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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. Liver Cancer Stem Cells

    OpenAIRE

    Sameh Mikhail; Aiwu Ruth He

    2011-01-01

    Hepatocellular carcinoma is the most common primary malignancy of the liver in adults. It is also the fifth most common solid cancer worldwide and the third leading cause of cancer-related death. Recent research supports that liver cancer is a disease of adult stem cells. From the models of experimental hepatocarcinogenesis, there may be at least three distinct cell lineages with progenitor properties susceptible to neoplastic transformation. Identification of specific cell surface markers fo...

  3. Report on Liver Cell Transplantation Using Human Fetal Liver Cells.

    Science.gov (United States)

    Pietrosi, Giada; Chinnici, Cinzia

    2017-01-01

    In an era of organ shortage, human fetuses donated after medically indicated abortion could be considered a potential liver donor for hepatic cell isolation. We investigated transplantation of fetal liver cells as a strategy to support liver functionality in end-stage liver disease. Here, we report our protocol of human fetal liver cells (hFLC) isolation in fetuses from 17 to 22 gestational weeks, and our clinical procedure of hFLC transplantation through the splenic artery.

  4. Heterologous corneal endothelial cell transplantation--human corneal endothelial cell transplantation in Lewis rats.

    OpenAIRE

    Tchah, H.

    1992-01-01

    A heterologous corneal endothelial transplantation was attempted using human endothelial cells and a Lewis rat penetrating keratoplasty model. Cultured human endothelial cells were seeded to a Lewis rat cornea, which was denuded of its endothelium. When grafted into the syngeneic Lewis rat, the graft remained clear for at least five days, and then became opaque and edematous because of immune rejection reaction. In contrast, corneas denuded of their endothelium became opaque and edematous imm...

  5. Cell surface engineering of Bacillus subtilis improves production yields of heterologously expressed α-amylases

    NARCIS (Netherlands)

    Cao, Haojie; van Heel, Auke J; Ahmed, Hifza; Mols, Maarten; Kuipers, Oscar P

    2017-01-01

    BACKGROUND: Bacillus subtilis is widely used as a cell factory for numerous heterologous proteins of commercial value and medical interest. To explore the possibility of further enhancing the secretion potential of this model bacterium, a library of engineered strains with modified cell surface

  6. [Genetic transformation and fate of heterological DNA in bacterial cells].

    Science.gov (United States)

    Piechowska, Mirosława

    2015-01-01

    Secretion of a metabolite enabling Streptococci to undergo genetic transformation was discovered. The metabolite combined with an optimization process were applied to increase the transformation yield about 20-fold. It was observed that large amounts of DNA exert a bactericidal effect, indicating the ability of at least 70% of cells to uptake the polymer. While studying the molecular mechanism of transformation of Bacillus subtilis it was shown that the uptaken DNA forms complexes with bacterial proteins, which hinders determination of its structure. A method was found to dissociate these complexes which enabled to determine the single-stranded structure of the uptaken DNA. Donor DNA fragments incorporated into the host DNA were of about 10 Da. Non-transforming DNA can be uptaken similarly but does not undergo incorporation into the host DNA. The selectivity of Bacillus subtilis receptors was determined towards DNA of phages containing modified bases: uracil, putrescinyl-thymine and its acetylated derivative, 5'-hydroxymethylcytosine and its glycosylated derivative and also towards double-stranded RNA of f2 phage. All these modifications were tolerated by the cellular receptors, with the exception of glycosylation and the 2'-OH group in RNA.

  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. 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...

  9. 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

    upon the induction of expression of two different heterologous killing genes in nonpathogenic Pseudomonas putida KT2440 derivatives have been analyzed, P. putida CMC4 and CMC12 carry in their chromosomes a fusion of the PAl-04/03 promoter to the Escherichia coli gef gene and the phi X174 lysis gene E......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...

  10. Epitope-Specific Vaccination Limits Clonal Expansion of Heterologous Naive T Cells during Viral Challenge

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    Lexus R. Johnson

    2016-10-01

    Full Text Available Despite robust secondary T cell expansion primed by vaccination, the impact on primary immune responses to heterotypic antigens remains undefined. Here we show that secondary expansion of epitope-specific memory CD8+ T cells primed by prior infection with recombinant pathogens limits the primary expansion of naive CD8+ T cells with specificity to new heterologous antigens, dampening protective immunity against subsequent pathogen challenge. The degree of naive T cell repression directly paralleled the magnitude of the recall response. Suppressed primary T cell priming reflects competition for antigen accessibility, since clonal expansion was not inhibited if the primary and secondary epitopes were expressed on different dendritic cells. Interestingly, robust recall responses did not impact antigen-specific NK cells, suggesting that adaptive and innate lymphocyte responses possess different activation requirements or occur in distinct anatomical locations. These findings have important implications in pathogen vaccination strategies that depend on the targeting of multiple T cell epitopes.

  11. 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

  12. HETEROLOGOUS IMMUNITY BETWEEN VIRUSES

    Science.gov (United States)

    Welsh, Raymond M.; Che, Jenny; Brehm, Michael A.; Selin, Liisa K.

    2010-01-01

    Summary Immune memory responses to previously encountered pathogens can sometimes alter the immune response to and the course of infection of an unrelated pathogen by a process known as heterologous immunity. This response can lead to enhanced or diminished protective immunity and altered immunopathology. Here we discuss the nature of T-cell cross-reactivity and describe matrices of epitopes from different viruses eliciting cross-reactive CD8+ T-cell responses. We examine the parameters of heterologous immunity mediated by these cross-reactive T cells during viral infections in mice and humans. We show that heterologous immunity can disrupt T-cell memory pools, alter the complexity of the T-cell repertoire, change patterns of T-cell immunodominance, lead to the selection of viral epitope-escape variants, alter the pathogenesis of viral infections, and, by virtue of the private specificity of T-cell repertoires within individuals, contribute to dramatic variations in viral disease. We propose that heterologous immunity is an important factor in resistance to and variations of human viral infections and that issues of heterologous immunity should be considered in the design of vaccines. PMID:20536568

  13. 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

  14. DNA Ploidy and Liver Cell Dysplasia in Liver Biopsies from Patients with Liver Cirrhosis

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    Sayed S El-Sayed

    2004-01-01

    Full Text Available There is controversy among pathologists when assessing the presence or absence of liver cell dysplasia in liver biopsies taken from cirrhotic patients. The objective of the present study was to determine the DNA ploidy pattern of hepatocytes of patients with liver cirrhosis and its relationship to liver cell dysplasia. A total of 48 male patients diagnosed with liver cirrhosis based on clinical, laboratory and histopathological criteria were included in the study. A liver biopsy was taken from each patient; one part of the biopsy was subjected to histopathology, and the other to flow cytometry. The histopathological examination revealed liver cell dysplasia in 60% of patients with liver cirrhosis (62% of them had large cell dysplasia [LCD] and 38% had small cell dysplasia [SCD]. Abnormal DNA content (aneuploidy was found in 81.5% of positive liver cell dysplasia specimens and found only in 11.1% of negative liver cell dysplasia specimens, with a statistically significant difference (P0.05 in comparison with SCD. In conclusion, SCD (similar to LCD is also associated with aneuploidy and elevated DNA index, and may carry the same risk for progression to hepatocellular carcinoma.

  15. Cell and Tissue Engineering for Liver Disease

    Science.gov (United States)

    Bhatia, Sangeeta N.; Underhill, Gregory H.; Zaret, Kenneth S.; Fox, Ira J.

    2015-01-01

    Despite the tremendous hurdles presented by the complexity of the liver’s structure and function, advances in liver physiology, stem cell biology and reprogramming, and the engineering of tissues and devices are accelerating the development of cell-based therapies for treating liver disease and liver failure. This State of the Art Review discusses both the near and long-term prospects for such cell-based therapies and the unique challenges for clinical translation. PMID:25031271

  16. Heterologous expression of NaV1.9 chimeras in various cell systems.

    Science.gov (United States)

    Goral, R Oliver; Leipold, Enrico; Nematian-Ardestani, Ehsan; Heinemann, Stefan H

    2015-12-01

    SCN11A encodes the voltage-gated sodium channel NaV1.9, which deviates most strongly from the other eight NaV channels expressed in mammals. It is characterized by resistance to the prototypic NaV channel blocker tetrodotoxin and exhibits slow activation and inactivation gating. Its expression in dorsal root ganglia neurons suggests a role in motor or pain signaling functions as also recently demonstrated by the occurrence of various mutations in human SCN11A leading to altered pain sensation syndromes. The systematic investigation of human NaV1.9, however, is severely hampered because of very poor heterologous expression in host cells. Using patch-clamp and two-electrode voltage-clamp methods, we show that this limitation is caused by the C-terminal structure of NaV1.9. A chimera of NaV1.9 harboring the C terminus of NaV1.4 yields functional expression not only in neuronal cells but also in non-excitable cells, such as HEK 293T or Xenopus oocytes. The major functional difference of the chimeric channel with respect to NaV1.9 is an accelerated activation and inactivation. Since the entire transmembrane domain is preserved, it is suited for studying pharmacological properties of the channel and the functional impact of disease-causing mutations. Moreover, we demonstrate how mutation S360Y makes NaV1.9 channels sensitive to tetrodotoxin and saxitoxin and that the unusual slow open-state inactivation of NaV1.9 is also mediated by the IFM (isoleucine-phenylalanine-methionine) inactivation motif located in the linker connecting domains III and IV.

  17. 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...... to 5 pg/cell/day. Time-course analysis of high- and low-producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO...

  18. Stem cells in liver disease

    NARCIS (Netherlands)

    Poll, D. van

    2008-01-01

    Failure of the liver, the largest vital organ in the body, unequivocally results in death. Hepatic failure most commonly evolves over a period of several years as a result of chronic liver disease, most often viral hepatitis or alcoholic liver damage. In rarer cases, the organ shuts down within

  19. 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.

  20. Characterization of an infectious pancreatic necrosis (IPN) virus carrier cell culture with resistance to superinfection with heterologous viruses.

    Science.gov (United States)

    García, Inmaculada; Galiana, Antonio; Falcó, Alberto; Estepa, Amparo; Perez, Luis

    2011-04-21

    A state of persistence of a non susceptible fish cell line with infectious pancreatic necrosis virus (IPNV) was established in vitro by experimental infection. The persistently infected culture showed sustained production of infectious virus and could be continuously passaged for months. A distinct feature of this culture is that only a very small fraction of the cells harbours virus replication, in contrast to other reported IPNV-persistently infected cells from salmonid fish, where nearly all the cells express viral antigens. In spite of the small number of detectable IPNV-infected cells, the carrier culture shows resistance to superinfection with homologous as well as heterologous viruses. Temperature shift-up experiments indicate that viral interference is due to continuous replication of IPNV in the culture. Quantitation of Mx gene expression suggested that the interference phenomenon could be mediated by the activation of the interferon (IFN) system. However, conditioned medium from the IPNV-infected cell cultures only marginally protected other cells against VHSV infection, indicating that other type I IFN-independent mechanism may be underlying the resistance of the persistently infected culture to infection with heterologous viruses. Our study defines a novel in vitro model of IPNV persistence and contributes to the understanding of the widespread distribution of aquabirnaviruses in marine and fresh water environments by establishing a carrier state in non susceptible fish species. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. Human herpesvirus 6 major immediate early promoter has strong activity in T cells and is useful for heterologous gene expression

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    Yamanishi Koichi

    2011-01-01

    Full Text Available Abstract Background Human herpesvirus-6 (HHV-6 is a beta-herpesvirus. HHV-6 infects and replicates in T cells. The HHV-6-encoded major immediate early gene (MIE is expressed at the immediate-early infection phase. Human cytomegalovirus major immediate early promoter (CMV MIEp is commercially available for the expression of various heterologous genes. Here we identified the HHV-6 MIE promoter (MIEp and compared its activity with that of CMV MIEp in various cell lines. Methods The HHV-6 MIEp and some HHV-6 MIEp variants were amplified by PCR from HHV-6B strain HST. These fragments and CMV MIEp were subcloned into the pGL-3 luciferase reporter plasmid and subjected to luciferase reporter assay. In addition, to investigate whether the HHV-6 MIEp could be used as the promoter for expression of foreign genes in a recombinant varicella-zoster virus, we inserted HHV-6 MIEp-DsRed expression casette into the varicella-zoster virus genome. Results HHV-6 MIEp showed strong activity in T cells compared with CMV MIEp, and the presence of intron 1 of the MIE gene increased its activity. The NF-κB-binding site, which lies within the R3 repeat, was critical for this activity. Moreover, the HHV-6 MIEp drove heterologous gene expression in recombinant varicella-zoster virus-infected cells. Conclusions These data suggest that HHV-6 MIEp functions more strongly than CMV MIEp in various T-cell lines.

  2. Cells and materials for liver tissue engineering.

    Science.gov (United States)

    Li, Yuan-Sheng; Harn, Horng-Jyh; Hsieh, Dean-Kuo; Wen, Tung-Chou; Subeq, Yi-Maun; Sun, Li-Yi; Lin, Shinn-Zong; Chiou, Tzyy-Wen

    2013-01-01

    Liver transplantation is currently the most efficacious treatment for end-stage liver diseases. However, one main problem with liver transplantation is the limited number of donor organs that are available. Therefore, liver tissue engineering based on cell transplantation that combines materials to mimic the liver is under investigation with the goal of restoring normal liver functions. Tissue engineering aims to mimic the interactions among cells with a scaffold. Particular materials or a matrix serve as a scaffold and provide a three-dimensional environment for cell proliferation and interaction. Moreover, the scaffold plays a role in regulating cell maturation and function via these interactions. In cultures of hepatic lineage cells, regulation of cell proliferation and specific function using biocompatible synthetic, biodegradable bioderived matrices, protein-coated materials, surface-modified nanofibers, and decellularized biomatrix has been demonstrated. Furthermore, beneficial effects of addition of growth factor cocktails to a flow bioreactor or coculture system on cell viability and function have been observed. In addition, a system for growing stem cells, liver progenitor cells, and primary hepatocytes for transplantation into animal models was developed, which produces hepatic lineage cells that are functional and that show long-term proliferation following transplantation. The major limitation of cells proliferated with matrix-based transplantation systems is the high initial cell loss and dysfunction, which may be due to the absence of blood flow and the changes in nutrients. Thus, the development of vascular-like scaffold structures, the formation of functional bile ducts, and the maintenance of complex metabolic functions remain as major problems in hepatic tissue engineering and will need to be addressed to enable further advances toward clinical applications.

