT. Dogs with positive evidence of mast cell infiltration to spleen, liver, or both had shorter survival times (34 versus 733 days) compared with dogs negative for mast cell infiltration at distant sites. Dogs with evidence of mast cell infiltration at distant sites have a shorter survival times than dogs without evidence of infiltration at distant sites. This study suggests that cytology of spleen and liver is indicated either for ultrasonographically normal or for ultrasonographically abnormal spleen and liver in dogs with cMCT.

  4. Heterologous Protection against Malaria after Immunization with Plasmodium falciparum Sporozoites.

    Directory of Open Access Journals (Sweden)

    Remko Schats

    Full Text Available Sterile protection in >90% of volunteers against homologous Plasmodium falciparum infection has been achieved only using the controlled human malaria infection (CHMI model. This efficient model involves whole parasite immunizations under chloroquine prophylaxis (CPS-immunization, requiring only 30-45 mosquitoes bites infected with P. falciparum-sporozoites. Given the large diversity of P. falciparum parasites, it is essential to assess protection against heterologous parasite strains.In an open-label follow-up study, 16 volunteers previously CPS-immunized and challenged with P. falciparum NF54 (West-Africa in a dose de-escalation and challenge trial were re-challenged with clone NF135.C10 (Cambodia at 14 months after the last immunization (NCT01660854.Two out of thirteen NF54 protected volunteers previously fully protected against NF54 were also fully protected against NF135.C10, while 11/13 showed a delayed patency (median prepatent period of 10.5 days (range 9.0-15.5 versus 8.5 days in 5 malaria-naïve controls (p = 0.0005. Analysis of patency by qPCR indicated a 91 to >99% estimated reduction of liver parasite load in 7/11 partially protected subjects. Three volunteers previously not protected against NF54, were also not protected against NF135.C10.This study shows that CPS-immunization can induce heterologous protection for a period of more than one year, which is a further impetus for clinical development of whole parasite vaccines.Clinicaltrials.gov NCT01660854.

  5. Cyclophilin A enhances cell proliferation and tumor growth of liver fluke-associated cholangiocarcinoma

    Directory of Open Access Journals (Sweden)

    Sawanyawisuth Kanlayanee

    2011-08-01

    Full Text Available Abstract Background Cyclophilin A (CypA expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of CypA in liver fluke-associated cholangiocarcinoma (CCA are not presently known. In this study, we investigated the expression of CypA in CCA tumor tissues and CCA cell lines as well as regulation mechanisms of CypA in tumor growth using CCA cell lines. Methods CypA expression was determined by real time RT-PCR, Western blot or immunohistochemistry. CypA silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell proliferation was assessed using MTS assay or Ki-67 staining. The effect of silencing CypA on CCA tumor growth was determined in nude mice. The effect of CypA knockdown on ERK1/2 activation was assessed by Western blot. Results CypA was upregulated in 68% of CCA tumor tissues. Silencing CypA significantly suppressed cell proliferation in several CCA cell lines. Likewise, inhibition of CypA peptidyl-prolyl cis-trans isomerase (PPIase activity using cyclosporin A (CsA decreased cell proliferation. In contrast, overexpression of CypA resulted in 30% to 35% increases in proliferation of CCA cell lines. Interestingly, neither silence nor overexpression of CypA affected cell proliferation of a non-tumor human cholangiocyte cell line, MMNK1. Suppression of CypA expression attenuated ERK1/2 activity in CCA M139 cells by using both transient and stable knockdown methods. In the in vivo study, there was a 43% reduction in weight of tumors derived from CypA-silenced CCA cell lines compared with control vector CCA tumors in mice; these tumors with stable CypA silencing showed a reduced cell proliferation. Conclusions CypA is upregulated in majority of CCA patients' tissues and confers a significant growth advantage in CCA cells. Suppression of CypA expression decreases proliferation of CCA cell lines in vitro and reduces tumor growth in the nude mouse model. Inhibition of Cyp

  6. Inhibition of type I NKT cells by retinoids or following sulfatide-mediated activation of type II NKT cells attenuates alcoholic liver disease

    Science.gov (United States)

    Maricic, Igor; Sheng, Huiming; Marrero, Idania; Seki, Ehikiro; Kisseleva, Tatiana; Chaturvedi, Som; Molle, Natasha; Mathews, K. Stephanie; Gao, Bin; Kumar, Vipin

    2015-01-01

    Innate immune mechanisms leading to liver injury following chronic alcohol ingestion are poorly understood. Natural killer T (NKT) cells, enriched in the liver and comprised of at least two distinct subsets, type I and type II, recognize different lipid antigens presented by CD1d molecules. We have investigated whether differential activation of NKT cell subsets orchestrates inflammatory events leading to alcoholic liver disease (ALD). We found that following chronic plus binge feeding of Lieber-DeCarli liquid diet in male C57BL/6 mice, type I but not type II NKT cells are activated leading to recruitment of inflammatory Gr-1highCD11b+ cells into liver. A central finding is that liver injury following alcohol feeding is dependent upon type I NKT cells. Thus liver injury is significantly inhibited in Jα18−/− mice deficient in type I NKT cells as well as following their inactivation by sulfatide-mediated activation of type II NKT cells. Furthermore we have identified a novel pathway involving all-trans retinoic acid (ATRA) and its receptor RARγ signaling that inhibits type I NKT cells and consequently ALD. A semi-quantitative PCR analysis of hepatic gene expression of some of the key proinflammatory molecules shared in human disease indicated that their upregulation in ALD is dependent upon type I NKT cells. Conclusion Type I but not type II NKT cells become activated following alcohol feeding. Type I NKT cells-induced inflammation and neutrophil recruitment results in liver tissue damage while type II NKT cells protect from injury in ALD. Inhibition of type I NKT cells by retinoids or by sulfatide prevents ALD. Since the CD1d pathway is highly conserved between mice and humans, NKT cell subsets might be targeted for potential therapeutic intervention in ALD. PMID:25477000

  7. Past Life and Future Effects—How Heterologous Infections Alter Immunity to Influenza Viruses

    Directory of Open Access Journals (Sweden)

    Aisha Souquette

    2018-05-01

    Full Text Available Influenza virus frequently mutates due to its error-prone polymerase. This feature contributes to influenza virus’s ability to evade pre-existing immunity, leading to annual epidemics and periodic pandemics. T cell memory plays a key protective role in the face of an antigenically distinct influenza virus strain because T cell targets are often derived from conserved internal proteins, whereas humoral immunity targets are often sites of increased mutation rates that are tolerated by the virus. Most studies of influenza T cell memory are conducted in naive, specific pathogen free mice and do not account for repetitive influenza infection throughout a lifetime, sequential acute heterologous infections between influenza infections, or heterologous chronic co-infections. By contrast to these mouse models, humans often experience numerous influenza infections, encounter heterologous acute infections between influenza infections, and are infected with at least one chronic virus. In this review, we discuss recent advances in understanding the effects of heterologous infections on the establishment and maintenance of CD8+ T cell immunological memory. Understanding the various factors that affect immune memory can provide insights into the development of more effective vaccines and increase reproducibility of translational studies between animal models and clinical results.

  8. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Anthony Skipper

    2016-01-01

    Full Text Available Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals. Many documented studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and several forms of cancer are attributed to overexposure to cadmium.  Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, its molecular mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG2 cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay. The result of MTT assay indicated that cadmium chloride induces toxicity to HepG2 cells in a concentration-dependent manner, showing a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant (p < 0.05 increase of hydroperoxide production, specifically at the highest concentration tested. Data obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG2 cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05 was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage, and programmed cell death in human liver carcinoma (HepG2 cells.

  9. A case of Esophageal small cell carcinoma with multiple liver metastases responding to chemotherapy with Irinotecan plus Cisplatin

    International Nuclear Information System (INIS)

    Endo, K.; Kohnoe, S.; Toh, Y.; Haraguchi, M.; Okamura, T.; Nishiyama, K.; Baba, H.; Maehara, Y.

    2005-01-01

    We report a case of small cell esophageal carcinoma (SCEC) with multiple liver metastases treated with some success by chemotherapy with irinotecan (CPT-11) plus cisplatin (CDDP). Radiologic and endoscopic examination of a 75-year-old man with multiple liver tumors disclosed a 4.0-cm type 2 tumor in the middle third of the esophagus. An endoscopically obtained biopsy specimen was diagnosed as undifferentiated small cell carcinoma. Multiple liver metastases were confirmed but lymph node metastases and distant metastases other than those in the liver were not detected. After six courses of chemotherapy with CPT-11 plus CDDP, the primary lesion showed complete response and liver metastases showed partial response. However, because all lesions almost immediately relapsed or progressed, arterial infusion chemotherapy for liver metastases and radiation for the primary lesion were given as second-line treatment. The primary lesion showed complete response with radiation. Arterial infusion chemotherapy prevented the progression of liver metastases once, but the patient died of liver failure at last. No distant lesions including metastatic lymph nodes were confirmed over the course of his illness, and the patient survived for a year after first diagnosis. Although the prognosis of SCEC is quite unfavorable due to highly aggressive behavior, a better prognosis is possible with effective chemotherapy and second-line treatment is important in improving prognosis

  10. Arctigenin protects against liver injury from acute hepatitis by suppressing immune cells in mice.

    Science.gov (United States)

    Cheng, Xixi; Wang, Huafeng; Yang, Jinlai; Cheng, Yingnan; Wang, Dan; Yang, Fengrui; Li, Yan; Zhou, Dongmei; Wang, Yanxia; Xue, Zhenyi; Zhang, Lijuan; Zhang, Qi; Yang, Luhong; Zhang, Rongxin; Da, Yurong

    2018-06-01

    As a phenylpropanoid and dibenzylbutyrolactone lignan present in medical plants, such as those used in traditional Chinese herbal medicine, including Arctium lappa (Niubang), arctigenin exhibits antimicrobial, anti-inflammatory, and anticancer activities. In this study, we investigated the protective role of arctigenin in Concanavalin A (ConA)-induced acute hepatitis in mice. Arctigenin remarkably reduced the congestion and necroinflammation of livers, and improved hepatic function (ALT and AST) in ConA-induced acute hepatitis in vivo. The infiltration of CD4 T, NKT and macrophages into the livers was found to be reduced with arctigenin treatment. Arctigenin suppressed ConA-induced T lymphocyte proliferations that might have resulted from enhanced IL-10 production by macrophages and CD4 T cells. These results suggested that arctigenin could be a powerful drug candidate for acute hepatitis through immune suppression. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  11. ADAM12 in human liver cancers: TGF-beta-regulated expression in stellate cells is associated with matrix remodeling

    DEFF Research Database (Denmark)

    Le Pabic, Hélène; Bonnier, Dominique; Wewer, Ulla M

    2003-01-01

    "A disintegrin and metalloproteinases" (ADAMs) form a family of cell-surface glycoproteins with potential protease and cell-adhesion activities. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT...... carcinomas (up to 3- and 6-fold, respectively) and liver metastases from colonic carcinomas (up to 40- and 60-fold, respectively). The up-regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12......-PCR, cDNA encoding sequences for ADAM9 and ADAM12 were identified in human activated hepatic stellate cells (HSCs). Northern blot analyses showed that HSCs, but not hepatocytes, expressed transcripts for ADAM9 messenger RNA (mRNA) and both the long and short forms of ADAM12. This expression...

  12. Immunohistochemical analyses of cell cycle progression and gene expression of biliary epithelial cells during liver regeneration after partial hepatectomy of the mouse.

    Science.gov (United States)

    Fukuda, Tatsuya; Fukuchi, Tomokazu; Yagi, Shinomi; Shiojiri, Nobuyoshi

    2016-05-20

    The liver has a remarkable regeneration capacity, and, after surgical removal of its mass, the remaining tissue undergoes rapid regeneration through compensatory growth of its constituent cells. Although hepatocytes synchronously proliferate under the control of various signaling molecules from neighboring cells, there have been few detailed analyses on how biliary cells regenerate for their cell population after liver resection. The present study was undertaken to clarify how biliary cells regenerate after partial hepatectomy of mice through extensive analyses of their cell cycle progression and gene expression using immunohistochemical and RT-PCR techniques. When expression of PCNA, Ki67 antigen, topoisomerase IIα and phosphorylated histone H3, which are cell cycle markers, was immunohistochemically examined during liver regeneration, hepatocytes had a peak of the S phase and M phase at 48-72 h after resection. By contrast, biliary epithelial cells had much lower proliferative activity than that of hepatocytes, and their peak of the S phase was delayed. Mitotic figures were rarely detectable in biliary cells. RT-PCR analyses of gene expression of biliary markers such as Spp1 (osteopontin), Epcam and Hnf1b demonstrated that they were upregulated during liver regeneration. Periportal hepatocytes expressed some of biliary markers, including Spp1 mRNA and protein. Some periportal hepatocytes had downregulated expression of HNF4α and HNF1α. Gene expression of Notch signaling molecules responsible for cell fate decision of hepatoblasts to biliary cells during development was upregulated during liver regeneration. Notch signaling may be involved in biliary regeneration.

  13. Honey can repairing damage of liver tissue due to protein energy malnutrition through induction of endogenous stem cells

    Directory of Open Access Journals (Sweden)

    R. Heru Prasetyo

    2017-06-01

    Full Text Available Aim: This study was to evaluate effect of honey in repairing damage of liver tissue due to energy protein malnutrition and in mobilization of endogenous stem cells. Materials and Methods: Male mice model of degenerative liver was obtained through food fasting but still have drinking water for 5 days. It caused energy protein malnutrition and damage of liver tissue. The administration of 50% (v/v honey was performed for 10 consecutive days, while the positive control group was fasted and not given honey and the negative control not fasted and without honey. Observations of regeneration the liver tissue based on histologically examination, observation of Hsp70 expression, and homing signal based on vascular endothelial growth factor-1 (VEGF-1 expression using immunohistochemistry technique. Observation on expression of CD34 and CD45 as the marker of auto mobilization of hematopoietic stem cells using flow cytometry technique. Results: There is regeneration of the liver tissue due to protein energy malnutrition, decrease of Hsp70 expression, increase of VEGF-1 expression, and high expression of CD34 and CD45. Conclusion: Honey can improve the liver tissue based on: (1 Mobilization of endogenous stem cells (CD34 and CD45; (2 Hsp70 and VEGF-1 expressions as regeneration marker of improvement, and (3 regeneration histologically of liver tissue.

  14. Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

    Directory of Open Access Journals (Sweden)

    Ruchi Sharma

    2010-01-01

    Full Text Available The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

  15. Honey can repairing damage of liver tissue due to protein energy malnutrition through induction of endogenous stem cells.

    Science.gov (United States)

    Prasetyo, R Heru; Hestianah, Eka Pramyrtha

    2017-06-01

    This study was to evaluate effect of honey in repairing damage of liver tissue due to energy protein malnutrition and in mobilization of endogenous stem cells. Male mice model of degenerative liver was obtained through food fasting but still have drinking water for 5 days. It caused energy protein malnutrition and damage of liver tissue. The administration of 50% (v/v) honey was performed for 10 consecutive days, while the positive control group was fasted and not given honey and the negative control not fasted and without honey. Observations of regeneration the liver tissue based on histologically examination, observation of Hsp70 expression, and homing signal based on vascular endothelial growth factor-1 (VEGF-1) expression using immunohistochemistry technique. Observation on expression of CD34 and CD45 as the marker of auto mobilization of hematopoietic stem cells using flow cytometry technique. There is regeneration of the liver tissue due to protein energy malnutrition, decrease of Hsp70 expression, increase of VEGF-1 expression, and high expression of CD34 and CD45. Honey can improve the liver tissue based on: (1) Mobilization of endogenous stem cells (CD34 and CD45); (2) Hsp70 and VEGF-1 expressions as regeneration marker of improvement, and (3) regeneration histologically of liver tissue.

  16. Induction of regulatory T cells by high-dose gp96 suppresses murine liver immune hyperactivation.

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    Xinghui Li

    Full Text Available Immunization with high-dose heat shock protein gp96, an endoplasmic reticulum counterpart of the Hsp90 family, significantly enhances regulatory T cell (Treg frequency and suppressive function. Here, we examined the potential role and mechanism of gp96 in regulating immune-mediated hepatic injury in mice. High-dose gp96 immunization elicited rapid and long-lasting protection of mice against concanavalin A (Con A-and anti-CD137-induced liver injury, as evidenced by decreased alanine aminotransaminase (ALT levels, hepatic necrosis, serum pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-6, and number of IFN-γ (+ CD4(+ and IFN-γ (+ CD8(+ T cells in the spleen and liver. In contrast, CD4(+CD25(+Foxp3(+ Treg frequency and suppressive function were both increased, and the protective effect of gp96 could be generated by adoptive transfer of Treg cells from gp96-immunized mice. In vitro co-culture experiments demonstrated that gp96 stimulation enhanced Treg proliferation and suppressive function, and up-regulation of Foxp3, IL-10, and TGF-β1 induced by gp96 was dependent on TLR2- and TLR4-mediated NF-κB activation. Our work shows that activation of Tregs by high-dose gp96 immunization protects against Con A- and anti-CD137-induced T cell-hepatitis and provides therapeutic potential for the development of a gp96-based anti-immune hyperactivation vaccine against immune-mediated liver destruction.

  17. Effects of the ionizing radiations, freezing and thawing duration on chicken liver cells quality

    International Nuclear Information System (INIS)

    Duarte, R.C.; Araujo, M.M.; Salum, D.C.; Marchioni, E.; Villavicencio, A.L.C.H.