  3. Stable heterologous expression of biologically active terpenoids in green plant cells

    DEFF Research Database (Denmark)

    Binti Khairul Ikram, Nur Kusaira; Zhan, Xin; Pan, Xiwu

    2015-01-01

    Plants biosynthesize a great diversity of biologically active small molecules of interest for fragrances, flavors, and pharmaceuticals. Among specialized metabolites, terpenoids represent the greatest molecular diversity. Many terpenoids are very complex, and total chemical synthesis often requires...... heterologous expression for production of valuable plant-derived natural products. In this review, our primary focus is on small terpenoids and we discuss the benefits of plant expression platforms and provide several successful examples of stable production of small terpenoids in plants....

  4. Human liver endothelial cells, but not macrovascular or microvascular endothelial cells, engraft in the mouse liver

    NARCIS (Netherlands)

    Filali, Ebtisam El; Hiralall, Johan K.; van Veen, Henk A.; Stolz, Donna B.; Seppen, Jurgen

    2013-01-01

    Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and

  5. Stem cells in acute liver failure.

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    Wesson, Russell N; Cameron, Andrew M

    2011-01-01

    Patients with acute liver failure are a particularly challenging group, with unique difficulties faced in treatment decisions. Life-saving therapy is available, but organ shortage, delays in transplantation, and complications in management result in a high mortality in this group of patients even after transplant. Any pharmacologic intervention that improved outcomes in this population of critically ill patients would be of great benefit. Based on available evidence, different scenarios of participation of HSCs in liver recovery are conceivable. Encouraging HSCs to differentiate into hepatocytes or supply paracrine and cellular level support to accelerate ongoing local repair mechanisms and assist a failing liver with inadequate mass and functional capacity might be directed to occur effectively in humans. Evidence within small animal models of liver injury and observations within the human population suggest that this might also be encouraged. The use of pharmacologic agents to mobilize hematopoietic stem cells is well established and effectively used in a different population of patients. As such, extending the use of these drugs, such as plerixafor, to the human population has a sound basis. However, there is a need for clarification of the mechanisms by which these cells exert their effect as well as which specific population of cells is involved in the regenerative process. To be clinically relevant in scenarios of acute liver failure, stem cell mobilizing strategies would have to impact survival when administered well after injury. Applications in other settings may also prove useful. Limits to liver resection exist where the size of the future liver remnant governs the extent of resection possible. Preexisting functional impairment may be restrictive, and strategies involving stem cells may assist the future liver remnant in both normal and functionally impaired livers. Benefit has already been reported from treatment with G-CSF in other injured tissues

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

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    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. A testis-mediated germline chimera production based on transfer of chicken testicular cells directly into heterologous testes.

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    Lee, Young Mok; Jung, Jin Gyoung; Kim, Jin Nam; Park, Tae Sub; Kim, Tae Min; Shin, Sang Su; Kang, Dae Kyung; Lim, Jeong Mook; Han, Jae Yong

    2006-09-01

    In this study, we proposed a testis-mediated germline chimera production system based on the transplantation of testicular cells directly into heterologous testes. The testicular cells of juvenile (4-wk-old) or adult (24-wk-old) Korean Ogol chickens with a recessive pigmentation inhibitory gene, with or without prior culture, were injected (2 x 10(7) cells/head) into the seminiferous tubules of juvenile or adult recipients with White Leghorn with a dominant pigmentation inhibitory gene in a 2 x 2 factorial arrangement. The localization of transplanted cells into the inner space of the seminiferous tubules was confirmed within 24 h after injection. Subsequent testcross analyses showed that 7.8% (5/64) of the recipients had chimeric status in their testes. The periods of time from transfer to hatching of the first progeny with black feathers were 38 and 45 days for adult cells transplanted into an adult recipient, 188 days for adult cells into a juvenile recipient, and 137 days for juvenile cells into a juvenile recipient. Culture of the testicular cells derived both colony-forming and monolayer-forming cells. The colony-forming cells were stained positively for periodic acid Schiff solution, and further reacted with anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibodies both before and after culture for 15 days. In conclusion, it may be possible to develop the testis-mediated germline chimera production technique, which extends the feasibility of genetic manipulations in avian species.

  8. Adult liver stem cells in hepatic regeneration and cancer

    NARCIS (Netherlands)

    Nantasanti, Sathidpak

    2015-01-01

    An alternative source of livers for transplantation in patients with (genetic) liver diseases and liver failure is needed because liver donors are scarce. HPC-derived hepatocyte-like cells could be one of the options. Because dogs and humans share liver-pathologies and disease-pathways, the dog is

  9. Mesenchymal stem cell therapy for liver fibrosis.

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    Eom, Young Woo; Shim, Kwang Yong; Baik, Soon Koo

    2015-09-01

    Currently, the most effective treatment for end-stage liver fibrosis is liver transplantation; however, transplantation is limited by a shortage of donor organs, surgical complications, immunological rejection, and high medical costs. Recently, mesenchymal stem cell (MSC) therapy has been suggested as an effective alternate approach for the treatment of hepatic diseases. MSCs have the potential to differentiate into hepatocytes, and therapeutic value exists in their immune-modulatory properties and secretion of trophic factors, such as growth factors and cytokines. In addition, MSCs can suppress inflammatory responses, reduce hepatocyte apoptosis, increase hepatocyte regeneration, regress liver fibrosis and enhance liver functionality. Despite these advantages, issues remain; MSCs also have fibrogenic potential and the capacity to promote tumor cell growth and oncogenicity. This paper summarizes the properties of MSCs for regenerative medicine and their therapeutic mechanisms and clinical application in the treatment of liver fibrosis. We also present several outstanding risks, including their fibrogenic potential and their capacity to promote pre-existing tumor cell growth and oncogenicity.

  10. Replacement of Diseased Mouse Liver by Hepatic Cell Transplantation

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    Rhim, Jonathan A.; Sandgren, Eric P.; Degen, Jay L.; Palmiter, Richard D.; Brinster, Ralph L.

    1994-02-01

    Adult liver has the unusual ability to fully regenerate after injury. Although regeneration is accomplished by the division of mature hepatocytes, the replicative potential of these cells is unknown. Here, the replicative capacity of adult liver cells and their medical usefulness as donor cells for transplantation were investigated by transfer of adult mouse liver cells into transgenic mice that display an endogenous defect in hepatic growth potential and function. The transplanted liver cell populations replaced up to 80 percent of the diseased recipient liver. These findings demonstrate the enormous growth potential of adult hepatocytes, indicating the feasibility of liver cell transplantation as a method to replace lost or diseased hepatic parenchyma.

  11. 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

    The dopamine transporter (DAT) belongs to SLC6 family of Na+/Cl- coupled transporters and mediates clearance of released dopamine (DA) from the synaptic cleft. To investigate the constitutive trafficking of heterologously expressed DAT we fused the N-terminus of DAT to the intracellular tail...... of the single-membrane spanning protein Tac, thereby creating an extracellular antibody epitope. Upon expression in HEK293 cells this Tac-DAT fusion protein displayed uptake properties similar to the wild type (WT) transporter. Additionally, Tac-DAT was, like the WT, internalized both in response to PMA...... 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...

  12. 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

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

    Science.gov (United States)

    Hu, Shumin; Kong, Jian; Sun, Zhilan; Han, Lanlan; Kong, Wentao; Yang, Pu

    2011-10-28

    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. 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. 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 application.

  14. 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. 

  15. OMOLOGICAL AND HETEROLOGICAL ANTIBODY AND T CELL IMMUNE RESPONSES TO LIVE ATTENUATED INFLUENZA VACCINE A (H5N2 AND A (H7N3

    Directory of Open Access Journals (Sweden)

    A. N. Naykhin

    2015-01-01

    Full Text Available From the beginning of 21th century outbreaks of H5, H7 and H9 avian flu are registered from time to time. These viruses are considered as one of the possible causes of the next pandemia. The development of avian influenza vaccines is one of the WHO priorities. The aim of this work was to study antibody and cellular immune responses to avian A (H5N2 and A (H7N3 live attenuated influenza vaccines (LAIVs. We examined serum antibodies (HAI assay, microneutralization assay, ELISA, local antibodies (ELISA and virus-specific CD4+ and CD8+ central memory and effector memory T cells. Two doses vaccination of healthy volunteers with A (H5N2 and A (H7N3 LAIVs induced homological antibody and cellular immune responses (i. e. serum and local antibody conversions, virus-specific memory T cell growth. These vaccines also stimulated heterological immunity (heterological serum and local antibodies and T cells. Heterological immune response intensity depended on antigenic structure of vaccine strain and heterological virus, particularly on HA type. 

  16. Liver cancer stem cells as an important target in liver cancer therapies.

    Science.gov (United States)

    Zou, Gang-Ming

    2010-02-01

    Hepatic cancer is one of most common cause of cancer-related death. Hepato-epithelial cancers are believed to originate from the malignant transformation of liver-resident stem/progenitor cells. Liver cancer stem cells have been characterized recently and the phenotype of liver cancer stem cells has been defined as CD133+ CD44+ cancer cells. Recently, it has been also demonstrated about the relevance of targeting liver cancer stem cells, due to cancer stem cells are related to cancer metastasis. These advances no doubt to bring the new strategy in liver cancer treatment and control in this disease. This review describes the current status and progress about cancer stem cell research in liver and discuss of the implications of these studies in new liver cancer treatment strategies.

  17. N-cadherin induces partial differentiation of cholinergic presynaptic terminals in heterologous cultures of brainstem neurons and CHO cells

    Directory of Open Access Journals (Sweden)

    Richard J Flannery

    2012-12-01

    Full Text Available N-cadherin is a calcium-sensitive cell adhesion molecule commonly expressed at synaptic junctions and contributes to formation and maturation of synaptic contacts. This study used heterologous cell cultures of brainstem cholinergic neurons and transfected Chinese Hamster Ovary (CHO cells to examine whether N-cadherin is sufficient to induce differentiation of cholinergic presynaptic terminals. Brainstem nuclei isolated from transgenic mice expressing EGFP under the control of choline acetyltransferase transcriptional regulatory elements (ChATBACEGFP were cultured as tissue explants for five days and cocultured with transfected CHO cells for an additional two days. Immunostaining for synaptic vesicle proteins SV2 and synapsin I revealed a ~3-fold increase in the area of SV2 immunolabeling over N-cadherin expressing CHO cells, and this effect was enhanced by coexpression of p120-catenin. Synapsin I immunolabeling per axon length was also increased on N-cadherin expressing CHO cells but required coexpression of p120-catenin. To determine whether N-cadherin induces formation of neurotransmitter release sites, whole-cell voltage-clamp recordings of CHO cells expressing alpha-3 and beta-4 nicotinic acetylcholine receptor (nAChR subunits in contact with cholinergic axons were used to monitor excitatory postsynaptic potentials (EPSPs and miniature EPSPs (mEPSPs. EPSPs and mEPSPs were not detected in both, control and in N-cadherin expressing CHO cells in the absence or presence of tetrodotoxin. These results indicate that expression of N-cadherin in non-neuronal cells is sufficient to initiate differentiation of presynaptic cholinergic terminals by inducing accumulation of synaptic vesicles; however, development of readily detectable mature cholinergic release sites and/or clustering of postsynaptic nAChR may require expression of additional synaptogenic proteins.

  18. Liver cell proliferation after partial hepatectomy in rats with liver metastases

    NARCIS (Netherlands)

    de Jong, KP; Brouwers, MAM; Huls, GA; Bun, JCAM; Wubbena, AS; Nieuwenhuis, P; Slooff, MJH; Dam, A.

    OBJECTIVE: To validate proliferating cell nuclear antigen (PCNA) expression and flow cytometry as proliferation markers in regenerating rat liver containing metastases. STUDY DESIGN: Rats containing colorectal liver metastases were killed at various days after 70% partial hepatectomy or a sham

  19. Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes

    OpenAIRE

    Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V. Bleu; Serrano, Elba E.

    2011-01-01

    The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. ...

  20. Modulation of benzylisoquinoline alkaloid biosynthesis by heterologous expression of CjWRKY1 in Eschscholzia californica cells.

    Directory of Open Access Journals (Sweden)

    Yasuyuki Yamada

    Full Text Available Transcription factors control many processes in plants and have high potentials to manipulate specialized metabolic pathways. Transcriptional regulation of the biosynthesis of monoterpenoid indole alkaloids (MIAs, nicotine alkaloids, and benzylisoquinoline alkaloids (BIAs has been characterized using Catharanthus roseus, Nicotiana and Coptis plants. However, metabolic engineering in which specific transcription factors are used in alkaloid biosynthesis is limited. In this study, we characterized the effects of ectopic expression of CjWRKY1, which is a transcriptional activator with many targets in BIA biosynthesis in Coptis japonica (Ranunculaceae and Eschscholzia californica (California poppy, Papaveraceae. Heterologous expression of CjWRKY1 in cultured California poppy cells induced increases in transcripts of several genes encoding BIA biosynthetic enzymes. Metabolite analyses indicated that the overexpression of the CjWRKY1 gene also induced increases in the accumulation of BIAs such as sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, and 10-hydroxychelerythrine in the culture medium. Previous characterization of EcbHLH1 and current results indicated that both transcription factors, WRKY1 and bHLH1, are substantially involved in the regulation of BIA biosynthesis. We discuss the function of CjWRKY1 in E. californica cells and its potential for metabolic engineering in BIA biosynthesis.

  1. Gene expression profiles in a porcine model of infarction: differential expression after intracoronary injection of heterologous bone marrow mesenchymal cells.

    Science.gov (United States)

    Martínez de Ilárduya, O; Barallobre Barreiro, J; Moscoso, I; Añón, P; Fraga, M; Centeno, A; López, E; Doménech, N

    2009-01-01

    Myocardial infarction is one of the main causes of mortality in developed countries. Injection of bone marrow mesenchymal stem cells (BMMSC) with the ability to regenerate lost cardiomyocytes is a promising therapy for heart failure. To evaluate this strategy, an in vivo porcine model of infarction was used. Gene expression profiles of 3 groups of pigs (n = 5 each) were analyzed and compared by real-time reverse transcription-polymerase chain reaction (RT-PCR). One of the groups underwent anterior descending coronary occlusion followed by BMMSC injection; a placebo group was injected with culture medium without cells after infarction; and a third group was formed by healthy pigs. Four weeks later, cells or medium was administered by intracoronary injection and, a month later, animals were sacrificed and samples collected. Genes related to cardiomyogenesis (Mef2C, Gata4, Nkx2.5), mobilization and homing of resident or circulating stem cells (Sdf1, Cxcr4, c-Kit), contractibility (Serca2a), and fibrosis (CollA1) were analyzed. Gene expression profiles changed in various heart areas in the 3 groups. Expression of genes related to cardiomyogenesis decreased in infarcted zones compared with homologous regions of healthy hearts. Sdf1 expression increased in the apex of infarcted hearts. Serca2a expression was reduced in the ventricles and atria of infarcted hearts. Also, increases in Cxcr4 and CollA1 expression were observed in infarcted hearts of cell-treated pigs compared with the placebo group. In conclusion, infarction induced changes in genes involved in various biological processes. Intracoronary injection of heterologous BMMSC resulted in localized changes in the expression of Cxcr4 and Col1A1.

  2. 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.

  3. Recent developments on human cell lines for the bioartificial liver

    NARCIS (Netherlands)

    Hoekstra, R.; Chamuleau, R. A. F. M.

    2002-01-01

    Most bioartificial liver (BAL) devices contain porcine primary hepatocytes as their biological component. However, alternatives are needed due to xenotransplantation associated risks. Human liver cell lines have excellent growth characteristics and are therefore candidates for application in BAL

  4. Characteristics of liver cancer stem cells and clinical correlations.

    Science.gov (United States)

    Cheng, Zhuo; Li, Xiaofeng; Ding, Jin

    2016-09-01

    Liver cancer is an aggressive malignant disease with a poor prognosis. Patients with liver cancer are usually diagnosed at an advanced stage and thus miss the opportunity for surgical resection. Chemotherapy and radiofrequency ablation, which target tumor bulk, have exhibited limited therapeutic efficacy to date. Liver cancer stem cells (CSCs) are a small subset of undifferentiated cells existed in liver cancer, which are considered to be responsible for liver cancer initiation, metastasis, relapse and chemoresistance. Elucidating liver CSC characteristics and disclosing their regulatory mechanism might not only deepen our understanding of the pathogenesis of liver cancer but also facilitate the development of diagnostic, prognostic and therapeutic approaches to improve the clinical management of liver cancer. In this review, we will summarize the recent advances in liver CSC research in terms of the origin, identification, regulation and clinical correlation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Stem Cell Therapies for Treatment of Liver Disease

    Directory of Open Access Journals (Sweden)

    Clara Nicolas

    2016-01-01

    Full Text Available Cell therapy is an emerging form of treatment for several liver diseases, but is limited by the availability of donor livers. Stem cells hold promise as an alternative to the use of primary hepatocytes. We performed an exhaustive review of the literature, with a focus on the latest studies involving the use of stem cells for the treatment of liver disease. Stem cells can be harvested from a number of sources, or can be generated from somatic cells to create induced pluripotent stem cells (iPSCs. Different cell lines have been used experimentally to support liver function and treat inherited metabolic disorders, acute liver failure, cirrhosis, liver cancer, and small-for-size liver transplantations. Cell-based therapeutics may involve gene therapy, cell transplantation, bioartificial liver devices, or bioengineered organs. Research in this field is still very active. Stem cell therapy may, in the future, be used as a bridge to either liver transplantation or endogenous liver regeneration, but efficient differentiation and production protocols must be developed and safety must be demonstrated before it can be applied to clinical practice.

  6. Role of Th17 cells in common liver diseases

    Directory of Open Access Journals (Sweden)

    WEI Linlin

    2013-06-01

    Full Text Available In recent years, it has been found that T helper type 17 (Th17 cells are a new subset of CD4+ Th cells. Th17 cells play an important role in the onset and development of many liver diseases and have become the research focus in immunology. This paper summarizes the studies on the relationship between Th17 cells and various liver diseases in order to provide a new idea for the study and treatment of liver diseases.

  7. Regulation of NK cell repertoire and function in the liver.

    Science.gov (United States)

    Krueger, Peter D; Lassen, Matthew G; Qiao, Huihong; Hahn, Young S

    2011-01-01

    NK cells represent a large proportion of the lymphocyte population in the liver and are involved in early innate immunity to pathogen infection. As a result of liver endothelial cell fenestrations, parenchymal cells are not separated by a basal membrane, and thereby pathogen-infected hepatocytes are extensively capable of interacting with innate immune cells including NK cells. In addition, hepatic NK cells interact with surrounding DC and alter their differentiation and function. Recent studies reveal that NK cells exhibit a regulatory function that modulates T cell responses through their interaction with DC and/or direct effect on T cells. Thus, NK cells play a central role, not only in innate immunity, but also in shaping the adaptive immune response. During pathogen infection, there is a remarkable increase of hepatic NK cells, possibly due to the expansion of resident liver NK cells and/or recruitement of NK cells from the blood. The liver microenvironment is believed to modulate hepatic NK cell function through the induction of activating/inhibitory receptor expression and inflammatory cytokine secretion. Particularly, the liver maintains intrahepatic NK cells in a functionally hyporesponsive state compared to splenic NK cells: liver NK cells displayed a dampened IFN-γ response to IL-12/IL-18 stimulation. Notably, the liver contains a significant population of functionally hyporesponsive NK cells that express high levels of the inhibitory receptor NKG2A and lack expression of MHC class I-binding Ly49 receptors. Importantly, adoptively transferred splenic NK cells that migrate to the liver displayed phenotypic and functional changes, supporting a view that the liver environment modifies NK cell receptor expression and functional responsiveness. In this article, we will review studies on the regulation of NK cell repertoire and function in the hepatic environment and the impact of liver NK cell immunoregulatory function on influencing adaptive immunity.

  8. 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.

  9. 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

    2016-02-19

    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.

  10. Thy-1 (CD90)-Positive Hepatic Progenitor Cells, Hepatoctyes, and Non-parenchymal Liver Cells Isolated from Human Livers.

    Science.gov (United States)

    Weiss, Thomas S; Dayoub, Rania

    2017-01-01

    In response to liver injury, hepatic cells, especially hepatocytes, can rapidly proliferate to repair liver damage. Additionally, it was shown that under certain circumstances liver resident cells with progenitor capabilities are involved in liver cell proliferation and differentiation. These hepatic progenitor cells (HPCs), known as oval cells in rodents, are derived from the canals of Hering, which are located in the periportal region of the liver. Regarding to different cell niches, which were defined for human HPCs, several markers have been used to identify these cells such as CD34, c-kit, OV-6, and Thy-1 (CD90). The latter was shown to be expressed on HPCs in human liver tissue with histological signs of regeneration. In this chapter we describe a detailed method for the isolation of Thy-1 positive cells from human resected liver tissue. Based on a procedure for isolating primary human hepatocytes and non-parenchymal cells (NPCs) we expanded this protocol to additional enzymatic dissociation, filtration, and centrifugation steps. This results in a bile duct cell enriched fraction of NPCs from which Thy-1 (CD90) positive cells were purified by Thy-1 positivity selection using MACS technique. Bipotential progenitor cells from human liver resections can be isolated using Thy-1 and was shown to be a suitable tool for the enrichment of liver resident progenitor cells for xenotransplantation.

  11. 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.

  12. 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

  13. 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......, whereas α-amylase tends to have a higher yield on substrate at low specific growth rates. Based on transcriptional analysis, we found that the difference in the production of the two proteins as function of the specific growth rate is mainly due to differences in endoplasmic reticulum processing, protein...... 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...

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

    Science.gov (United States)

    Liu, Zihe; Hou, Jin; Martínez, José L; Petranovic, Dina; Nielsen, Jens

    2013-10-01

    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.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, whereas α-amylase tends to have a higher yield on substrate at low specific growth rates. Based on transcriptional analysis, we found that the difference in the production of the two proteins as function of the specific growth rate is mainly due to differences in endoplasmic reticulum processing, protein 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 for higher specific growth rates, the two strains respond differently to changes in the specific growth rate.

  15. Unsolved Puzzles Surrounding HCV Immunity: Heterologous Immunity Adds Another Dimension

    Science.gov (United States)

    Gupta, Nancy; Li, Wen; Vedi, Satish; Kumar, Rakesh

    2017-01-01

    Chronic infection with hepatitis C virus (HCV) afflicts 3% of the world’s population and can lead to serious and late-stage liver diseases. Developing a vaccine for HCV is challenging because the correlates of protection are uncertain and traditional vaccine approaches do not work. Studies of natural immunity to HCV in humans have resulted in many enigmas. Human beings are not immunologically naïve because they are continually exposed to various environmental microbes and antigens, creating large populations of memory T cells. Heterologous immunity occurs when this pool of memory T cells cross-react against a new pathogen in an individual. Such heterologous immunity could influence the outcome when an individual is infected by a pathogen. We have recently made an unexpected finding that adenoviruses, a common environmental pathogen and an experimental vaccine vector, can induce robust cross-reactive immune responses against multiple antigens of HCV. Our unique finding of previously uncharacterized heterologous immunity against HCV opens new avenues to understand HCV pathogenesis and develop effective vaccines. PMID:28749434

  16. Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin.

    Science.gov (United States)

    Hirt, H; Erlandsen, S L; Dunny, G M

    2000-04-01

    Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.

  17. Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*

    Science.gov (United States)

    Galiullina, Raisa A.; Kasperkiewicz, Paulina; Chichkova, Nina V.; Szalek, Aleksandra; Serebryakova, Marina V.; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B.

    2015-01-01

    Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. PMID:26283788

  18. Susceptibility of human liver cells to porcine endogenous retrovirus.

    Science.gov (United States)

    Lin, Xinzi; Qi, Lin; Li, Zhiguo; Chi, Hao; Lin, Wanjun; Wang, Yan; Jiang, Zesheng; Pan, Mingxin; Gao, Yi

    2013-12-01

    The risk of porcine endogenous retrovirus infection is a major barrier for pig-to-human xenotransplant. Porcine endogenous retrovirus, present in porcine cells, can infect many human and nonhuman primate cells in vitro, but there is no evidence available about in vitro infection of human liver cells. We investigated the susceptibility of different human liver cells to porcine endogenous retrovirus. The supernatant from a porcine kidney cell line was added to human liver cells, including a normal hepatocyte cell line (HL-7702 cells), primary hepatocytes (Phh cells), and a liver stellate cell line (Lx-2 cells), and to human embryonic kidney cells as a reference control. Expression of the porcine endogenous retrovirus antigen p15E in the human cells was evaluated with polymerase chain reaction, reverse transcription-polymerase chain reaction, and Western blot. The porcine endogenous retrovirus antigen p15E was not expressed in any human liver cells (HL-7702, Phh, or Lx-2 cells) that had been exposed to supernatants from porcine kidney cell lines. Porcine endogenous retrovirus-specific fragments were amplified in human kidney cells. Human liver cells tested were not susceptible to infection by porcine endogenous retrovirus. Therefore, not all human cells are susceptible to porcine endogenous retrovirus.

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

    DEFF Research Database (Denmark)

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

    The plant cell wall (CW) consists of numerous complex and uniqe carbohydrate polymer structures. Although the structure (sugar composition) of the various plant CW components are known in some detail, functional characterisation of of the more than 300 glycosyltransferases (GTs), that are believed...... 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...... UDP-xylose on to the monosaccharide sugar fucose. Partly based on these data, the two genes were proposed to function in the biosynthesis of pectic rhamnogalacturonan II (RG-II) and designated RhamnoGalacturonan XylosylTransferase 1 and -2 (RGXT1 and -2), accordingly (Egelund et al. 2006, The Plant...

  20. Hepatic stellate cells in liver development, regeneration, and cancer

    Science.gov (United States)

    Yin, Chunyue; Evason, Kimberley J.; Asahina, Kinji; Stainier, Didier Y.R.

    2013-01-01

    Hepatic stellate cells are liver-specific mesenchymal cells that play vital roles in liver physiology and fibrogenesis. They are located in the space of Disse and maintain close interactions with sinusoidal endothelial cells and hepatic epithelial cells. It is becoming increasingly clear that hepatic stellate cells have a profound impact on the differentiation, proliferation, and morphogenesis of other hepatic cell types during liver development and regeneration. In this Review, we summarize and evaluate the recent advances in our understanding of the formation and characteristics of hepatic stellate cells, as well as their function in liver development, regeneration, and cancer. We also discuss how improved knowledge of these processes offers new perspectives for the treatment of patients with liver diseases. PMID:23635788

  1. Isolation and Culture of Single Cell Types from Rat Liver.

    Science.gov (United States)

    Zhang, Qidi; Qu, Ying; Li, Zhenghong; Zhang, Qingqing; Xu, Mingyi; Cai, Xiaobo; Li, Fei; Lu, Lungen

    2016-01-01

    There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future. © 2016 S. Karger AG, Basel.

  2. Role of NKT cells in autoimmune liver disease.

    Science.gov (United States)

    Santodomingo-Garzon, Tania; Swain, Mark G

    2011-10-01

    The three main broad categories of autoimmune liver disease are autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC). The etiologies of these diseases are still incompletely understood, but seem to involve a combination of immune, genetic and environmental factors. Although each of these diseases has relatively distinct clinical, serologic and histological profiles, all of them share common pathways of immune-mediated liver injury. The development of autoimmune liver diseases is thought to be due to an imbalance of proinflammatory and anti-inflammatory immune responses within the liver, with proinflammatory immune responses being upregulated and anti-inflammatory ones downregulated. The available evidence, suggest that during autoimmune responses within the liver, "self" antigens are presented by antigen presenting cells (APCs) which then activate, directly and/or indirectly, NKT cells and other innate immune cells within the liver. Importantly, the hepatic innate immune system plays an increasingly recognized role in the development and propagation of autoimmune liver injury. NKT cells predominantly reside in the liver sinusoids, and through their ability to rapidly produce a wide variety of cytokines (e.g. Th1, TH2, Th17 cytokine patterns), are a critical checkpoint that bridges innate and adaptive immune responses. Specifically, activated NKT cells are capable of transactivating other innate and adaptive immune cells within the liver to amplify and regulate subsequent immune responses within the liver. It has been hypothesized that NKT cells in the setting of autoimmune liver disease can play diverse roles, including driving both anti-inflammatory and proinflammatory responses, as well as regulating the hepatic recruitment of other types of immunoregulatory cells, including regulatory T cells. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Analysis of the CD8+T cell anti-HIV activity in heterologous cell co-cultures reveals the benefit of multiple HLA class I matches.