    2009-01-01

    All food storage processes modify the food. Irradiation reduces and could stop cell division, avoid infestation, reduce contamination and delay food decomposition. The cold chain is a succession of steps which maintains the food at low temperature. Defrosted food shall never be frozen again, the best way being to consume it quickly then avoiding multiplication and acceleration of microbial growth, which causes decay and nutrients damage. The Comet Assay indicates DNA damage and can then be used to control the overall quality of the food and in a certain extent to evaluate the damage caused by irradiation and storage on liver chicken cells. In this work, different thawing temperatures and radiation doses were checked to establish a 'DNA damage index' by using the Comet Assay. Samples were irradiated in a 60 Co irradiator with 1.5, 3.0 and 4.5 kGy radiation doses. Our results showed that no intact cells were detected in frozen samples: however, irradiated liver samples in natura showed some intact cells depending on the applied radiation doses.

  18. Stem cell injury and restitution after ionizing irradiation in intestine, liver, salivary gland, mesenteric lymph node

    International Nuclear Information System (INIS)

    Lee, Jae Hyun; Cho, Kyung Ja; Lee, Sun Joo; Jang, Won Suk

    1998-01-01

    There is little information about radiation injury on stem cell resident in other organs. In addition there is little experimental model in which radiation plays a role on proliferation stem cell in adult organ. This study was carried out to evaluate the early response of tissue injury and restitution in intestine, liver, salivary gland and lymph node, and to develop in vivo model to investigate stem cell biology by irradiation. The study is to assay the early response to radiation and setup an animal model for radiation effect on cellular response. Duodenal intestine, liver, submandibular salivary gland and mesenteric lymph node were selected to compare apoptosis and proliferating cell nuclear antigen (PCNA) expression to radiosensitivity. For the effect of radiation on cellular responses, rats were irradiated during starvation. Conclusionly, this study showed the value of apoptosis in detection system for evaluating cellular damage against radiation injury. Because apoptosis was regularly inducted depending on tissue-specific pattern, dose and time sequence as well as cellular activity. Furthermore in vivo model in the study will be helped in the further study to elucidate the relationship between radiation injury and starvation or malnutrition. (author). 22 refs., 6 figs

  19. The Relationship between fenestrations, sieve plates and rafts in liver sinusoidal endothelial cells.

    Directory of Open Access Journals (Sweden)

    Dmitri Svistounov

    Full Text Available Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished.

  20. Effects of the ionizing radiations, freezing and thawing duration on chicken liver cells quality

    Energy Technology Data Exchange (ETDEWEB)

    Duarte, R.C. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Av. Professor Lineu Prestes 2242 Zip code, 05508-000 Cidade Universitaria Sao Paulo, SP-Brazil (Brazil)], E-mail: renatocduarte@yahoo.com.br; Araujo, M.M.; Salum, D.C. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Av. Professor Lineu Prestes 2242 Zip code, 05508-000 Cidade Universitaria Sao Paulo, SP-Brazil (Brazil); Marchioni, E. [IPHC-UMR 7178-Faculte de Pharmacie, 74 route du Rhin, 67400 Illkirch (France); Villavicencio, A.L.C.H. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Av. Professor Lineu Prestes 2242 Zip code, 05508-000 Cidade Universitaria Sao Paulo, SP-Brazil (Brazil)], E-mail: villavic@ipen.br

    2009-07-15

    All food storage processes modify the food. Irradiation reduces and could stop cell division, avoid infestation, reduce contamination and delay food decomposition. The cold chain is a succession of steps which maintains the food at low temperature. Defrosted food shall never be frozen again, the best way being to consume it quickly then avoiding multiplication and acceleration of microbial growth, which causes decay and nutrients damage. The Comet Assay indicates DNA damage and can then be used to control the overall quality of the food and in a certain extent to evaluate the damage caused by irradiation and storage on liver chicken cells. In this work, different thawing temperatures and radiation doses were checked to establish a 'DNA damage index' by using the Comet Assay. Samples were irradiated in a {sup 60}Co irradiator with 1.5, 3.0 and 4.5 kGy radiation doses. Our results showed that no intact cells were detected in frozen samples: however, irradiated liver samples in natura showed some intact cells depending on the applied radiation doses.

  1. A Novel Small-molecule WNT Inhibitor, IC-2, Has the Potential to Suppress Liver Cancer Stem Cells.

    Science.gov (United States)

    Seto, Kenzo; Sakabe, Tomohiko; Itaba, Noriko; Azumi, Junya; Oka, Hiroyuki; Morimoto, Minoru; Umekita, Yoshihisa; Shiota, Goshi

    2017-07-01

    The presence of cancer stem cells (CSCs) contributes to metastasis, recurrence, and resistance to chemo/radiotherapy in hepatocellular carcinoma (HCC). The WNT signaling pathway is reportedly linked to the maintenance of stemness of CSCs. In the present study, in order to eliminate liver CSCs and improve the prognosis of patients with HCC, we explored whether small-molecule compounds targeting WNT signaling pathway suppress liver CSCs. The screening was performed using cell proliferation assay and reporter assay. We next investigated whether these compounds suppress liver CSC properties by using flow cytometric analysis and sphere-formation assays. A mouse xenograft model transplanted with CD44-positive HuH7 cells was used to examine the in vivo antitumor effect of IC-2. In HuH7 human HCC cells, 10 small-molecule compounds including novel derivatives, IC-2 and PN-3-13, suppressed cell viability and WNT signaling activity. Among them, IC-2 significantly reduced the CD44-positive population, also known as liver CSCs, and dramatically reduced the sphere-forming ability of both CD44-positive and CD44-negative HuH7 cells. Moreover, CSC marker-positive populations, namely CD90-positive HLF cells, CD133-positive HepG2 cells, and epithelial cell adhesion molecule-positive cells, were also reduced by IC-2 treatment. Finally, suppressive effects of IC-2 on liver CSCs were also observed in a xenograft model using CD44-positive HuH7 cells. The novel derivative of small-molecule WNT inhibitor, IC-2, has the potential to suppress liver CSCs and can serve as a promising therapeutic agent to improve the prognosis of patients with HCC. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  2. FXR blocks the growth of liver cancer cells through inhibiting mTOR-s6K pathway

    International Nuclear Information System (INIS)

    Huang, Xiongfei; Zeng, Yeting; Wang, Xinrui; Ma, Xiaoxiao; Li, Qianqian; Li, Ningbo; Su, Hongying; Huang, Wendong

    2016-01-01

    The nuclear receptor Farnesoid X Receptor (FXR) is likely a tumor suppressor in liver tissue but its molecular mechanism of suppression is not well understood. In this study, the gene expression profile of human liver cancer cells was investigated by microarray. Bioinformatics analysis of these data revealed that FXR might regulate the mTOR/S6K signaling pathway. This was confirmed by altering the expression level of FXR in liver cancer cells. Overexpression of FXR prevented the growth of cells and induced cell cycle arrest, which was enhanced by the mTOR/S6K inhibitor rapamycin. FXR upregulation also intensified the inhibition of cell growth by rapamycin. Downregulation of FXR produced the opposite effect. Finally, we found that ectopic expression of FXR in SK-Hep-1 xenografts inhibits tumor growth and reduces expression of the phosphorylated protein S6K. Taken together, our data provide the first evidence that FXR suppresses proliferation of human liver cancer cells via the inhibition of the mTOR/S6K signaling pathway. FXR expression can be used as a biomarker of personalized mTOR inhibitor treatment assessment for liver cancer patients. -- Highlights: •FXR inhibits the proliferation of liver cancer cells by prolonging G0/G1 phase. •Microarray results indicate that mTOR-S6k signaling is involved in cellular processes in which FXR plays an important role. •FXR blocks the growth of liver cancer cells via the inhibition of the mTOR/S6K signaling pathway in vitro and in vivo.

  3. FXR blocks the growth of liver cancer cells through inhibiting mTOR-s6K pathway

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xiongfei, E-mail: xiongfeihuang@hotmail.com [Department of Pathology and Institute of Oncology, Preclinical School, Fujian Medical University, Fuzhou 350108, Fujian (China); Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, Fuzhou 350108, Fujian (China); Zeng, Yeting [Department of Pathology and Institute of Oncology, Preclinical School, Fujian Medical University, Fuzhou 350108, Fujian (China); Wang, Xinrui [Department of Biochemistry and Molecular Biology, Fujian Medical University, Fuzhou 350108, Fujian (China); Ma, Xiaoxiao [Department of Diabetes Complications and Metabolism, Diabetes & Metabolism Research Institute, Beckman Research Institute, City of Hope, CA 91010 (United States); Li, Qianqian; Li, Ningbo; Su, Hongying [Department of Pathology and Institute of Oncology, Preclinical School, Fujian Medical University, Fuzhou 350108, Fujian (China); Huang, Wendong [Department of Diabetes Complications and Metabolism, Diabetes & Metabolism Research Institute, Beckman Research Institute, City of Hope, CA 91010 (United States)

    2016-05-27

    The nuclear receptor Farnesoid X Receptor (FXR) is likely a tumor suppressor in liver tissue but its molecular mechanism of suppression is not well understood. In this study, the gene expression profile of human liver cancer cells was investigated by microarray. Bioinformatics analysis of these data revealed that FXR might regulate the mTOR/S6K signaling pathway. This was confirmed by altering the expression level of FXR in liver cancer cells. Overexpression of FXR prevented the growth of cells and induced cell cycle arrest, which was enhanced by the mTOR/S6K inhibitor rapamycin. FXR upregulation also intensified the inhibition of cell growth by rapamycin. Downregulation of FXR produced the opposite effect. Finally, we found that ectopic expression of FXR in SK-Hep-1 xenografts inhibits tumor growth and reduces expression of the phosphorylated protein S6K. Taken together, our data provide the first evidence that FXR suppresses proliferation of human liver cancer cells via the inhibition of the mTOR/S6K signaling pathway. FXR expression can be used as a biomarker of personalized mTOR inhibitor treatment assessment for liver cancer patients. -- Highlights: •FXR inhibits the proliferation of liver cancer cells by prolonging G0/G1 phase. •Microarray results indicate that mTOR-S6k signaling is involved in cellular processes in which FXR plays an important role. •FXR blocks the growth of liver cancer cells via the inhibition of the mTOR/S6K signaling pathway in vitro and in vivo.