    Science.gov (United States)

    Killian, M Scott; Teque, Fernando; Sudhagoni, Ramu

    2018-02-01

    CD8 + T lymphocytes can reduce the production of human immunodeficiency virus 1 (HIV-1) by CD4 + T cells by cytotoxic and non-cytotoxic mechanisms. To investigate the involvement of human leukocyte antigen (HLA) class I compatibility in anti-HIV responses, we co-cultured primary CD8 + T cells, isolated from the peripheral blood of HIV-1-infected individuals, with panels of autologous and heterologous acutely HIV-1-infected primary CD4 + T cells. Altogether, CD8 + T cell anti-HIV activity was evaluated in more than 200 co-cultures. Marked heterogeneity in HIV-1 replication levels was observed among the co-cultures sharing a common CD8 + T cell source. The co-cultures that exhibited greater than 50% reduction in HIV production were found to have significantly increased numbers of matching HLA class I alleles (Yates chi-square = 54.21; p T cells from HIV controllers and asymptomatic viremic individuals, matching HLA-B and/or HLA-C alleles were more predictive of strong anti-HIV activity than matching HLA-A alleles. Overall, HLA class I genotype matches were more closely associated with CD8 + T cell anti-HIV activity than supertype pairings. Antibodies against HLA class I and CD3 reduced the CD8 + T cell anti-HIV activity. Stimulated CD8 + T cells exhibited increased anti-HIV activity and reduced dependency on HLA compatibility. These findings provide evidence that the maximal suppression of HIV replication by CD8 + T cells requires the recognition of multiple epitopes. These studies provide insight for HIV vaccine development, and the analytic approach can be useful for the functional characterization of HLA class I alleles and tentative HLA class I supertypes.

  4. Simultaneous characterization of progenitor cell compartments in adult human liver.

    Science.gov (United States)

    Porretti, Laura; Cattaneo, Alessandra; Colombo, Federico; Lopa, Raffaella; Rossi, Giorgio; Mazzaferro, Vincenzo; Battiston, Carlo; Svegliati-Baroni, Gianluca; Bertolini, Francesco; Rebulla, Paolo; Prati, Daniele

    2010-01-01

    The human liver is a complex tissue consisting of epithelial, endothelial, hematopoietic, and mesenchymal elements that probably derive from multiple lineage-committed progenitors, but no comprehensive study aimed at identifying and characterizing intrahepatic precursors has yet been published. Cell suspensions for this study were obtained by enzymatic digestion of liver specimens taken from 20 patients with chronic liver disease and 13 multiorgan donors. Stem and progenitor cells were first isolated, amplified, and characterized ex vivo according to previously validated methods, and then optimized flow cytometry was used to assess their relative frequencies and characterize their immunophenotypes in the clinical specimens. Stem and progenitor cells committed to hematopoietic, endothelial, epithelial, and mesenchymal lineages were clearly identifiable in livers from both healthy and diseased subjects. Within the mononuclear liver cell compartment, epithelial progenitors [epithelial cell adhesion molecule (EpCAM)(+)/CD49f(+)/CD29(+)/CD45(-)] accounted for 2.7-3.5% whereas hematopoietic (CD34(+)/CD45(+)), endothelial [vascular endothelial growth factor-2 (KDR)(+)/CD146(+)/CD45(-)], and mesenchymal [CD73(+)/CD105(+)/CD90 (Thy-1)(+)/CD45 (-)] stem cells and progenitors accounted for smaller fractions (0.02-0.6%). The patients' livers had higher percentages of hematopoietic and endothelial precursors than those of the donors. In conclusion, we identified and characterized precursors committed to four different lineages in adult human liver. We also optimized a flow cytometry approach that will be useful in exploring the contribution of these cells to the pathogenesis of liver disease.

  5. Macrophages and dendritic cells in the development of liver injury leading to liver failure.

    Science.gov (United States)

    Ananiev, J; Penkova, M; Tchernev, G; Chokoeva, A A; Philipov, S; Tana, C; Gulubova, M; Wollina, U

    2014-01-01

    Liver failure (LF) continues to be a serious problem due to different underlying disorders. Not only hepatocytes but Kupffer cells (KCs) and dendritic cells (DCs) are of importance in this instance. We wanted to investigate the possible role of KCs and liver DCs in the development of liver injury in patients with liver failure. Liver specimens from 23 patients who died after liver failure were examined for the presence and distribution of CD68-positive KCs and CD83-positive DCs by immunohistochemistry. The distribution of the CD83-positive DC in the sinusoidal and the periportal spaces was not even. While 39.1% of patients had a high sinusoidal density of CD83-positive cells, 60.9% demonstrated a high density of CD83-positive cells in the periportal tract. The number of CD83-positive DCs in periportal tracts in patients with advanced liver fibrosis (n=5) were high, while those with mild liver fibrosis (n=18) had low numbers of mature dendritic cells (χ2=4.107; p=0.043). In addition, all patients with intensive fibrosis had low counts of CD68-positive KC’s in portal tracts vs patients with mild fibrosis of which 67% had high counts (χ2=6.97; p=0.008). In seven of the patients with moderate steatosis (87.5%) low numbers of CD68-positive KCs were found in sinusoids, in contrast to those with severe steatosis, where 12 patients (80%) had high KC counts (χ2=13.4; p less than 0.001). The distribution and number of CD68-positive KC and CD83-positive DC reflect the progression of liver fibrosis leading to liver failure.

  6. Liver Cancer: Molecular Characterization, Clonal Evolution and Cancer Stem Cells

    Directory of Open Access Journals (Sweden)

    Germana Castelli

    2017-09-01

    Full Text Available Liver cancer is the second most common cause of cancer-related death. The major forms of primary liver cancer are hepatocellular carcinoma (HCC and intrahepatic cholangiocarcinoma (iCCA. Both these tumors develop against a background of cirrhotic liver, non-alcoholic fatty liver disease, chronic liver damage and fibrosis. HCC is a heterogeneous disease which usually develops within liver cirrhosis related to various etiologies: hepatitis B virus (HBV infection (frequent in Asia and Africa, hepatitis C virus (HCV, chronic alcohol abuse, or metabolic syndrome (frequent in Western countries. In cirrhosis, hepatocarcinogenesis is a multi-step process where pre-cancerous dysplastic macronodules transform progressively into HCC. The patterns of genomic alterations observed in these tumors were recently identified and were instrumental for the identification of potential targeted therapies that could improve patient care. Liver cancer stem cells are a small subset of undifferentiated liver tumor cells, responsible for cancer initiation, metastasis, relapse and chemoresistance, enriched and isolated according to immunophenotypic and functional properties: cell surface proteins (CD133, CD90, CD44, EpCAM, OV-6, CD13, CD24, DLK1, α2δ1, ICAM-1 and CD47; the functional markers corresponding to side population, high aldehyde dehydrogenase (ALDH activity and autofluorescence. The identification and definition of liver cancer stem cells requires both immunophenotypic and functional properties.

  7. A Transcriptomic Signature of Mouse Liver Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Adam M. Passman

    2016-01-01

    Full Text Available Liver progenitor cells (LPCs can proliferate extensively, are able to differentiate into hepatocytes and cholangiocytes, and contribute to liver regeneration. The presence of LPCs, however, often accompanies liver disease and hepatocellular carcinoma (HCC, indicating that they may be a cancer stem cell. Understanding LPC biology and establishing a sensitive, rapid, and reliable method to detect their presence in the liver will assist diagnosis and facilitate monitoring of treatment outcomes in patients with liver pathologies. A transcriptomic meta-analysis of over 400 microarrays was undertaken to compare LPC lines against datasets of muscle and embryonic stem cell lines, embryonic and developed liver (DL, and HCC. Three gene clusters distinguishing LPCs from other liver cell types were identified. Pathways overrepresented in these clusters denote the proliferative nature of LPCs and their association with HCC. Our analysis also revealed 26 novel markers, LPC markers, including Mcm2 and Ltbp3, and eight known LPC markers, including M2pk and Ncam. These markers specified the presence of LPCs in pathological liver tissue by qPCR and correlated with LPC abundance determined using immunohistochemistry. These results showcase the value of global transcript profiling to identify pathways and markers that may be used to detect LPCs in injured or diseased liver.

  8. Cell therapy for liver diseases: current medicine and future promises.

    Science.gov (United States)

    Alejandra, Meza-Ríos; Juan, Armendáriz-Borunda; Ana, Sandoval-Rodríguez

    2015-06-01

    Liver diseases are a major health problem worldwide since they usually represent the main causes of death in most countries, causing excessive costs to public health systems. Nowadays, there are no efficient current therapies for most hepatic diseases and liver transplant is infrequent due to the availability of organs, cost and risk of transplant rejection. Therefore, alternative therapies for liver diseases have been developed, including cell-based therapies. Stem cells (SCs) are characterized by their self-renewing capacity, unlimited proliferation and differentiation under certain conditions into tissue- or organ-specific cells with special functions. Cell-based therapies for liver diseases have been successful in experimental models, showing anti-inflammatory, antifibrogenic and regenerative effects. Nowadays, clinical trials using SCs for liver pathologies are increasing in number, and those that have reached publication have achieved favorable effects, encouraging us to think that SCs will have a potential clinical use in a short time.

  9. Cancer stem cells in the development of liver cancer

    Science.gov (United States)

    Yamashita, Taro; Wang, Xin Wei

    2013-01-01

    Liver cancer is an aggressive disease with a poor outcome. Several hepatic stem/progenitor markers are useful for isolating a subset of liver cells with stem cell features, known as cancer stem cells (CSCs). These cells are responsible for tumor relapse, metastasis, and chemoresistance. Liver CSCs dictate a hierarchical organization that is shared in both organogenesis and tumorigenesis. An increased understanding of the molecular signaling events that regulate cellular hierarchy and stemness, and success in defining key CSC-specific genes, have opened up new avenues to accelerate the development of novel diagnostic and treatment strategies. This Review highlights recent advances in understanding the pathogenesis of liver CSCs and discusses unanswered questions about the concept of liver CSCs. PMID:23635789

  10. Mesenchymal Stem Cell-Dependent Modulation of Liver Diseases.

    Science.gov (United States)

    Gazdic, Marina; Arsenijevic, Aleksandar; Markovic, Bojana Simovic; Volarevic, Ana; Dimova, Ivanka; Djonov, Valentin; Arsenijevic, Nebojsa; Stojkovic, Miodrag; Volarevic, Vladislav

    2017-01-01

    Acute liver failure and cirrhosis display sequential and overlapping severe pathogenic processes that include inflammation, hepatocyte necrosis, and fibrosis, carrying a high mortality rate. Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells with immunonodulatory characteristics. MSCs are considered to act through multiple mechanisms to coordinate a dynamic, integrated response to liver inflammation and fibrosis, which prevents the progressive distortion of hepatic architecture. Accordingly, MSCs as well as their products have been investigated as a novel therapeutic approach for the treatment of inflammatory and fibrotic liver diseases. In this review, we highlight the current findings on the MSC-based modulation of liver inflammation and fibrosis, and the possible use of MSCs in the therapy of immune-mediated liver pathology. We briefly describe the cellular and molecular mechanisms involved in MSC-dependent modulation of cytokine production, phenotype and function of liver infiltrated inflammatory cells and compare effects of engrafted MSCs versus MSC-generated conditioned medium (MSC-CM) in the therapy of acute liver injury. In order to elucidate therapeutic potential of MSCs and their products in modulation of chronic liver inflammation and fibrosis, we present the current findings regarding pathogenic role of immune cells in liver fibrosis and describe mechanisms involved in MSC-dependent modulation of chronic liver inflammation with the brief overview of on-going and already published clinical trials that used MSCs for the treatment of immune mediated chronic liver diseases. The accumulating evidence shows that MSCs had a significant beneficial effect in the treatment of immune-mediated liver diseases.

  11. Epigenetic regulation of hepatic stellate cell activation and liver fibrosis.

    Science.gov (United States)

    El Taghdouini, Adil; van Grunsven, Leo A

    2016-12-01

    Chronic liver injury to hepatocytes or cholangiocytes, when left unmanaged, leads to the development of liver fibrosis, a condition characterized by the excessive intrahepatic deposition of extracellular matrix proteins. Activated hepatic stellate cells constitute the predominant source of extracellular matrix in fibrotic livers and their transition from a quiescent state during fibrogenesis is associated with important alterations in their transcriptional and epigenetic landscape. Areas covered: We briefly describe the processes involved in hepatic stellate cell activation and discuss our current understanding of alterations in the epigenetic landscape, i.e DNA methylation, histone modifications and the functional role of non-coding RNAs that accompany this key event in the development of chronic liver disease. Expert commentary: Although great progress has been made, our understanding of the epigenetic regulation of hepatic stellate cell activation is limited and, thus far, insufficient to allow the development of epigenetic drugs that can selectively interrupt liver fibrosis.

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

    Indian Academy of Sciences (India)

    Eight liver cell types were isolated using the methods of Percoll density gradient centrifugation and immunomagnetic beads to explore effects of histidine catabolites on rat liver regeneration. Rat Genome 230 2.0 Array was used to detect the expression profiles of genes associated with metabolism of histidine and its ...

  13. Liver X receptor α is essential for the capillarization of liver sinusoidal endothelial cells in liver injury.

    Science.gov (United States)

    Xing, Yan; Zhao, Tingting; Gao, Xiaoyan; Wu, Yuzhang

    2016-02-18

    Liver X receptors (LXRs) play essential roles in lipogenesis, anti-inflammatory action and hepatic stellate cells (HSCs) activation in the liver. However, the effects of LXRs on the capillarization of liver sinusoidal endothelial cells (LSECs) in liver fibrosis remain undetermined. Here, we demonstrated that LXRα plays an important role in LSECs capillarization in a manner that involved Hedgehog (Hh) signaling. We found that LXRα expression in LSECs was increased in the carbon tetrachloride (CCl4)-induced fibrosis model. LXRα deletion markedly exacerbated CCl4-induced lesions assessed by histopathology, as well as inflammation and collagen deposition. Furthermore, capillarization of the sinusoids was aggravated in CCl4 -treated LXRα-deficient mice, as evidenced by increased CD34 expression, the formation of continuous basement membranes and aggravation of the loss of fenestrae. In vitro, LXR agonist could maintain freshly isolated LSECs differentiation on day 3. Furthermore, LXRα deletion led to increased expression of Hedgehog (Hh)-regulated gene in LSECs in the injured liver. Conversely, the LXR agonist could inhibit the Hh pathway in cultured LSECs. These responses indicated that LXRα suppressed the process of LSECs capillarization by repressing Hh signaling. Overall, our findings suggest that LXRα, by restoring the differentiation of LSECs, may be critical for the regression of liver fibrosis.

  14. Signals and Cells Involved in Regulating Liver Regeneration

    Directory of Open Access Journals (Sweden)

    Liang-I. Kang

    2012-12-01

    Full Text Available Liver regeneration is a complex phenomenon aimed at maintaining a constant liver mass in the event of injury resulting in loss of hepatic parenchyma. Partial hepatectomy is followed by a series of events involving multiple signaling pathways controlled by mitogenic growth factors (HGF, EGF and their receptors (MET and EGFR. In addition multiple cytokines and other signaling molecules contribute to the orchestration of a signal which drives hepatocytes into DNA synthesis. The other cell types of the liver receive and transmit to hepatocytes complex signals so that, in the end of the regenerative process, complete hepatic tissue is assembled and regeneration is terminated at the proper time and at the right liver size. If hepatocytes fail to participate in this process, the biliary compartment is mobilized to generate populations of progenitor cells which transdifferentiate into hepatocytes and restore liver size.

  15. Therapeutic Implications of Mesenchymal Stem Cells in Liver Injury

    Directory of Open Access Journals (Sweden)

    Maria Ausiliatrice Puglisi

    2011-01-01

    Full Text Available Mesenchymal stem cells (MSCs, represent an attractive tool for the establishment of a successful stem-cell-based therapy of liver diseases. A number of different mechanisms contribute to the therapeutic effects exerted by MSCs, since these cells can differentiate into functional hepatic cells and can also produce a series of growth factors and cytokines able to suppress inflammatory responses, reduce hepatocyte apoptosis, regress liver fibrosis, and enhance hepatocyte functionality. To date, the infusion of MSCs or MSC-conditioned medium has shown encouraging results in the treatment of fulminant hepatic failure and in end-stage liver disease in experimental settings. However, some issues under debate hamper the use of MSCs in clinical trials. This paper summarizes the biological relevance of MSCs and the potential benefits and risks that can result from translating the MSC research to the treatment of liver diseases.

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

    NARCIS (Netherlands)

    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

  17. Unbalanced fermentation of glycerol in Escherichia coli via heterologous production of an electron transport chain and electrode interaction in microbial electrochemical cells.

    Science.gov (United States)

    Sturm-Richter, Katrin; Golitsch, Frederik; Sturm, Gunnar; Kipf, Elena; Dittrich, André; Beblawy, Sebastian; Kerzenmacher, Sven; Gescher, Johannes

    2015-06-01

    Microbial electrochemical cells are an emerging technology for achieving unbalanced fermentations. However, organisms that can serve as potential biocatalysts for this application are limited by their narrow substrate spectrum. This study describes the reprogramming of Escherichia coli for the efficient use of anodes as electron acceptors. Electron transfer into the periplasm was accelerated by 183% via heterologous expression of the c-type cytochromes CymA, MtrA and STC from Shewanella oneidensis. STC was identified as a target for heterologous expression via a two-stage screening approach. First, mass spectroscopic analysis revealed natively expressed cytochromes in S. oneidensis. Thereafter, the corresponding genes were cloned and expressed in E. coli to quantify periplasmic electron transfer activity using methylene blue. This redox dye was further used to expand electron transfer to carbon electrode surfaces. The results demonstrate that E. coli can be reprogrammed from glycerol fermentation to respiration upon production of the new electron transport chain. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Development of Liver Decellularized Extracellular Matrix Bioink for Three-Dimensional Cell Printing-Based Liver Tissue Engineering.

    Science.gov (United States)

    Lee, Hyungseok; Han, Wonil; Kim, Hyeonji; Ha, Dong-Heon; Jang, Jinah; Kim, Byoung Soo; Cho, Dong-Woo

    2017-04-10

    The liver is an important organ and plays major roles in the human body. Because of the lack of liver donors after liver failure and drug-induced liver injury, much research has focused on developing liver alternatives and liver in vitro models for transplantation and drug screening. Although numerous studies have been conducted, these systems cannot faithfully mimic the complexity of the liver. Recently, three-dimensional (3D) cell printing technology has emerged as one of a number of innovative technologies that may help to overcome this limitation. However, a great deal of work in developing biomaterials optimized for 3D cell printing-based liver tissue engineering remains. Therefore, in this work, we developed a liver decellularized extracellular matrix (dECM) bioink for 3D cell printing applications and evaluated its characteristics. The liver dECM bioink retained the major ECM components of the liver while cellular components were effectively removed and further exhibited suitable and adjustable properties for 3D cell printing. We further studied printing parameters with the liver dECM bioink to verify the versatility and fidelity of the printing process. Stem cell differentiation and HepG2 cell functions in the liver dECM bioink in comparison to those of commercial collagen bioink were also evaluated, and the liver dECM bioink was found to induce stem cell differentiation and enhance HepG2 cell function. Consequently, the results demonstrate that the proposed liver dECM bioink is a promising bioink candidate for 3D cell printing-based liver tissue engineering.

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

    Science.gov (United States)

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

    2016-09-01

    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. © 2016 by the Society for Experimental Biology and Medicine.

  20. Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus.

    Science.gov (United States)

    Vázquez-Gómez, Elizabeth; Arias, Hugo R; Feuerbach, Dominik; Miranda-Morales, Marcela; Mihailescu, Stefan; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof; García-Colunga, Jesús

    2014-10-05

    The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Histopathology and cell culture characteristics of liver cells from grc- and grc+ rats given diethylnitrosamine.

    Science.gov (United States)

    Smith, G J; Kunz, H W; Dunsford, H A; Gill, T J

    1990-04-01

    The histopathological response and cell culture characteristics of liver cells from the R16 (grc-) strain of rats, which carries an MHC-linked deletion, were examined one week after a single intraperitoneal injection of 200 mg/kg body weight diethylnitrosamine (DEN) and were compared with the response of liver cells from wild type (grc+) rats. The DEN exposure induced hydropic/vacuolar changes in the parenchymal cells and a limited proliferation of oval cells in the periportal areas of the livers of both grc+ and grc- rats. Primary culture of collagenase-digested livers consisted of parenchymal, bile ductular and oval-related cells as determined by cell-specific immunohistochemistry. Subpassaged cells from grc+ rats exhibited oval cell ultrastructural morphology, inducible histochemical staining for gamma-glutamyl transpeptidase (GGT), and DEN-associated onset of anchorage-independent growth. Primary cultures of liver cells from R16 rats consistently failed to form cell strains upon subpassage.

  2. Antitumor Immunity Produced by the Liver Kupffer Cells, NK Cells, NKT Cells, and CD8+ CD122+ T Cells

    Directory of Open Access Journals (Sweden)

    Shuhji Seki

    2011-01-01

    Full Text Available Mouse and human livers contain innate immune leukocytes, NK cells, NKT cells, and macrophage-lineage Kupffer cells. Various bacterial components, including Toll-like receptor (TLR ligands and an NKT cell ligand (α-galactocylceramide, activate liver Kupffer cells, which produce IL-1, IL-6, IL-12, and TNF. IL-12 activates hepatic NK cells and NKT cells to produce IFN-γ, which further activates hepatic T cells, in turn activating phagocytosis and cytokine production by Kupffer cells in a positive feedback loop. These immunological events are essentially evoked to protect the host from bacterial and viral infections; however, these events also contribute to antitumor and antimetastatic immunity in the liver by activated liver NK cells and NKT cells. Bystander CD8+CD122+ T cells, and tumor-specific memory CD8+T cells, are also induced in the liver by α-galactocylceramide. Furthermore, adoptive transfer experiments have revealed that activated liver lymphocytes may migrate to other organs to inhibit tumor growth, such as the lungs and kidneys. The immunological mechanism underlying the development of hepatocellular carcinoma in cirrhotic livers in hepatitis C patients and liver innate immunity as a double-edged sword (hepatocyte injury/regeneration, septic shock, autoimmune disease, etc. are also discussed.

  3. Liver stem cell therapy for cirrhosis: advances in cell classification and selection

    Directory of Open Access Journals (Sweden)

    XU Ying

    2015-08-01

    Full Text Available Liver cirrhosis is the final outcome of various chronic liver diseases. In recent years, along with the rapid development of molecular biology and cell biological engineering technology, liver regeneration based on stem cell transplantation technique has become a new research hotspot for treatment of acute and chronic hepatic failure. Here we review basic and clinical studies on different types of stem cells for the treatment of liver cirrhosis and optimal choices of the stem cell type used for transplantation based on specificity of patients' particular status. A large number of experimental studies reveal that the technology of stem cell for the treatment of liver cirrhosis has gradually become mature with a broad prospect of application in the field of liver regeneration. This technology that holds enormous potential for treatment will bring hopes to the patients with end-stage liver cirrhosis.

  4. 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

  5. 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.

  6. 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.

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

    Science.gov (United States)

    Jiang, Lili; Teng, Gladys M K; Chan, Elaine Y M; Au, Shannon W N; Wise, Helen; Lee, Susanna S T; Cheung, Wing-Tai

    2012-01-01

    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.

  8. Specific cross-immunity between Hymenolepis nana and H. diminuta: effects of transfer of immunity with homologous and heterologous immune mesenteric lymph node cells in BALB/c mice.

    Science.gov (United States)

    Palmas, C; Ecca, A R; Gabriele, F

    1993-12-01

    In BALB/c mice, Hymenolepis nana and H. diminuta stimulate a strong response. Accelerated expulsion of adult worms, as well as protection against larvae, occurred anamnestically when homologous or heterologous mesenteric lymph node cells from immune mice were transferred before challenge. The results further support the hypothesis that worm expulsion is a thymus dependent phenomenon. The extensive cross reactivity found is discussed in relation to the distinctive characteristics of the two worms. These results strongly suggest that there are antigenic similarities between the two parasites and that the accelerated expulsion of heterologous challenge infections is the result of a specific response to shared antigens.

  9. CORRELATED MORPHOMETRIC AND BIOCHEMICAL STUDIES ON THE LIVER CELL

    Science.gov (United States)

    Weibel, Ewald R.; Stäubli, Willy; Gnägi, Hans Rudolf; Hess, Felix A.

    1969-01-01

    The basic morphological properties of liver cells are defined in the form of a morphometric model to permit integrated quantitative characterization of functionally important parameters. Stereologic methods which allow efficient and reliable quantitative evaluation of sectioned liver tissue are presented. Material, obtained by a rigorous three-stage sampling procedure from five normal rat livers, is systematically subjected to this analysis at four levels of magnification. This yields quantitative data which are expressed as "densities," i.e. content per 1 ml of tissue, as "specific dimensions" related to 100 g body weight, and as absolute dimensions per average "mononuclear" hepatocyte. Base line data relating to the normal rat liver are presented for the entire spectrum of parameters. As examples, 1 ml of liver tissue contains 169 x 106 hepatocyte nuclei, some 90 x 106 nuclei of other cells, and 280 x 109 mitochondria. Hepatocyte cytoplasm accounts for 77% of liver volume, and the mitochondria for 18%. The surface area of endoplasmic reticulum membranes in 1 ml of liver tissue measures 11 m2 of which are ⅔ of the rough form carrying some 2 x 1013 ribosomes. The surface area of mitochondrial cristae in the unit volume is estimated at 6 m2. The validity and applicability of the method are discussed, and the data are compared with available information from other studies. PMID:4891915

  10. 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.

  11. Heterologous vaccine effects

    NARCIS (Netherlands)

    Saadatian-Elahi, M.; Aaby, P.; Shann, F.; Netea, M.G.; Levy, O.; Louis, J.; Picot, V.; Greenberg, M.; Warren, W.

    2016-01-01

    The heterologous or non-specific effects (NSEs) of vaccines, at times defined as "off-target effects" suggest that they can affect the immune response to organisms other than their pathogen-specific intended purpose. These NSEs have been the subject of clinical, immunological and epidemiological

  12. Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver

    DEFF Research Database (Denmark)

    Oertel, Michael; Menthena, Anuradha; Chen, Yuan-Qing

    2008-01-01

    . Rat ED14 FLSPC are alpha-fetoprotein(+)/cytokeratin-19(+) or alpha-fetoprotein(+)/cytokeratin-19(-) and contain all of the normal liver repopulation capacity found in fetal liver. Hematopoietic stem cells, a major component in crude fetal liver cell preparations that engraft in other organs......BACKGROUND & AIMS: Previously, we showed high-level, long-term liver replacement after transplantation of unfractionated embryonic day (ED) 14 fetal liver stem/progenitor cells (FLSPC). However, for clinical applications, it will be essential to transplant highly enriched cells, while maintaining...... and characteristic properties in vitro and their proliferative and differentiation potential in vivo after transplantation into normal adult rat liver. RESULTS: Rat ED14 FLSPC were purified to 95% homogeneity and exhibited cell culture and gene expression characteristics expected for hepatic stem/progenitor cells...

  13. Nonparenchymal liver cells and granulomas during lamprey biliary atresia.

    Science.gov (United States)

    Youson, J H; Sargent, P A; Yamamoto, K; Ogilvie, D; Fisher, M M

    1987-06-01

    Transmission (thin sections and freeze-fracture replicas) and scanning electron microscopy were used to describe the nonparenchymal liver cells during the seven (1-7) stages of metamorphosis in the sea lamprey, Petromyzon marinus L., when bile ducts and canaliculi degenerate. The biliary atresia is accompanied by an increased diameter of fenestrae in the endothelium, an active phagocytosis by Kupffer cells in the sinusoids, and large lipid inclusions in perisinusoidal lipocytes (fat-storing or Ito cells). Plasma-like cells and foci of nonparenchymal cells (granulomas) are present in the liver interstitium during at least four stages of metamorphosis. The fenestrae in the sinusoidal wall are wider (up to 2.8-micron diameter) than normally reported for vertebrate livers but are likely a reflection of the morphogenetic and physiological events and consequences of the biliary atresia. Kupffer cells are involved in an extensive erythrophagocytosis, the storage of iron, and perhaps the incorporation of cellular components from hepatocytes. Lipocytes are the vitamin A-storing cells of the transforming liver and may be responsible for some perisinusoidal fibrosis. Granulomas are present during stages 3-6 and are focal areas where mononuclear leukocytes (lymphocytes and plasmalike cells), macrophages, and neutrophils have infiltrated the hepatic parenchyma. The function of the granulomas is not known; but their presence may be related to the porous nature of the sinusoidal wall, the tissue degeneration, and/or the physiological change (e.g., bile stasis) during biliary atresia.