  4. Adaptation in the innate immune system and heterologous innate immunity.

    Science.gov (United States)

    Martin, Stefan F

    2014-11-01

    The innate immune system recognizes deviation from homeostasis caused by infectious or non-infectious assaults. The threshold for its activation seems to be established by a calibration process that includes sensing of microbial molecular patterns from commensal bacteria and of endogenous signals. It is becoming increasingly clear that adaptive features, a hallmark of the adaptive immune system, can also be identified in the innate immune system. Such adaptations can result in the manifestation of a primed state of immune and tissue cells with a decreased activation threshold. This keeps the system poised to react quickly. Moreover, the fact that the innate immune system recognizes a wide variety of danger signals via pattern recognition receptors that often activate the same signaling pathways allows for heterologous innate immune stimulation. This implies that, for example, the innate immune response to an infection can be modified by co-infections or other innate stimuli. This "design feature" of the innate immune system has many implications for our understanding of individual susceptibility to diseases or responsiveness to therapies and vaccinations. In this article, adaptive features of the innate immune system as well as heterologous innate immunity and their implications are discussed.

  5. Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2

    Directory of Open Access Journals (Sweden)

    Hui Xu

    2015-01-01

    Full Text Available The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA and Csac_1078 (celB from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA and TM0070 (xynB, resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization.

  6. A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination.

    Science.gov (United States)

    León-Ortiz, Ana María; Panier, Stephanie; Sarek, Grzegorz; Vannier, Jean-Baptiste; Patel, Harshil; Campbell, Peter J; Boulton, Simon J

    2018-01-18

    Erroneous DNA repair by heterologous recombination (Ht-REC) is a potential threat to genome stability, but evidence supporting its prevalence is lacking. Here we demonstrate that recombination is possible between heterologous sequences and that it is a source of chromosomal alterations in mitotic and meiotic cells. Mechanistically, we find that the RTEL1 and HIM-6/BLM helicases and the BRCA1 homolog BRC-1 counteract Ht-REC in Caenorhabditis elegans, whereas mismatch repair does not. Instead, MSH-2/6 drives Ht-REC events in rtel-1 and brc-1 mutants and excessive crossovers in rtel-1 mutant meioses. Loss of vertebrate Rtel1 also causes a variety of unusually large and complex structural variations, including chromothripsis, breakage-fusion-bridge events, and tandem duplications with distant intra-chromosomal insertions, whose structure are consistent with a role for RTEL1 in preventing Ht-REC during break-induced replication. Our data establish Ht-REC as an unappreciated source of genome instability that underpins a novel class of complex genome rearrangements that likely arise during replication stress. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Orphan nuclear receptor NR4A2 inhibits hepatic stellate cell proliferation through MAPK pathway in liver fibrosis.

    Science.gov (United States)

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Li, Bin; Gan, Run; Guo, Cheng

    2015-01-01

    Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis, which is a pathological process characterized by extracellular matrix accumulation. NR4A2 is a nuclear receptor belonging to the NR4A subfamily and vital in regulating cell growth, metabolism, inflammation and other biological functions. However, its role in HSCs is unclear. We analyzed NR4A2 expression in fibrotic liver and stimulated HSCs compared with control group and studied the influence on cell proliferation, cell cycle, cell apoptosis and MAPK pathway after NR4A2 knockdown. NR4A2 expression was examined by real-time polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence analyses. NR4A2 expression was significantly lower in fibrotic liver tissues and PDGF BB or TGF-β stimulated HSCs compared with control group. After NR4A2 knockdown α-smooth muscle actin and Col1 expression increased. In addition, NR4A2 silencing led to the promotion of cell proliferation, increase of cell percentage in S phase and reduced phosphorylation of ERK1/2, P38 and JNK in HSCs. These results indicate that NR4A2 can inhibit HSC proliferation through MAPK pathway and decrease extracellular matrix in liver fibrogenesis. NR4A2 may be a promising therapeutic target for liver fibrosis.

  8. The IL-33-ST2-MyD88 axis promotes regulatory T cell proliferation in the murine liver.

    Science.gov (United States)

    Xu, Lei; Li, Wei; Wang, Xiaofan; Zhang, Lina; Qi, Qianqian; Dong, Liyang; Wei, Chuan; Pu, Yanan; Li, Yalin; Zhu, Jifeng; Zhou, Sha; Liu, Feng; Chen, Xiaojun; Su, Chuan

    2018-05-05

    Hepatic Foxp3 + regulatory T (Treg) cells are crucial for maintaining local immune homeostasis in the liver. However, the environmental cues required for hepatic Treg cell homeostasis are unclear. In this study, we showed that the IL-33 receptor ST2 was preferentially expressed on Treg cells in the mouse liver, but it was more lowly expressed in the spleen, mesenteric lymph nodes, and blood. More importantly, we found that IL-33 promoted the proliferation of hepatic Treg cells through myeloid differentiation factor MyD88 signaling concomitant with increased CDK4 and cyclin D1 expression. These results suggested that IL-33 is a potential tissue-specific factor controlling Treg cell homeostasis via increased Treg proliferation in the liver. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  9. Silent information regulator 1 (SIRT1) ameliorates liver fibrosis via promoting activated stellate cell apoptosis and reversion

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yuting, E-mail: wuyuting1302@sina.com; Liu, Xuejiao; Zhou, Qun; Huang, Cheng; Meng, Xiaoming; Xu, Fengyun; Li, Jun, E-mail: lj@ahmu.edu.cn

    2015-12-01

    SIRT1 (silent information regulator 1), a conserved NAD +-dependent histone deacetylase, is closely related with various biological processes. Moreover, the important role of SIRT1 in alcoholic liver disease, nonalcoholic fatty liver and HCC had been widely reported. Recently, a novel role of SIRT1 was uncovered in organ fibrosis diseases. Here, we investigated the inhibitory effect of SIRT1 in liver fibrogenesis. SIRT1 protein was dramatically decreased in CCl4-treated mice livers. Stimulation of LX-2 cells with TGF-β1 also resulted in a significant suppression of SIRT1 protein. Nevertheless, TGF-β1-induced LX-2 cell activation was inhibited by SIRT1 plasmid, and this was accompanied by up-regulation of cell apoptosis-related proteins. Overexpression of SIRT1 also attenuated TGF-β1-induced expression of myofibroblast markers α-SMA and COL1a. However, the important characteristic of the recovery of liver fibrosis is not only the apoptosis of activated stellate cells but also the reversal of the myofibroblast-like phenotype to a quiescent-like phenotype. Restoration of SIRT1 protein was observed in the in vivo spontaneously liver fibrosis reversion model and in vitro MDI (isobutylmethylxanthine, dexamethasone, and insulin)-induced reversed stellate cells, and forced expression of SIRT1 also promoted the reversal of activated stellate cells. Furthermore, lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was increased in liver fibrosis. RNAi-mediated suppression of MALAT1 resulted in a decrease of myofibroblast markers and restoration of SIRT1 protein. These observations suggested that SIRT1 contributed to apoptosis and reversion of activated LX-2 cells and SIRT1 might be regulated by MALAT1 in liver fibrosis. Therefore, SIRT1 could be considered as a valuable therapeutic target for translational studies of liver fibrosis. - Highlights: • This is the first report of SIRT1 expression and function in liver fibrogenesis and reversion.

  10. Overcoming tumor resistance by heterologous adeno-poxvirus combination therapy

    Directory of Open Access Journals (Sweden)

    Markus Vähä-Koskela

    2014-01-01

    Full Text Available Successful cancer control relies on overcoming resistance to cell death and on activation of host antitumor immunity. Oncolytic viruses are particularly attractive in this regard, as they lyse infected tumor cells and trigger robust immune responses during the infection. However, repeated injections of the same virus promote antiviral rather than antitumor immunity and tumors may mount innate antiviral defenses to restrict oncolytic virus replication. In this article, we have explored if alternating the therapy virus could circumvent these problems. We demonstrate in two virus-resistant animal models a substantial delay in antiviral immune- and innate cellular response induction by alternating injections of two immunologically distinct oncolytic viruses, adenovirus, and vaccinia virus. Our results are in support of clinical development of heterologous adeno-/vaccinia virus therapy of cancer.