  14. Liver biopsy findings in patients with hematopoietic cell transplantation.

    Science.gov (United States)

    Eskandari, Farzan; Rowan, Daniel J; Hari, Parameswaran; Kapke, Jonathan; Schneidewend, Robert; E Hagen, Catherine; Oshima, Kiyoko

    2017-08-01

    Liver dysfunction is a frequent complication after hematopoietic cell transplantation. Liver biopsy has an important role for confirming the diagnosis of graft-versus-host disease (GVHD) or other liver diseases. The histological features of GVHD are not specific, and GVHD and other coexisting diseases may be present in the same biopsy, which makes the histologic interpretation of the liver biopsy more complex and challenging. The aim of the study is to improve the present diagnostic criteria. Fifty-two liver biopsies were studied. Most biopsies (47, 92%) showed some features of GVHD. Five (9.6%) had no GVHD, 20 (38.5%) had possible GVHD, and 27 (51.9%) had likely GVHD. Histologic features were analyzed semi-quantitatively and scored. Bile duct damage and intraepithelial lymphocytes were significantly more frequent in likely GVHD groups. Bile duct injury score calculated as the sum of bile duct damage and intraepithelial lymphocytes score was 2.3 in no GVHD and possible GVHD groups, and 4.2 in likely GVHD group (Pliver injury (8, 16%) and sinusoidal obstruction syndrome (6, 12%) are particularly important causes of liver dysfunction. Moderate degree of bile duct injury and intraepithelial lymphocytes were the most helpful histologic findings to confirm the diagnosis of GVHD. In addition, it is important for the pathologist to be aware of the etiologies of liver dysfunction other than GVHD. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes.

    Science.gov (United States)

    Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V Bleu; Serrano, Elba E

    2011-10-01

    The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology.

  16. PC61 (anti-CD25) treatment inhibits influenza A virus-expanded regulatory T cells and severe lung pathology during a subsequent heterologous lymphocytic choriomeningitis virus infection.

    Science.gov (United States)

    Kraft, Anke R M; Wlodarczyk, Myriam F; Kenney, Laurie L; Selin, Liisa K

    2013-12-01

    Prior immunity to influenza A virus (IAV) in mice changes the outcome to a subsequent lymphocytic choriomeningitis virus (LCMV) infection and can result in severe lung pathology, similar to that observed in patients that died of the 1918 H1N1 pandemic. This pathology is induced by IAV-specific memory CD8(+) T cells cross-reactive with LCMV. Here, we discovered that IAV-immune mice have enhanced CD4(+) Foxp3(+) T-regulatory (Treg) cells in their lungs, leading us to question whether a modulation in the normal balance of Treg and effector T-cell responses also contributes to enhancing lung pathology upon LCMV infection of IAV-immune mice. Treg cell and interleukin-10 (IL-10) levels remained elevated in the lungs and mediastinal lymph nodes (mLNs) throughout the acute LCMV response of IAV-immune mice. PC61 treatment, used to decrease Treg cell levels, did not change LCMV titers but resulted in a surprising decrease in lung pathology upon LCMV infection in IAV-immune but not in naive mice. Associated with this decrease in pathology was a retention of Treg in the mLN and an unexpected partial clonal exhaustion of LCMV-specific CD8(+) T-cell responses only in IAV-immune mice. PC61 treatment did not affect cross-reactive memory CD8(+) T-cell proliferation. These results suggest that in the absence of IAV-expanded Treg cells and in the presence of cross-reactive memory, the LCMV-specific response was overstimulated and became partially exhausted, resulting in a decreased effector response. These studies suggest that Treg cells generated during past infections can influence the characteristics of effector T-cell responses and immunopathology during subsequent heterologous infections. Thus, in humans with complex infection histories, PC61 treatment may lead to unexpected results.

  17. [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.

  18. 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

  19. 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

  20. Side Population Cells Derived from Adult Human Liver Generate Hepatocyte-like Cells In Vitro

    OpenAIRE

    HUSSAIN, SUNNY ZAHEED; Strom, Stephen C.; Kirby, Martha R.; Burns, Sean; Langemeijer, Saskia; Ueda, Takahiro; HSIEH, MATTHEW; Tisdale, John F.

    2005-01-01

    We sought to determine whether hepatic side population (SP) cells derived from adult human liver possess the potential of a novel candidate hepatic stem cell. Human cadaveric donor liver was subjected to collagenase perfusion and hepatocytes were separated from nonparenchymal cells by differential centrifugation. SP cells were isolated from the nonparenchymal portion after Hoechst 33342 staining. Since CD45 is a panleukocyte antigen, CD45-negative SP cells were separated from the vast majorit...

  1. Growth factors and hepatic progenitor cells in liver regeneration : translating bench to bedside

    NARCIS (Netherlands)

    Kruitwagen, H.S.

    2017-01-01

    Upon severe acute or chronic liver injury, hepatic progenitor cells (HPCs) become activated. HPCs are adult stem cells of the liver and are considered a reserve population acting as second line of defense in liver regeneration. However, in many cases of severe liver disease this repair mechanism

  2. Isolation, propagation, and characterization of rat liver serosal mesothelial cells.

    Science.gov (United States)

    Faris, R A; McBride, A; Yang, L; Affigne, S; Walker, C; Cha, C J

    1994-12-01

    Although rat liver epithelial cell (RLEC) lines have been developed by a number of laboratories, the identity of the clonogenic nonparenchymal progenitors is unknown. To provide insight into the derivation of RLEC, we immunoisolated serosal liver mesothelial cells (LMC) and bile duct epithelial cells and attempted to propagate each epithelial cell population using culture conditions routinely employed to establish RLEC lines. Briefly, the selective reactivity of LMC with two bile duct cell surface markers, OC.2 and BD.2, was exploited to develop an immunocytochemical technique to isolate LMC. Livers were collagenase dissociated, the mesothelial capsule was "peeled" and digested with pronase to destroy contaminating hepatocytes, and rare biliary ductal epithelial cells were immunodepleted using OC.2. LMC were subsequently isolated by selective binding to magnetic beads adsorbed with BD.2 and cultured in supplemented Waymouths 752/1 media containing 10% fetal calf serum. Proliferating BD.2+ LMC rapidly formed epithelial-like monolayers that could be continuously subcultured after trypsinization. In contrast, attempts to establish cell lines from purified OC.2+ bile duct epithelial cells were unsuccessful. Results from reverse transcriptase polymerase chain reaction analysis confirmed that LMC expressed Wilms' tumor transcripts, a lineage marker for mesodermally-derived cells. In summary, our findings clearly demonstrate that LMC can be continuously propagated using culture conditions routinely employed to establish RLEC lines, an observation that supports the contention that some RLEC lines may be derived from LMC.

  3. Molecular Biology of Liver Cancer Stem Cells

    National Research Council Canada - National Science Library

    Oishi, Naoki; Yamashita, Taro; Kaneko, Shuichi

    2014-01-01

    .... The concept of cancer stem cells (CSCs) is based primarily on the clinical and experimental observations that indicate the existence of a subpopulation of cells with the capacity to self-renew and differentiate as well as show increased...

  4. [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(Pcell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells(Pcells. 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 proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. The pharmacodynamic mechanism may be related to the expressions of key factors in pathways related with proliferation and apoptosis mediated by the three decoctions. Copyright© by the Chinese Pharmaceutical Association.

  5. 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.

  6. A more desirable balanced polyunsaturated fatty acid composition achieved by heterologous expression of Δ15/Δ4 desaturases in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Guiming Zhu

    Full Text Available Arachidonic (ARA, eicosapentaenoic (EPA and docosahexaenoic (DHA acids are the most biologically active polyunsaturated fatty acids, but their biosyntheses in mammals are very limited. The biosynthesis of DHA is the most difficult, because this undergoes the Sprecher pathway--a further elongation step from docosapentaenoic acid (DPA, a Δ6-desaturase acting on a C24 fatty acid substrate followed by a peroxisomal chain shortening step. This paper reports the successful heterologous expression of two non-mammalian genes (with modification of codon usage, coding for Euglena gracilis Δ4-desaturase and Siganus canaliculatus Δ4-desaturase respectively, in mammalian cells (HEK293 cell line. Both of the Δ4-desaturases can efficiently function, directly converting DPA into DHA. Moreover, the cooperation of the E. gracilis Δ4-desaturase with C. elegans Δ15-desaturase (able to convert a number of n-6 PUFAs to their corresponding n-3 PUFAs in transgenic HEK293 cells made a more desirable fatty acid composition--a drastically reduced n-6/n-3 PUFAs ratio and a high level of DHA as well as EPA and ARA. Our findings provide a basis for potential applications of the gene constructs for expression of Δ15/Δ4-desaturases in transgenic livestock to produce such a fatty acid profile in the related products, which certainly will bring benefit to human health.

  7. Human fetal liver cells for regulated ex vivo erythropoietin gene therapy

    Directory of Open Access Journals (Sweden)

    Ebtisam El Filali

    2014-01-01

    Full Text Available Possible risks and lack of donor livers limit application of liver transplantation. Liver cell transplantation is, at this moment, not a feasible alternative because engraftment in the liver is poor. Furthermore, there is also shortage of cells suitable for transplantation. Fetal liver cells are able to proliferate in cell culture and could therefore present an alternative source of cells for transplantation. In this study, we investigated the utility of human fetal liver cells for therapeutic protein delivery. We transplanted human fetal liver cells in immunodeficient mice but were not able to detect engraftment of human hepatocytes. In contrast, transplantation of human adult hepatocytes led to detectable engraftment of hepatocytes in murine liver. Transplantation of fetal liver cells did lead to abundant reconstitution of murine liver with human endothelium, indicating that endothelial cells are the most promising cell type for ex vivo liver cell gene therapy. Human liver endothelial cells were subsequently transduced with a lentiviral autoregulatory erythropoietin expression vector. After transplantation in immunodeficient mice, these cells mediated long-term regulation of murine hematocrits. Our study shows the potential of human liver endothelial cells for long-term regulated gene therapy.

  8. 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.

  9. Fetal liver cell-containing hybrid organoids improve cell viability and albumin production upon transplantation.

    Science.gov (United States)

    Ye, Jingjia; Shirakigawa, Nana; Ijima, Hiroyuki

    2016-06-01

    Cell transplantation is a potential alternative for orthotopic liver transplantation because of the chronic donor shortage. Functional liver tissue is needed for cell transplantations. However, large functional liver tissue is difficult to construct because of the high oxygen consumption of hepatocytes. In our previous study, we developed a novel method to generate hybrid organoids. In this study, we used fetal liver cells (FLCs) to construct a hybrid organoid. Nucleus numbers, angiogenesis, and albumin production were measured in transplanted samples. Higher cell viability and larger liver tissue was found in FLC-containing samples than in hepatocyte-containing samples. Furthermore, the therapeutic efficiency of FLC-containing samples was evaluated by transplantation into Nagase analbuminemia rats. As a result, an increase in albumin concentration was found in rat blood. In summary, transplantation of a FLC-containing hybrid organoid is a potential approach for cell transplantation. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  10. Differentiation of hamster liver oval cell following Clonorchis sinensis infection.

    Science.gov (United States)

    Yoon, B I; Jung, S Y; Hur, K; Lee, J H; Joo, K H; Lee, Y S; Kim, D Y

    2000-12-01

    Oval cells which appear in the liver after hepatic injuries are suspected to be progenitor cells for both hepatocytes and bile duct cells. Oval cell isolated from the livers of the hamsters treated with diethylnitrosamine and 2-acetylaminofluorene and infected with Clonorchis sinensis (CS). cultured for 2 weeks and evaluated for differentiation and plasticity by electron microscopy and immunohistochemistry. In the CS-uninfected group, glycogen granules and peroxisomes were noted in the cells that were cultured for 2 weeks. Starting at 1 week postculture, immunoreactivity of the cells to cytokeratin 19 markedly decreased but that to albumin and alpha-fetoprotein gradually increased. This means that oval cells isolated from hamsters that were not infected with CS differentiated toward hepatocyte lineage. However, in the CS-infected group, cultured cells contained numerous rough endoplasmic reticulum and showed immunoreactivity that was generally in reverse to that of CS-uninfected group, meaning that cells isolated following CS infection were primed by CS and differentiated toward bile duct cell lineage. The results of this study suggested that oval cells are indeed bipolar progenitor cells for hepatocytes and bile duct cells and can differentiate toward either lineage depending upon the priming factor.

  11. Cell Death and Cell Death Responses in Liver Disease: Mechanisms and Clinical Relevance

    Science.gov (United States)

    Luedde, Tom; Kaplowitz, Neil; Schwabe, Robert F.

    2015-01-01

    Summary Hepatocellular death is present in almost all types of human liver disease and is used as a sensitive parameter for the detection of acute and chronic liver disease of viral, toxic, metabolic, or autoimmune origin. Clinical data and animal models suggest that hepatocyte death is the key trigger of liver disease progression, manifested by the subsequent development of inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Modes of hepatocellular death differ substantially between liver diseases. Different modes of cell death such as apoptosis, necrosis, and necroptosis trigger specific cell death responses and promote progression of liver disease through distinct mechanisms. In this review, we first discuss molecular mechanisms by which different modes of cell death, damage-associated molecular patterns, and specific cell death responses contribute to the development of liver disease. We then review the clinical relevance of cell death, focusing on biomarkers; the contribution of cell death to drug-induced, viral, and fatty liver disease and liver cancer; and evidence for cell death pathways as therapeutic targets. PMID:25046161

  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. 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 ...

  15. Expression of neural cell adhesion molecule in human liver development and in congenital and acquired liver diseases.

    Science.gov (United States)

    Libbrecht, L; Cassiman, D; Desmet, V; Roskams, T

    2001-09-01

    In the liver, neural cell adhesion molecule (NCAM) is a marker of immature cells committed to the biliary lineage and is expressed by reactive bile ductules in human liver diseases. We investigated the possible role of NCAM in the development of intrahepatic bile ducts and aimed at determining whether immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases. Therefore, we performed immunohistochemistry for NCAM and bile duct cell markers cytokeratin 7 and cytokeratin 19 on frozen sections of 85 liver specimens taken from 14 fetuses, 10 donor livers, 18 patients with congenital liver diseases characterized by ductal plate malformations (DPMs), and 43 cirrhotic explant livers. Duplicated ductal plates and incorporating bile ducts during development showed a patchy immunoreactivity for NCAM, while DPMs were continuously positive for NCAM. Bile ducts showing complete or patchy immunoreactivity for NCAM were found in cirrhotic livers, with higher frequency in biliary than in posthepatitic cirrhosis. Our results suggest that NCAM may have a function in the development of the intrahepatic bile ducts and that NCAM-positive immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases.

  16. 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.

  17. Flow cytometry and monoclonal antibodies identify normal liver cell populations antigenically related to oval cells.

    Science.gov (United States)

    Agelli, M; Halay, E D

    1995-01-01

    Oval cells, a non-parenchymal cell population induced to rapidly proliferate in animals treated with carcinogens, are thought to be related to the hypothesized liver stem cells. In normal liver there are poorly defined cells antigenically related to oval cells. These oval cell antigen positive (OCAP) cells present in normal animals are thought to include hepatocyte and bile duct cell precursors. To isolate them, we modified the existing protocols designed for oval cells and used it on normal neonatal rat livers. Using flow cytometry, the percentage of normal liver OCAP-cells varied with the monoclonal antibody (MoAb) to the different oval cell membrane markers used: 12% (MoAb 18.2), 23% (MoAb 270.38), 27% (MoAb 18.11), 31% (MoAb 18.13), and 37% (MoAb 374.3). Macrophages consisted 10% of the cells (MoAb MCA 275); hepatocytes were essentially absent ( < 1%, MoAb 236.4). Our results demonstrate that is possible to obtain significant numbers of normal cells antigenically related to oval cells and that using different MoAbs, different cell populations can be sorted for use in experimental studies testing liver progenitor cell hypothesis.

  18. Retinol esterification in cultured rat liver cells.

    Science.gov (United States)

    Drevon, C A; Blomhoff, R; Rasmussen, M; Kindberg, G M; Berg, T; Norum, K R

    1985-01-01

    Retinol esterification was examined in cultured hepatocytes and stellate cells from the rat. Esterification of [3H]retinol was linear for 2 h in both cell types. By increasing the concentration of retinol in the medium, there was a marked increase in retinol esterification in both cell types. The capacity for esterification of retinol was in the same order of magnitude in the two cell types at 3.5 microM-retinol in the medium. This represents a rate of retinol esterification which far exceeds that required to esterify the amount of retinol absorbed in the intestine. It was demonstrated in particulate homogenates from cultured hepatocytes that the esterification of retinol was dependent on acyl-CoA. Addition of 25-hydroxycholesterol or mevalonolactone promoted an increase in cholesterol esterification, whereas retinol esterification was unaffected, suggesting that cholesterol and retinol are esterified by two different enzymes. Some 80% of vitamin A in cultured hepatocytes is retinyl esters, mostly retinyl palmitate. By adding 87 microM-retinol in the medium the cells accumulated 100-fold free retinol and 2.5-3.0-fold retinyl esters within 1 h. When retinol-loaded cells were incubated without retinol, there was a marked decrease especially in free but also in esterified retinol. In the presence of 1 mM-oleic acid in the medium the amount of retinyl oleate was twice that in control cells. PMID:4062867

  19. Evaluation of encapsulated liver cell spheroids in a fluidised-bed bioartificial liver for treatment of ischaemic acute liver failure in pigs in a translational setting.

    Directory of Open Access Journals (Sweden)

    Clare Selden

    Full Text Available Liver failure is an increasing problem. Donor-organ shortage results in patients dying before receiving a transplant. Since the liver can regenerate, alternative therapies providing temporary liver-support are sought. A bioartificial-liver would temporarily substitute function in liver failure buying time for liver regeneration/organ-procurement. Our aim: to develop a prototype bioartificial-liver-machine (BAL comprising a human liver-derived cell-line, cultured to phenotypic competence and deliverable in a clinical setting to sites distant from its preparation. The objective of this study was to determine whether its use would improve functional parameters of liver failure in pigs with acute liver failure, to provide proof-of-principle. HepG2 cells encapsulated in alginate-beads, proliferated in a fluidised-bed-bioreactor providing a biomass of 4-6 × 10(10cells, were transported from preparation-laboratory to point-of-use operating theatre (6000 miles under perfluorodecalin at ambient temperature. Irreversible ischaemic liver failure was induced in anaesthetised pigs, after portal-systemic-shunt, by hepatic-artery-ligation. Biochemical parameters, intracranial pressure, and functional-clotting were measured in animals connected in an extracorporeal bioartificial-liver circuit. Efficacy was demonstrated comparing outcomes between animals connected to a circuit containing alginate-encapsulated cells (Cell-bead BAL, and those connected to circuit containing alginate capsules without cells (Empty-bead BAL. Cells of the biomass met regulatory standards for sterility and provenance. All animals developed progressive liver-failure after ischaemia induction. Efficacy of BAL was demonstrated since animals connected to a functional biomass (+ cells had significantly smaller rises in intracranial pressure, lower ammonia levels, more bilirubin conjugation, improved acidosis and clotting restoration compared to animals connected to the circuit without

  20. 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...

  1. Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice.

    Science.gov (United States)

    Coombes, J D; Swiderska-Syn, M; Dollé, L; Reid, D; Eksteen, B; Claridge, L; Briones-Orta, M A; Shetty, S; Oo, Y H; Riva, A; Chokshi, S; Papa, S; Mi, Z; Kuo, P C; Williams, R; Canbay, A; Adams, D H; Diehl, A M; van Grunsven, L A; Choi, S S; Syn, W K

    2015-07-01

    Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-β signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis. Published by the BMJ Publishing Group Limited. For permission to use (where not already

  2. Drug-induced and postnatal hypothyroidism impairs the accumulation of diacylglycerol in liver and liver cell plasma membranes

    Directory of Open Access Journals (Sweden)

    Kavok Nataliya S

    2002-08-01

    Full Text Available Abstract Background Thyroid hormones are well known modulators of signal transduction. The effect of hyper- and hypo-thyroidism on diacylglycerol/protein kinase C (DAG/PKC signaling in cardiomiocytes has been determined. Triiodothyronine (T3 has been shown to prevent the α1-adrenoreceptor-mediated activation of PKC but does not alter the stimulation of enzyme and hepatic metabolism by phorbol ethers. It has been suggested that the elevation of endogenous DAG in senescent or hypothyroid cells changes the PKC-dependent response of cells to phorbol esters and hormones. In the present study, was examined the formation of DAG and activation of PKC in liver cells from rats of different thyroid status. Results The results obtained provide the first demonstration of DAG accumulation in liver and cell plasma membranes at age- and drug-dependent thyroid gland malfunction. The experiments were performed in either the [14C]CH3COOH-labeled rat liver, liver slices or hepatocytes labeled by [14C] oleic acid and [3H]arachidonic acid or [14C]palmitic acid as well as in the isolated liver cell plasma membranes of 90- and 720-day-old rats of different thyroid status. The decrease of T4 and T3 levels in blood serum of 720-day-old rats and mercazolil-treated animals was associated with increases of both the DAG mass in liver and liver cell plasma membranes and newly synthesized [14C]DAG level in liver and isolated hepatocytes. Hypothyroidism decreased PKC activity in both membrane and cytosol as well as phospholipid and triacylglycerol synthesis in liver. These hypothyroidism effects were restored in liver by injection of T4. T4 administration to the intact animals of different ages decreased the DAG level in liver and isolated plasma membranes and the content of newly synthesized DAG in liver. The reduction of DAG level in liver was not associated with increasing free fatty acid level. DAG labeling ratio 14C/3H in liver slices of rats of different thyroid state

  3. Fetal liver hepatic progenitors are supportive stromal cells for hematopoietic stem cells.

    Science.gov (United States)

    Chou, Song; Lodish, Harvey F

    2010-04-27

    Previously we showed that the ~2% of fetal liver cells reactive with an anti-CD3epsilon monoclonal antibody support ex vivo expansion of both fetal liver and bone marrow hematopoietic stem cells (HSCs); these cells express two proteins important for HSC ex vivo expansion, IGF2, and angiopoietin-like 3. Here we show that these cells do not express any CD3 protein and are not T cells; rather, we purified these HSC-supportive stromal cells based on the surface phenotype of SCF(+)DLK(+). Competitive repopulating experiments show that SCF(+)DLK(+) cells support the maintenance of HSCs in ex vivo culture. These are the principal fetal liver cells that express not only angiopoietin-like 3 and IGF2, but also SCF and thrombopoietin, two other growth factors important for HSC expansion. They are also the principal fetal liver cells that express CXCL12, a factor required for HSC homing, and also alpha-fetoprotein (AFP), indicating that they are fetal hepatic stem or progenitor cells. Immunocytochemistry shows that >93% of the SCF(+) cells express DLK and Angptl3, and a portion of SCF(+) cells also expresses CXCL12. Thus SCF(+)DLK(+) cells are a highly homogenous population that express a complete set of factors for HSC expansion and are likely the primary stromal cells that support HSC expansion in the fetal liver.

  4. 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.

  5. 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.

  6. Osteopontin Neutralization Abrogates the Liver Progenitor Cell Response and Fibrogenesis in Mice

    Science.gov (United States)

    Coombes, J; Swiderska-Syn, M; Dollé, L; Reid, D; Eksteen, B; Claridge, L; Briones-Orta, MA; Shetty, S; Oo, YH; Riva, A; Chokshi, S; Papa, S; Mi, Z; Kuo, PC; Williams, R; Canbay, A; Adams, DH; Diehl, AM; van Grunsven, LA; Choi, SS; Syn, WK

    2015-01-01

    Background Chronic liver injury triggers a progenitor-cell repair-response, and liver fibrosis occurs when repair becomes de-regulated. Previously, we reported that reactivation of the Hedgehog (Hh) pathway promotes fibrogenic liver-repair. Osteopontin (OPN) is a Hh-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesized that OPN may modulate liver progenitor-cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralization on murine liver fibrosis. Methods Liver progenitors (603B and BMOL) were treated with OPN-neutralizing aptamers in the presence or absence of TGF–β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralization (using OPN-aptamers or OPN-neutralizing antibodies) on liver progenitor-cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3, 5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by qRTPCR, Sirius-Red staining, hydroxyproline assay, and semi-quantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. Results OPN is over-expressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound-healing by modulating TGF-β signaling. In vivo, OPN-neutralization attenuates the liver progenitor-cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. Conclusions OPN upregulation during liver injury is a conserved repair-response, and influences liver progenitor-cell function. OPN-neutralization abrogates the liver progenitor-cell response and fibrogenesis in mouse models of liver fibrosis. PMID:24902765

  7. Myostatin inhibits proliferation and insulin-stimulated glucose uptake in mouse liver cells.

    Science.gov (United States)

    Watts, Rani; Ghozlan, Mostafa; Hughey, Curtis C; Johnsen, Virginia L; Shearer, Jane; Hittel, Dustin S

    2014-06-01

    Although myostatin functions primarily as a negative regulator of skeletal muscle growth and development, accumulating biological and epidemiological evidence indicates an important contributing role in liver disease. In this study, we demonstrate that myostatin suppresses the proliferation of mouse Hepa-1c1c7 murine-derived liver cells (50%; p myostatin-responsive transcript in skeletal muscle, revealed a significant downregulation (25% and 50%, respectively; p myostatin-treated mice and liver cells. The importance of Malat1 in liver cell proliferation was confirmed via arrested liver cell proliferation (p Myostatin also significantly blunted insulin-stimulated glucose uptake and Akt phosphorylation in liver cells while increasing the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS), a protein that is essential for cancer cell proliferation and insulin-stimulated glucose transport. Together, these findings reveal a plausible mechanism by which circulating myostatin contributes to the diminished regenerative capacity of the liver and diseases characterized by liver insulin resistance.

  8. 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.

  9. Stem cell-based regenerative opportunities for the liver: State of the art and beyond.

    Science.gov (United States)

    Tsolaki, Eleftheria; Yannaki, Evangelia

    2015-11-21

    The existing mismatch between the great demand for liver transplants and the number of available donor organs highlights the urgent need for alternative therapeutic strategies in patients with acute or chronic liver failure. The rapidly growing knowledge on stem cell biology and the intrinsic repair processes of the liver has opened new avenues for using stem cells as a cell therapy platform in regenerative medicine for hepatic diseases. An impressive number of cell types have been investigated as sources of liver regeneration: adult and fetal liver hepatocytes, intrahepatic stem cell populations, annex stem cells, adult bone marrow-derived hematopoietic stem cells, endothelial progenitor cells, mesenchymal stromal cells, embryonic stem cells, and induced pluripotent stem cells. All these highly different cell types, used either as cell suspensions or, in combination with biomaterials as implantable liver tissue constructs, have generated great promise for liver regeneration. However, fundamental questions still need to be addressed and critical hurdles to be overcome before liver cell therapy emerges. In this review, we summarize the state-of-the-art in the field of stem cell-based therapies for the liver along with existing challenges and future perspectives towards a successful liver cell therapy that will ultimately deliver its demanding goals.

  10. 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.

  11. Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling

    NARCIS (Netherlands)

    Spee, B.|info:eu-repo/dai/nl/304830925; Carpino, G.; Schotanus, B.A.|info:eu-repo/dai/nl/304836524; Katoonizadeh, A.; Vander Borght, S.; Gaudio, E.; Roskams, T.

    2010-01-01

    Hepatology Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling Bart Spee1, Guido Carpino2, Baukje A Schotanus3, Azeam Katoonizadeh1, Sara Vander Borght1, Eugenio Gaudio4, Tania Roskams1 + Author Affiliations 1Department of

  12. Glucocorticoids Have Opposing Effects on Liver Fibrosis in Hepatic Stellate and Immune Cells

    OpenAIRE

    Kim, Kang Ho; Lee, Jae Man; Zhou, Ying; Harpavat, Sanjiv; Moore, David D.

    2016-01-01

    Liver fibrosis is a reversible wound-healing process that is protective in the short term, but prolonged fibrotic responses lead to excessive accumulation of extracellular matrix components that suppresses hepatocyte regeneration, resulting in permanent liver damage. Upon liver damage, nonparenchymal cells including immune cells and hepatic stellate cells (HSCs) have crucial roles in the progression and regression of liver fibrosis. Here, we report differential roles of the glucocorticoid rec...

  13. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Stefania Bruno

    2016-01-01

    Full Text Available Human liver stem cells (HLSCs are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs, and dendritic cells (DCs in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2 and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs, HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

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

    DEFF Research Database (Denmark)

    Petersen, Bent Larsen; Egelund, Jack; Damager, Iben

    2009-01-01

    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-α-D-xylose to L-fucose and derivatives hereof. We have now examined expression of RGX...... nmol conversion of substrate • min-1 • µl 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-α-D-xylose to L-fucose and derivatives hereof. We have now examined expression of RGXT1...