  11. Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/Bcl-2 ratio

    Directory of Open Access Journals (Sweden)

    Azimahtol Hawariah LP

    2007-04-01

    Full Text Available Abstract Background Zerumbone is a cytotoxic component isolated from Zingiber zerumbet Smith, a herbal plant which is also known as lempoyang. This new anticancer bioactive compound from Z. zerumbet was investigated for its activity and mechanism in human liver cancer cell lines. Results Zerumbone significantly showed an antiproliferative activity upon HepG2 cells with an IC50 of 3.45 ± 0.026 μg/ml. Zerumbone was also found to inhibit the proliferation of non-malignant Chang Liver and MDBK cell lines. However the IC50 obtained was higher compared to the IC50 for HepG2 cells (> 10 μg/ml. The extent of DNA fragmentation was evaluated by the Tdt-mediated dUTP nick end labelling assay which showed that, zerumbone significantly increased apoptosis in HepG2 cells in a time-course manner. In detail, the apoptotic process triggered by zerumbone involved the up-regulation of pro-apoptotic Bax protein and the suppression of anti-apoptotic Bcl-2 protein expression. The changes that occurred in the levels of this antagonistic proteins Bax/Bcl-2, was independent of p53 since zerumbone did not affect the levels of p53 although this protein exists in a functional form. Western blotting analysis for Bax protein was further confirmed qualitatively with an immunoassay that showed the distribution of Bax protein in zerumbone-treated cells. Conclusion Therefore, zerumbone was found to induce the apoptotic process in HepG2 cells through the up and down regulation of Bax/Bcl-2 protein independently of functional p53 activity.

  12. In situ targeting of dendritic cells sets tolerogenic environment and ameliorates CD4+ T-cell response in the postischemic liver.

    Science.gov (United States)

    Funken, Dominik; Ishikawa-Ankerhold, Hellen; Uhl, Bernd; Lerchenberger, Maximilian; Rentsch, Markus; Mayr, Doris; Massberg, Steffen; Werner, Jens; Khandoga, Andrej

    2017-11-01

    CD4 + T cells recruited to the liver play a key role in the pathogenesis of ischemia/reperfusion (I/R) injury. The mechanism of their activation during alloantigen-independent I/R is not completely understood. We hypothesized that liver-resident dendritic cells (DCs) interact with CD4 + T cells in the postischemic liver and that modulation of DCs or T-cell-DC interactions attenuates liver inflammation. In mice, warm hepatic I/R (90/120-240 min) was induced. Tolerogenic DCs were generated in situ by pretreatment of animals with the vitamin D analog paricalcitol. A mAb-CD44 was used for blockade of CD4 + T-cell-DC interactions. As shown by 2-photon in vivo microscopy as well as confocal microscopy, CD4 + T cells were closely colocalized with DCs in the postischemic liver. Pretreatment with paricalcitol attenuated I/R-induced maturation of DCs (flow cytometry), CD4 + T-cell recruitment into the liver (intravital microscopy), and hepatocellular/microvascular damage (intravital microscopy, alanine aminotransferase/aspartate aminotransferase, histology). However, interruption of T-cell-DC interaction increased proinflammatory DC maturation and even enhanced tissue damage. Simultaneous treatment with an anti-CD44mAb completely abolished the beneficial effect of paricalcitol on T-cell migration and tissue injury. Our study demonstrates for the first time that hepatic DCs interact with CD4 + T cells in the postischemic liver in vivo ; modulation of DCs and/or generation of tolerogenic DCs attenuates intrahepatic CD4 + T-cell recruitment and reduces I/R injury; and interruption of CD44-dependent CD4 + T-cell-DC interactions enhances tissue injury by preventing the modulatory effect of hepatic DCs on T cells, especially type 1 T helper effector cells. Thus, hepatic DCs are strongly involved in the promotion of CD4 + T-cell-dependent postischemic liver inflammation.-Funken, D., Ishikawa-Ankerhold, H., Uhl, B., Lerchenberger, M., Rentsch, M., Mayr, D., Massberg, S., Werner, J

  13. Hepatic stellate cell-targeted imatinib nanomedicine versus conventional imatinib: A novel strategy with potent efficacy in experimental liver fibrosis.

    Science.gov (United States)

    El-Mezayen, Nesrine S; El-Hadidy, Wessam F; El-Refaie, Wessam M; Shalaby, Th I; Khattab, Mahmoud M; El-Khatib, Aiman S

    2017-11-28

    Liver fibrosis is a global health problem without approved treatment. Imatinib inhibits two key profibrotic pathways; platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-β) and thus can be used to treat liver fibrosis. However, conventional imatinib therapy is hampered by low concentration at target tissue and increased toxicity to other tissues especially heart, lung and liver. Since hepatic stellate cells (HSCs) are the main contributors to liver fibrosis pathogenesis and sole hepatic vitamin A (V A ) storage cells, they can be actively targeted by coupling liposomes to V A . In this study, novel V A -coupled imatinib-loaded liposomes (ILC) were prepared and optimized regarding V A -coupling efficiency, imatinib entrapment efficiency, and particle size. Preferential accumulation of the selected formula in liver was proved by tracing intraperitoneally (i.p.)-injected V A -coupled liposomes loaded with Nile Red (LCNR) to rats with CCl 4 -induced liver fibrosis using live animal imaging. Co-localization of LCNR with immunofluorescently-labeled PDGFR-β in frozen liver tissue sections confirmed HSCs targeting. ILC bio-distribution, following single i.p. injection, revealed 13.5 folds higher hepatic accumulation than conventional imatinib in addition to limited bio-distribution to other organs including heart and lung reflecting diminished adverse effects. ILC therapy resulted in a potent inhibition of phosphorylated PDGFR-β expression when compared to conventional imatinib. Subsequently, there was a statistically significant improvement in liver function tests and reversal of hepatotoxicity along with liver fibrosis. Anti-fibrotic effect was evident from histopathologic Ishak score reduction as well as normalization of the level of profibrotic mediators (hydroxyproline, TGF-B and matrix metalloproteinase-2). Thus, HSC-targeted imatinib therapy shows outstanding anti-fibrotic effects with reduced cytotoxicity compared to conventional

  14. Bone marrow-derived mesenchymal stem cells effectively regenerate fibrotic liver in bile duct ligation rat model.

    Science.gov (United States)

    Mohamed, Hoda E; Elswefy, Sahar E; Rashed, Laila A; Younis, Nahla N; Shaheen, Mohamed A; Ghanim, Amal M H

    2016-03-01

    Mesenchymal stem cells (MSCs) have attracted lots of attention for the treatment of acute liver failure and end-stage liver diseases. This study aimed at investigating the fundamental mechanism by which bone marrow-derived MSCs (BM-MSCs) induce liver regeneration of fibrotic liver in rats. Rats underwent bile duct ligation (BDL) surgery and four weeks later they were treated with either BM-MSCs (3 × 10(6) cells /rat, once, tail vein injection) or silymarin (100 mg/kg, daily, orally) for four weeks. Liver function tests and hepatic oxidative stress were determined. Hepatic injury and fibrosis were assessed by H and E, Sirus red staining and immunohistochemical expression of α-smooth muscle actin (α-SMA). Hepatocyte growth factor (HGF) and the gene expression of cytokeratin-19 (CK-19) and matrix metalloproteinase-2 (MMP-2) in liver tissue were determined. BDL induced cholestatic liver injury characterized by elevated ALT and AST activities, bilirubin and decreased albumin. The architecture damage was staged as Metavir score: F3, A3. Fibrosis increased around proliferating bile duct as indicated by sirus red staining and α-SMA immunostaining. Fibrogenesis was favored over fibrolysis and confirmed by decreased HGF with increased expression of CK-19, but decreased MMP-2 expression. BM-MSCs treatment restored deteriorated liver functions and restored the histological changes, resolved fibrosis by improving liver regenerative capabilities (P liver regenerative capabilities can be stimulated by BM-MSCs via augmentation of HGF that subsequently up-regulate MMP-2 mRNA while downregulating CK-19 mRNA. © 2016 by the Society for Experimental Biology and Medicine.

  15. CAR-mediated repression of Foxo1 transcriptional activity regulates the cell cycle inhibitor p21 in mouse livers

    International Nuclear Information System (INIS)

    Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O.

    2014-01-01

    Highlights: • CAR activation decreased the level of Foxo1 in mouse livers. • CAR activation decreased the level of p21 in mouse livers. • CAR activation inhibited Foxo1 transcriptional activity in mouse livers. - Abstract: 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments

  16. The novel protein C9orf116 promotes rat liver cell line BRL-3A proliferation.

    Directory of Open Access Journals (Sweden)

    Chunyan Zhang

    Full Text Available Our previous study has proved that the chromosome 9 open reading frame 116 (C9orf116 (NM_001106564.1 was significantly up-regulated in the proliferation phase of liver regeneration. To study its possible physiological function, we analyzed the effect of C9orf116 on BRL-3A cells via over-expression and interference technique. MTT results showed that the cell viability of the interference group was significantly lower than the control group at 48h after transfection (P<0.05, whereas it was significantly higher in the over-expression group (P<0.05. The flow cytometry results showed that C9orf116 knockdown or over-expression had little effect on BRL-3A cell apoptosis. However, the number of cells in division phase (G2/M was significantly reduced in the interference group (P<0.05, but significantly increased in the over-expression group (P<0.01. Furthermore, the expressions of cell proliferation-related genes CCNA2, CCND1 and MYC both at mRNA and protein levels were down-regulated in the interference group and up-regulated in the over-expression group. Therefore, we concluded that C9orf116 may promote cell proliferation by modulating cell cycle transition and the expression of key genes CCNA2, CCND1 and MYC in BRL-3A cells.

  17. Exogenous regucalcin suppresses the growth of human liver cancer HepG2 cells in vitro.

    Science.gov (United States)

    Yamaguchi, Masayoshi; Murata, Tomiyasu

    2018-04-05

    Regucalcin, which its gene is localized on the X chromosome, plays a pivotal role as a suppressor protein in signal transduction in various types of cells and tissues. Regucalcin gene expression has been demonstrated to be suppressed in various tumor tissues of animal and human subjects, suggesting a potential role of regucalcin in carcinogenesis. Regucalcin, which is produced from the tissues including liver, is found to be present in the serum of human subjects and animals. This study was undertaken to determine the effects of exogenous regucalcin on the proliferation in cloned human hepatoma HepG2 cells in vitro. Proliferation of HepG2 cells was suppressed after culture with addition of regucalcin (0.01 – 10 nM) into culture medium. Exogenous regucalcin did not reveal apoptotic cell death in HepG2 cells in vitro. Suppressive effects of regucalcin on cell proliferation were not enhanced in the presence of various signaling inhibitors including tumor necrosis factor-α (TNF-α), Bay K 8644, PD98059, staurosporine, worthomannin, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or gemcitabine, which were found to suppress the proliferation. In addition, exogenous regucalcin suppressed the formation of colonies of cultured hepatoma cells in vitro. These findings demonstrated that exogenous regucalcin exhibits a suppressive effect on the growth of human hepatoma HepG2 cells, proposing a strategy with the gene therapy for cancer treatment.

  18. Studying circulation times of liver cancer cells by in vivo flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Liu, G; Li, Y; Fan, Z; Guo, J; Tan, X; Wei, X, E-mail: xwei@fudan.edu.cn [Institutes of Biomedical Sciences, Fudan University, 138 Yi Xue Yuan Road, Shanghai, 200032 (China)

    2011-02-01

    Hepatocellular carcinoma (HCC) may metastasize to lung kidney and many other organs. The survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. A recently developed 'in vivo flow cytometer' combined with real-time confocal fluorescence imaging are used to assess spreading and the circulation kinetics of liver tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labeled cells in vivo in real time without extracting blood sample. We have measured the depletion kinetics of two related human HCC cell lines high-metastatic HCCLM3 cells and low-metastatic HepG2 cells which were from the same origin and obtained by repetitive screenings in mice. >60% HCCLM3 cells are depleted within the first hour. Interestingly the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.

  19. Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium.

    Science.gov (United States)

    Liu, Chieh-Lun; Watson, Aaron M; Place, Allen R; Jagus, Rosemary

    2017-05-25

    Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL) has been adapted to a taurine-free medium by gradual accommodation to a commercially available synthetic medium, UltraMEM™-ITES. Here we show that ZFL cells are able to synthesize taurine and be maintained in medium without taurine. This has allowed for the investigation of the effects of taurine supplementation on cell growth, cellular amino acid pools, as well as the expression of the taurine biosynthetic pathway and taurine transporter genes in a defined fish cell type. After taurine supplementation, cellular taurine levels increase but hypotaurine levels stay constant, suggesting little suppression of taurine biosynthesis. Cellular methionine levels do not change after taurine addition, consistent with maintenance of taurine biosynthesis. The addition of taurine to cells grown in taurine-free medium has little effect on transcript levels of the biosynthetic pathway genes for cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), or cysteamine dioxygenase (ADO). In contrast, supplementation with taurine causes a 30% reduction in transcript levels of the taurine transporter, TauT. This experimental approach can be tailored for the development of cell lines from aquaculture species for the elucidation of their taurine biosynthetic capacity.

  20. Unscheduled DNA synthesis and elimination of DNA damage in liver cells of. gamma. -irradiated senescent mice

    Energy Technology Data Exchange (ETDEWEB)

    Gaziev, A.I.; Malakhova, L.V. (AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki)

    1982-10-01

    The level of 'spontaneous' and ..gamma..-radiation-induced DNA synthesis which is not inhibited with hydroxyurea (unscheduled synthesis) is considerably lower in hepatocytes of 18-22-month-old mice than that of 1.5-2-month-old mice. The dose-dependent increase (10-300 Gy) of unscheduled DNA synthesis (UDS) in hepatocytes of senescent mice is higher than in young animals. The elimination of damage in DNA of ..gamma..-irradiated hepatocytes (100 Gy) was examined by using an enzyme system (M. luteus extract and DNA-polymerase I of E. coli). It was found that the rate of elimination of the DNA damage in hepatocytes of 20-month-old mice is lower than that of 2-month-old mice although the activities of DNA-polymerase ..beta.. and apurinic endonuclease remain equal in the liver of both senescent and young mice. However, the nucleoids from ..gamma..-irradiated liver nuclei of 2-month-old mice are relaxed to a greater extent (as judged by the criterion of ethidium-binding capacity) than those of 20-month-old mice. The results suggest that there are limitations in the functioning of repair enzymes and in their access to damaged DNA sites in the chromatin of senescent mouse liver cells.

  1. STEREOLOGICAL ESTIMATION OF ITO CELLS FROM RAT LIVER USING THE OPTICAL FRACTIONATOR - A PRELIMINARY REPORT

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    Ricardo Marcos

    2011-05-01

    Full Text Available In the last two decades, much light has been shed on hepatic fibrosis, and the activation / proliferation of Ito cells (IC emerged to play a central role. Therefore, it is essential to have solid quantitative data in nonpathological statuses; yet, this data is scarce and confined to "number per area" or semiquantitative information. Moreover, the supposed heterogeneous distribution of IC in the hepatic lobule was never analysed with design-based (unbiased stereology. In the present study, the total number (N of IC in rat liver was estimated for the first time, by combining immunocytochemistry with the optical fractionator. Quantification was extended to the hepatocytes, to disclose the IC index, an often-used ratio in hepatology. Systematic uniform random liver sections were obtained from male Wistar rats (n = 3, and immunostained against glial fibrillary acidic protein (GFAP, a known specific marker for hepatic IC. For the first time, these were marked against GFAP in thick (30 μm paraffin sections. The estimated N of IC was 224E06; with a coefficient of error of 0.04 or 0.06, depending on the particular equation used (based on the so-called "quadratic approximation". The IC index was 91 IC/1000 hepatocytes. Concerning the lobular heterogeneity, it was proved the liver harbours a larger total number of periportal IC and hepatocytes.

  2. Effects of ethanol on calcium transport across the liver cell plasma membrane

    International Nuclear Information System (INIS)

    Bernstein, J.; Santacana, G.

    1987-01-01

    The effect of ethanol on calcium transport by the liver cell was studied by using a rat liver slice preparation. Ethanol was shown to decrease by about 30% the rate constant for 45 Ca efflux from the intracellular compartment. This inhibitory effect of ethanol was not observed in the absence of Ca 2+ or Na + from the incubation medium. Ethanol was also shown to greatly increase non-insulin calcium uptake by liver slices. This effect of ethanol appeared to be dose dependent and was not observed in the absence of Na + from the incubation medium. The ability of ethanol to increase calcium uptake by the hepatocyte was completely blocked by 1 mM Amiloride. Amiloride, however, did not affect the increased entry of either Na + or Ca 2+ produced by 10 mM Ouabain, a specific inhibitor of the sodium pump. Carbon tetrachloride (CCl 4 ), a well known hepatotoxin, also increased calcium uptake by the hepatocyte. Amiloride, however, was not able to block the CCl 4 -induced calcium uptake. These results suggest that ethanol activates a Na + entry pathway, probably represented by a Na + /H + exchanger, which in turn stimulates an entry of Ca 2+ through a Na + /Ca 2+ exchange mechanism located in the plasma membrane of the hepatocyte

  3. Autologous bone marrow mononuclear cell transplantation in patients with decompensated alcoholic liver disease: a randomized controlled trial.

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    Laurent Spahr

    Full Text Available OBJECTIVE: Impaired liver regeneration is associated with a poor outcome in patients with decompensated alcoholic liver disease (ALD. We assessed whether autologous bone marrow mononuclear cell transplantation (BMMCT improved liver function in decompensated ALD. DESIGN: 58 patients (mean age 54 yrs; mean MELD score 19, all with cirrhosis, 81% with alcoholic steatohepatitis at baseline liver biopsy were randomized early after hospital admission to standard medical therapy (SMT alone (n = 30, including steroids in patients with a Maddrey's score ≥32, or combined with G-CSF injections and autologous BMMCT into the hepatic artery (n = 28. Bone marrow cells were harvested, isolated and reinfused the same day. The primary endpoint was a ≥3 points decrease in the MELD score at 3 months, corresponding to a clinically relevant improvement in liver function. Liver biopsy was repeated at week 4 to assess changes in Ki67+/CK7+ hepatic progenitor cells (HPC compartment. RESULTS: Both study groups were comparable at baseline. After 3 months, 2 and 4 patients died in the BMMCT and SMT groups, respectively. Adverse events were equally distributed between groups. Moderate alcohol relapse occurred in 31% of patients. The MELD score improved in parallel in both groups during follow-up with 18 patients (64% from the BMMCT group and 18 patients (53% from the SMT group reaching the primary endpoint (p = 0.43 (OR 1.6, CI 0.49-5.4 in an intention to treat analysis. Comparing liver biopsy at 4 weeks to baseline, steatosis improved (p<0.001, and proliferating HPC tended to decrease in both groups (-35 and -33%, respectively. CONCLUSION: Autologous BMMCT, compared to SMT is a safe procedure but did not result in an expanded HPC compartment or improved liver function. These data suggest either insufficient regenerative stimulation after BMMCT or resistance to liver regenerative drive in patients with decompensated alcoholic cirrhosis. TRIAL REGISTRATION

  4. DNA alkylation and tumor induction in regenerating rat liver after cell cycle-related continuous N-nitrosodimethylamine infusion

    Energy Technology Data Exchange (ETDEWEB)

    Rabes, H.M.; Kerler, R.; Wilhelm, R.

    1983-01-01

    Synchronized regenerating rat liver after partial hepatectomy was used to study cell cycle-related DNA base alkylation and liver carcinogenesis. A continuous iv infusion of (/sup 14/C)N-nitrosodimethylamine (DMN) at a dose of 0.5 mg/kg/hour was given to inbred male Wistar Af/Han rats over a period of 8 hours either during the G1 phase, hydroxyurea-synchronized DNA synthesis, or the G2+M-phase of regenerating liver or to untreated rats (G0-phase liver--carcinogen dose, 1.5 mg/kg/hour). Two hours after the end of the infusion, the amount of 7-methylguanine was highest in the G0-phase liver, with a decrease in the G1 phase, the S-phase, and the G2+M-phase. After continuous DMN exposure, the O/sub 6/-methylguanine:7-methylguanine ratio was lower in the S-phase and G2+M-phase livers than in the G0-phase and G1-phase livers, indicating an increased O/sub 6/-methylguanine repair during DNA synthesis and the G2+M-phase. Liver tumors in rats treated by continuous DMN infusion either during the G0 phase or the S-phase developed only after carcinogen exposure during DNA synthesis.

  5. Effect of polysaccharides from Angelica sinensis on Bcl-2 and Bax protein expression of irradiated liver cells

    International Nuclear Information System (INIS)

    Sun Yuanlin; Tang Jian; Gu Xiaohong; Li Deyuan

    2009-01-01

    Objective: To investigate the effect of polysaccharides from Angelica sinensis (ASP3) on Bcl-2 and Bax protein expression of irradiated liver cells from mice. Methods: Bcl-2 and Bax protein expression of liver cells in vitro exposed to 2.0 Gy rays were examined by using immunohistochemistry method. Results: The expression of apoptosis-accelerating protein Bax in the irradiation group was enhanced obviously (70.83%), while apoptosis inhibiting protein Bcl-2 tended to decline (55.60%), with the statistically significant difference (P <0.01) compared with that of the control. ASP3 pretreatment could regulate Bcl-2 and Bax protein expression of liver cells, inhibiting Bax protein expression(64.14/58.37%) and increasing Bcl-2 protein expression(59.21%/ 67.45%). The differences between the high dosage (100 mg/L of ASP3) and the irradiation group were statistically significant (P<0.05). Conclusions: ASP3 pretreatment could prohibit the apoptosis of radiation- damaged liver cells due to abnormal expression of Bcl-2 and Bax, and reduce the cell apoptosis by increasing Bcl-2/Bax protein expression so as to enhance the radiation endurance of liver cells. (authors)

  6. Alleviation of lipopolysaccharide/d-galactosamine-induced liver injury in leukocyte cell-derived chemotaxin 2 deficient mice

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    Akinori Okumura

    2017-12-01

    Full Text Available Leukocyte cell-derived chemotaxin 2 (LECT2 is a secreted pleiotropic protein that is mainly produced by the liver. We have previously shown that LECT2 plays an important role in the pathogenesis of inflammatory liver diseases. Lipopolysaccharide/d-galactosamine (LPS/d-GalN-induced acute liver injury is a known animal model of fulminant hepatic failure. Here we found that this hepatic injury was alleviated in LECT2-deficient mice. The levels of TNF-α and IFN-γ, which mediate this hepatitis, had significantly decreased in these mice, with the decrease in IFN-γ production notably greater than that in TNF-α. We therefore analyzed IFN-γ-producing cells in liver mononuclear cells. Flow cytometric analysis showed significantly reduced IFN-γ production in hepatic NK and NKT cells in LECT2-deficient mice compared with in wild-type mice. We also demonstrated a decrease in IFN-γ production in LECT2-deficient mice after systemic administration of recombinant IL-12, which is known to induce IFN-γ in NK and NKT cells. These results indicate that a decrease of IFN-γ production in NK and NKT cells was involved in the alleviation of LPS/d-GalN-induced liver injury in LECT2-deficient mice.

  7. Dysregulation of the DNA Damage Response and KMT2A Rearrangement in Fetal Liver Hematopoietic Cells.

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    Mai Nanya

    Full Text Available Etoposide, a topoisomerase 2 (TOP2 inhibitor, is associated with the development of KMT2A (MLL-rearranged infant leukemia. An epidemiological study suggested that in utero exposure to TOP2 inhibitors may be involved in generation of KMT2A (MLL rearrangement. The present study examined the mechanism underlying the development of KMT2A (MLL-rearranged infant leukemia in response to in utero exposure to etoposide in a mouse model. Fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells. Etoposide-induced Kmt2a breakage was detected in fetal liver hematopoietic stem cells using a newly developed chromatin immunoprecipitation (ChIP assay. Assessment of etoposide-induced chromosomal translocation by next-generation RNA sequencing (RNA-seq identified several chimeric fusion messenger RNAs that were generated by etoposide treatment. However, Kmt2a (Mll-rearranged fusion mRNA was detected in Atm-knockout mice, which are defective in the DNA damage response, but not in wild-type mice. The present findings suggest that in utero exposure to TOP2 inhibitors induces Kmt2a rearrangement when the DNA damage response is defective.

  8. Chitosan nanoparticles from marine squid protect liver cells against N-diethylnitrosoamine-induced hepatocellular carcinoma.

    Science.gov (United States)

    Subhapradha, Namasivayam; Shanmugam, Vairamani; Shanmugam, Annaian

    2017-09-01

    Rationale of this study was framed to investigate the protective effect and anti-cancer property of nanoparticles based on chitosan isolated from squid, Sepioteuthis lessoniana, on hepatic cells in N-Nitrosodiethylamine-induced hepatocellular carcinoma in rats. The results conferred that the chitosan nanoparticle supplementation had a protective effect on liver cells by reducing the levels of marker enzymes and bilirubin and thus increasing the albumin levels. The level of reduced glutathione, ascorbic acid and α-tocopherol significantly increased in both post- and pre-treatment with chitosan nanoparticles. The levels of antioxidant enzymes were enhanced and lipid peroxidation products were diminished while treating nitrosodiethylamine-induced hepatocellular carcinoma with chitosan nanoparticles. Supplementation of chitosan nanoparticles had potent anti-hyperlipidemic property that was evidenced by monitoring the serum lipid levels and its components. Animals pre-treated with chitosan nanoparticles along with nitrosodiethylamine showed a significant reduction in the total cholesterol and triglycerides levels with increase in the levels of phospholipids and free fatty acids. Chitosan nanoparticles treated rats showed significant increment in high-density lipoprotein cholesterol and reduction in low-density lipoprotein and very low-density lipoprotein cholesterol when compared with levels in nitrosodiethylamine-induced hepatocellular carcinoma. Nitrosodiethylamine-induced carcinoma changes on circulation and hepatic antioxidant defense mechanism were regulated by chitosan nanoparticles, concluding that the chitosan nanoparticles have a potent protective effect on liver cells which might be due to its robust antioxidant and anti-lipidemic property. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Betatrophin: A liver-derived hormone for the pancreatic β-cell proliferation

    OpenAIRE

    Raghow, Rajendra

    2013-01-01

    The pancreatic β-cell failure which invariably accompanies insulin resistance in the liver and skeletal muscle is a hallmark of type-2 diabetes mellitus (T2DM). The persistent hyperglycemia of T2DM is often treated with anti-diabetic drugs with or without subcutaneous insulin injections, neither of which mimic the physiological glycemic control seen in individuals with fully functional pancreas. A sought after goal for the treatment of T2DM has been to harness the regenerative potential of pa...

  10. Impacts of low doses of pesticide mixtures on liver cell defence systems

    OpenAIRE

    Zucchini-Pascal, Nathalie; Dupont, Gwendoline; Razpotnik, Andrej; Fouche, Edwin; De Sousa, Georges; Rahmani, Roger

    2012-01-01

    Low amounts of residual pesticides are present in the environment, often as mixtures of chemicals which contaminate drinking water and food, being a source of chronic exposure for humans and a growing matter of concern in public health policy. Despite of the needs and growing investigation, little is known about the impact of low doses and mixtures of these chemicals on human health. The purpose of this study was to enlighten if modifications of liver cell metabolic- and/or defence-related ca...

  11. Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites.

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    Ian A Cockburn

    2010-05-01

    Full Text Available Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization--a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

  12. Intermittent Hypoxia Disrupts Glucose Homeostasis in Liver Cells in an Insulin-Dependent and Independent Manner

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    Chen Juan Gu

    2018-05-01

    Full Text Available Background/Aims: Obstructive sleep apnea is associated with diabetes and insulin resistance, but the underlying mechanisms remain unclear. The purpose of the current study was to determine the molecular effects of intermittent hypoxia (IH on hepatic insulin signaling and glucose homeostasis, and whether c-Jun NH2-terminal-kinase (JNK contributed to metabolic responses to IH in liver cells. Methods: The human HepG2 cells and rat FAO cells were exposed to 10, 30, 120, 240 or 360 cycles of IH (1% O2 for 60 s followed by 21% O2 for 60s, 7.5 cycles per hour or normoxia as a control. In a subgroup, we exposed cells to 360 cycles of IH with the JNK inhibitor SP600125. After IH exposure, cell glycogen content and glucose output were measured using colorimetric assay kits. Canonical insulin signaling and gluconeogenic genes were measured by western blot and quantitative polymerase chain reaction. Results: IH decreased insulin-stimulated protein kinase B (AKT/glycogen synthase kinase-3β (GSK-3β phosphorylation in a time-dependent manner, while inhibiting forkhead box protein O1 (FOXO1 expression and phosphoenolpyruvate carboxykinase (PEPCK transcription independent of insulin signaling. JNK inhibitor SP600125 partially restored AKT/ GSK-3β phosphorylation and glycogen synthesis, but did not affect other IH-induced glucose metabolic changes. Conclusion: IH in vitro impaired insulin signal transduction in liver cells as assessed by inhibited AKT/GSK-3β phosphorylation via JNK activation. IH inhibited FOXO1 and gluconeogenesis in an insulin-independent manner.

  13. Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

    Science.gov (United States)

    Klepeisz, Philip; Sagmeister, Sandra; Haudek-Prinz, Verena; Pichlbauer, Melanie; Grasl-Kraupp, Bettina; Gerner, Christopher

    2013-01-01

    Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs) have concentrated on alterations induced in hepatocytes (HCs). A potential role of non-parenchymal liver cells (NPCs) in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB) which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme. PMID:24204595

  14. Phenobarbital induces alterations in the proteome of hepatocytes and mesenchymal cells of rat livers.

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    Philip Klepeisz

    Full Text Available Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs have concentrated on alterations induced in hepatocytes (HCs. A potential role of non-parenchymal liver cells (NPCs in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme.

  15. Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury

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    Burra Patrizia

    2012-07-01

    Full Text Available Abstract Background Hepatocytes and stem cells transplantation may be an alternative to liver transplantation in acute or chronic liver disease. We aimed to evaluate the therapeutic potential of mesenchymal stem cells from human umbilical cord (UCMSCs, a readily available source of mesenchymal stem cells, in the CCl4-induced acute liver injury model. Methods Mesenchymal stem cells profile was analyzed by flow cytometry. In order to evaluate the capability of our UCMSCs to differentiate in hepatocytes, cells were seeded on three different supports, untreated plastic support, MatrigelTM and human liver acellular matrix. Cells were analyzed by immunocitochemistry for alpha-fetoprotein and albumin expression, qPCR for hepatocyte markers gene expression, Periodic Acid-Schiff staining for glycogen storage, ELISA for albumin detection and colorimetric assay for urea secretion. To assess the effects of undifferentiated UCMSCs in hepatic regeneration after an acute liver injury, we transplanted them via tail vein in mice injected intraperitoneally with a single dose of CCl4. Livers were analyzed by histological evaluation for damage quantification, immunostaining for Kupffer and stellate cells/liver myofibroblasts activation and for UCMSCs homing. Pro- and anti-inflammatory cytokines gene expression was evaluated by qPCR analysis and antioxidant enzyme activity was measured by catalase quantification. Data were analyzed by Mann–Whitney U-test, Kruskal-Wallis test and Cuzick’s test followed by Bonferroni correction for multiple comparisons. Results We have standardized the isolation procedure to obtain a cell population with hepatogenic properties prior to in vivo transplantation. When subjected to hepatogenic differentiation on untreated plastic support, UCMSCs differentiated in hepatocyte-like cells as demonstrated by their morphology, progressive up-regulation of mature hepatocyte markers, glycogen storage, albumin and urea secretion. However

  16. [Overexpression of liver kinase B1 inhibits the proliferation of lung cancer cells].

    Science.gov (United States)

    Li, Yang; Zhang, Libin; Wang, Ping

    2017-01-01

    Objective To explore the effect of overexpressed liver kinase B1(LKB1) on the proliferation of lung cancer cell lines. Methods The expression levels of LKB1 and PTEN in A549, NCI-H23, NCI-H157, XWLC-05, NCI-H446 lung cancer cells were detected by immunocytochemistry (ICC) and Western blotting. Plasmid pcDNA3.1 + -LKB1 and empty vector pcDNA3.1 + -null were separately transfected into the above five cell lines, and then the expression of LKB1 mRNA and protein were determined by quantitative real-time PCR and Western blotting, respectively. Finally, CCK-8 assay was used to analyze the proliferation ability of the transfected cells. Results LKB1 and PTEN were positive in NCI-H23 cells; LKB1 was negative while PTEN was positive in A549 and NCI-H446 cells; both LKB1 and PTEN were negative in NCI-H157 and XWLC-05 cells. Quantitative real-time PCR and Western blotting showed that the expression level of LKB1 significantly increased in the above cell lines transfected with plasmid pcDNA3.1 + -LKB1 compared with the ones with empty vector pcDNA3.1 + -null. Besides, CCK-8 assay showed that the overexpression of LKB1 in the lung cancer cells transfected with pcDNA3.1 + -LKB1 had an obvious inhibitory effect on cell proliferation. Conclusion The expression of LKB1 is down-regulated in most of the lung cell lines to different extent and the over-expression of LKB1 can remarkably inhibit the proliferation ability of lung cancer cell lines.

  17. Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines

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    Travert Carine

    2010-10-01

    Full Text Available Abstract Background Worldwide used pesticides containing different adjuvants like Roundup formulations, which are glyphosate-based herbicides, can provoke some in vivo toxicity and in human cells. These pesticides are commonly found in the environment, surface waters and as food residues of Roundup tolerant genetically modified plants. In order to know their effects on cells from liver, a major detoxification organ, we have studied their mechanism of action and possible protection by precise medicinal plant extracts called Dig1. Methods The cytotoxicity pathways of four formulations of glyphosate-based herbicides were studied using human hepatic cell lines HepG2 and Hep3B, known models to study xenobiotic effects. We monitored mitochondrial succinate dehydrogenase activity and caspases 3/7 for cell mortality and protection by Dig1, as well as cytochromes P450 1A1, 1A2, 3A4 and 2C9 and glutathione-S-transferase to approach the mechanism of actions. Results All the four Roundup formulations provoke liver cell death, with adjuvants having stronger effects than glyphosate alone. Hep3B are 3-5 times more sensitive over 48 h. Caspases 3/7 are greatly activated in HepG2 by Roundup at non-cytotoxic levels, and some apoptosis induction by Roundup is possible together with necrosis. CYP3A4 is specifically enhanced by Roundup at doses 400 times less than used in agriculture (2%. CYP1A2 is increased to a lesser extent together with glutathione-S-transferase (GST down-regulation. Dig 1, non cytotoxic and not inducing caspases by itself, is able to prevent Roundup-induced cell death in a time-dependant manner with an important efficiency of up to 89%, within 48 h. In addition, we evidenced that it prevents Caspases 3/7 activation and CYP3A4 enhancement, and not GST reduction, but in turn it slightly inhibited CYP2C9 when added before Roundup. Conclusion Roundup is able to provoke intracellular disruption in hepatic cell lines at different levels, but a

  18. Transient mTOR inhibition facilitates continuous growth of liver tumors by modulating the maintenance of CD133+ cell populations.

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    Zhaojuan Yang