  15. 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.

  16. 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

  17. Squamous cell carcinoma antigen in human liver carcinogenesis.

    Science.gov (United States)

    Guido, M; Roskams, T; Pontisso, P; Fassan, M; Thung, S N; Giacomelli, L; Sergio, A; Farinati, F; Cillo, U; Rugge, M

    2008-04-01

    Squamous cell carcinoma antigen (SCCA) is a serine protease inhibitor that can be overexpressed in hepatocellular carcinoma (HCC) at both molecular and protein level, but no data are available on its expression in pre-malignant stages. To assess SCCA expression by immunohistochemistry in HCC and its nodular precursors in cirrhotic livers. 55 nodules from 42 explanted livers were evaluated: 7 large regenerative nodules (LRNs), 7 low-grade dysplastic nodules (LG-DNs), 10 high-grade DNs (HG-DNs), and 31 HCC. SCCA expression was semiquantitatively scored on a four-tiered scale. SCCA hepatocyte immunostaining was always restricted to the cytoplasm, mainly exhibiting a granular pattern. Stain intensity varied, ranging from weak to very strong. Within the nodules, positive cells were unevenly distributed, either scattered or in irregular clusters. The prevalence of SCCA expression was 29% in LRNs, 100% in DNs and 93% in HCC. A significant difference emerged in both prevalence and score for LRNs versus LG-DNs (pHCC (p = 0.000). A barely significant difference (p = 0.49) was observed between LG-DNs and HG-DNs, while no difference in SCCA expression was detected between HG-DNs and HCC. Cirrhotic tissue adjacent to the nodules was positive in 96% of cases, with a significant difference in the score (p = 0.000) between hepatocytes adjacent to HCC and those surrounding LRNs. This study provides the first evidence that aberrant SCCA expression is an early event in liver cell carcinomatous transformation.

  18. Heterologous production of fungal secondary metabolites in Aspergilli

    DEFF Research Database (Denmark)

    Anyaogu, Diana Chinyere; Mortensen, Uffe Hasbro

    2015-01-01

    Fungal natural products comprise a wide range of compounds. Some are medically attractive as drugs and drug leads, some are used as food additives, while others are harmful mycotoxins. In recent years the genome sequence of several fungi has become available providing genetic information of a lar...... pathway discovery. Heterologous expression of the biosynthetic pathway in model systems or cell factories facilitates product discovery, elucidation, and production. This review summarizes the recent strategies for heterologous expression of fungal biosynthetic pathways in Aspergilli....

  19. Cloning, heterologous expression and characterization of ascorbate peroxidase (APX) gene in laticifer cells of rubber tree (Hevea brasiliensis Muell. Arg.).

    Science.gov (United States)

    Chao, Jinquan; Zhang, Shixin; Chen, Yueyi; Tian, Wei-Min

    2015-12-01

    Ascorbate peroxidases (APXs) are a kind of crucial enzymes for removing reactive oxygen species (ROS) in plant cell. In the present study, a full-length cDNA encoding an APX, designated HbAPX, was isolated from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbAPX was 1174-bp in length and contained a 912-bp open reading frame (ORF) encoding a putative protein of 304 amino acids. The predicted molecular mass of HbAPX was 27.6 kDa (kDa) with an isoelectric point (pI) of 6.73. The phylogenetic analysis showed that HbAPX belonged to the cytosolic subgroup and was more relative to PtAPX and MdAPX2. By using PlantCare online analysis, such cis-acting elements as W-box and MRE were detected in the promoter region of HbAPX. Overproduction of recombinant HbAPX protein either in Escherichia coli or yeast enhanced their tolerance to such abiotic stresses as Cu(2+), Zn(2+), Na(2+) and hydrogen peroxide (H2O2). Ethrel application significantly down-regulated the expression of HbAPX and inhibited the activity of HbAPX in vivo. The ethrel-caused down-regulation of HbAPX may disturb the redox homeostasis in laticifer cells of rubber tree. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  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. 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.

  2. Hepatic Stellate Cells Support Hematopoiesis and are Liver-Resident Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Claus Kordes

    2013-02-01

    Full Text Available Background/Aims: Hematopoiesis can occur in the liver, when the bone marrow fails to provide an adequate environment for hematopoietic stem cells. Hepatic stellate cells possess characteristics of stem/progenitor cells, but their contribution to hematopoiesis is not known thus far. Methods: Isolated hepatic stellate cells from rats were characterized with respect to molecular markers of bone marrow mesenchymal stem cells (MSC and treated with adipocyte or osteocyte differentiation media. Stellate cells of rats were further co-cultured with murine stem cell antigen-1+ hematopoietic stem cells selected by magnetic cell sorting. The expression of murine hematopoietic stem cell markers was analyzed by mouse specific quantitative PCR during co-culture. Hepatic stellate cells from eGFP+ rats were transplanted into lethally irradiated wild type rats. Results: Desmin-expressing stellate cells were associated with hematopoietic sites in the fetal rat liver. Hepatic stellate cells expressed MSC markers and were able to differentiate into adipocytes and osteocytes in vitro. Stellate cells supported hematopoietic stem/progenitor cells during co-culture similar to bone marrow MSC, but failed to differentiate into blood cell lineages after transplantation. Conclusion: Hepatic stellate cells are liver-resident MSC and can fulfill typical functions of bone marrow MSC such as the differentiation into adipocytes or osteocytes and support of hematopoiesis.

  3. Novel therapeutic Strategies for Targeting Liver Cancer Stem Cells

    Science.gov (United States)

    Oishi, Naoki; Wang, Xin Wei

    2011-01-01

    The cancer stem cell (CSC) hypothesis was first proposed over 40 years ago. Advances in CSC isolation were first achieved in hematological malignancies, with the first CSC demonstrated in acute myeloid leukemia. However, using similar strategies and technologies, and taking advantage of available surface markers, CSCs have been more recently demonstrated in a growing range of epithelial and other solid organ malignancies, suggesting that the majority of malignancies are dependent on such a compartment. Primary liver cancer consists predominantly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). It is believed that hepatic progenitor cells (HPCs) could be the origin of some HCCs and ICCs. Furthermore, stem cell activators such as Wnt/β-catenin, TGF-β, Notch and Hedgehog signaling pathways also expedite tumorigenesis, and these pathways could serve as molecular targets to assist in designing cancer prevention strategies. Recent studies indicate that additional factors such as EpCAM, Lin28 or miR-181 may also contribute to HCC progression by targeting HCC CSCs. Various therapeutic drugs that directly modulate CSCs have been examined in vivo and in vitro. However, CSCs clearly have a complex pathogenesis, with a considerable crosstalk and redundancy in signaling pathways, and hence targeting single molecules or pathways may have a limited benefit for treatment. Many of the key signaling molecules are shared by both CSCs and normal stem cells, which add further challenges for designing molecularly targeted strategies specific to CSCs but sparing normal stem cells to avoid side effects. In addition to the direct control of CSCs, many other factors that are needed for the maintenance of CSCs, such as angiogenesis, vasculogenesis, invasion and migration, hypoxia, immune evasion, multiple drug resistance, and radioresistance, should be taken into consideration when designing therapeutic strategies for HCC. Here we provide a brief review of

  4. Embryonic liver cells and permanent lines as models for hepatocyte and bile duct cell differentiation.

    Science.gov (United States)

    Strick-Marchand, Hélène; Weiss, Mary C

    2003-01-01

    Analysis of liver cells during development is facilitated by the possibility of complementing in vivo analysis with experiments on cultured cells. In this review, we discuss results from several laboratories concerning bipotential hepatic stem cells from mouse (HBC-3, H-CFU-C, MMH and BMEL), rat (rhe14321) and primate (IPFLS) embryos. Several groups have used fluorescence-activated cell sorting to identify clonogenic bipotential cells; others have derived bipotential cell lines by plating liver cell suspensions and cloning. The bipotential cells, which probably originate from hepatoblasts, can differentiate as hepatocytes or bile duct cells, and undergo morphogenesis in culture. Disparities in differentiation can be explained by distinct medium compositions, extracellular matrix coated culture surfaces, and gene expression detection methods. Potential applications of these cell lines are discussed.

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

    African Journals Online (AJOL)

    kemrilib

    growth of the liver from increased mitosis in the hepatocytes and a stretching of the liver capsule. Metastasis to the liver accounts for a very small percentage in Nigerians. A nodular liver is thus likely to be from a primary liver pathology. [Afr J Health Sci. 2007;14:14-18]. Introduction. It strikes all age groups and both genders ...

  6. Primary liver tumour of intermediate (hepatocyte-bile duct cell) phenotype: a progenitor cell tumour?

    Science.gov (United States)

    Robrechts, C; De Vos, R; Van den Heuvel, M; Van Cutsem, E; Van Damme, B; Desmet, V; Roskams, T

    1998-08-01

    A 57-year-old female patient presented with painless obstructive jaundice and mild mesogastric pain; she was in good general condition on admission. Abdominal ultrasonography revealed diffuse tumoral invasion of the liver, suggesting diffuse metastases. A liver biopsy showed a tumour with a trabecular growth pattern, composed of uniform relatively small cells, very suggestive of an endocrine carcinoma. Additional immunohistochemical stains, however, did not show any endocrine differentiation, but showed positivity for both hepatocyte-type cytokeratins (cytokeratin 8 and 18) and bile duct-type cytokeratins (cytokeratin 7 and 19). In addition, parathyroid hormone-related peptide, shown to be a good marker for cholangiocarcinoma, was immunoreactive. Electron microscopy revealed tumour cells with an intermediate phenotype: the cells clearly showed hepatocyte features on one hand and bile duct cell features on the other hand. Nine days after admission, the patient died due to liver failure and hepatic encephalopathy. Autopsy excluded another primary tumour site. Overall, this tumour was a primary liver tumour with an intermediate phenotype and with a very rapid clinical course. The intermediate (between hepatocyte and bile duct cell) phenotype suggests an immature progenitor cell origin, which is concordant with a rapid clinical course. This type of tumour has not been described previously and provides additional evidence for the existence of progenitor cells in human liver.

  7. Molecular Imaging of Stem Cell Transplantation for Liver Diseases: Monitoring, Clinical Translation, and Theranostics

    OpenAIRE

    Wang, Ping; Petrella, Francesco; Nicosia, Luca; Bellomi, Massimo; Rizzo, Stefania

    2016-01-01

    Stem cell transplantation has been investigated to rescue experimental liver failure and is promising to offer an alternative therapy to liver transplantation for liver diseases treatment. Several clinical studies in this field have been carried out, but the therapeutic benefit of this treatment is still controversial. A major obstacle to developing stem cell therapies in clinic is being able to visualize the cells in vivo. Imaging modalities allow optimization of delivery, detecting cell sur...

  8. Diagnosis and significance of liver metastases in small cell carcinoma of the lung

    Energy Technology Data Exchange (ETDEWEB)

    Mulshine, J.L.; Makuch, R.W.; Johnston-Early, A.; Matthews, M.J.; Carney, D.N.; Ihde, D.C.; Cohen, M.H.; Bates, H.R.; Dunnick, N.R.; Minna, J.D.

    1984-07-01

    One hundred fifty-seven consecutive patients with small cell lung cancer seen at the National Cancer Institute over a four-year period underwent a series of pretherapy liver staging procedures to determine optimal means of detection and prognostic implications of hepatic metastases. Liver evaluation included physical examination, liver function tests, and liver scan (radionuclide or computerized tomography (CT)), as well as percutaneous and/or peritoneoscopy-directed liver biopsy when possible (74%). Liver metastases were detected in 26% of patients. Peritoneoscopy was the most sensitive method of liver evaluation and increased the detection of liver metastases when done in a sequential fashion after percutaneous liver biopsy from 18 to a total of 27 patients. Of the noninvasive procedures, radionuclide and CT liver scan were the most accurate concurring with liver biopsy in 87% of patients but permitting correct discrimination of stage in excess of 96% of patients. The accuracy of this noninvasive procedure was enhanced by an algorithm combining the results of radionuclide liver scan with liver function tests to detect patients with high or low likelihood of liver involvement. The survival and response of patients with liver metastases was significantly worse than those without such metastases with no three-year disease-free survivors among patients with liver metastases.

  9. Porcine Adipose-Derived Mesenchymal Stem Cells Retain Their Stem Cell Characteristics and Cell Activities While Enhancing the Expression of Liver-Specific Genes after Acute Liver Failure.

    Science.gov (United States)

    Hu, Chenxia; Zhou, Ning; Li, Jianzhou; Shi, Ding; Cao, Hongcui; Li, Jun; Li, Lanjuan

    2016-01-05

    Acute liver failure (ALF) is a kind of complicated syndrome. Furthermore, adipose-derived mesenchymal stem cells (ADMSCs) can serve as a useful cell resource for autotransplantation due to their abundance and micro-invasive accessability. However, it is unknown how ALF will influence the characteristics of ADMSCs and whether ADMSCs from patients suffering from end-stage liver diseases are potential candidates for autotransplantation. This study was designed to compare various properties of ALF-derived ADMSCs with normal ADMSCs in pig models, with regard to their cellular morphology, cell proliferative ability, cell apoptosis, expression of surface antigens, mitochondrial and lysosomal activities, multilineage potency, and expression of liver-specific genes. Our results showed that ALF does not influence the stem cell characteristics and cell activities of ADMSCs. Intriguingly, the expression levels of several liver-specific genes in ALF-derived ADMSCs are higher than in normal ADMSCs. In conclusion, our findings indicate that the stem cell characteristics and cell activities of ADMSCs were not altered by ALF and these cells can serve as a new source for regenerative medicine.

  10. Porcine Adipose-Derived Mesenchymal Stem Cells Retain Their Stem Cell Characteristics and Cell Activities While Enhancing the Expression of Liver-Specific Genes after Acute Liver Failure

    Directory of Open Access Journals (Sweden)

    Chenxia Hu

    2016-01-01

    Full Text Available Acute liver failure (ALF is a kind of complicated syndrome. Furthermore, adipose-derived mesenchymal stem cells (ADMSCs can serve as a useful cell resource for autotransplantation due to their abundance and micro-invasive accessability. However, it is unknown how ALF will influence the characteristics of ADMSCs and whether ADMSCs from patients suffering from end-stage liver diseases are potential candidates for autotransplantation. This study was designed to compare various properties of ALF-derived ADMSCs with normal ADMSCs in pig models, with regard to their cellular morphology, cell proliferative ability, cell apoptosis, expression of surface antigens, mitochondrial and lysosomal activities, multilineage potency, and expression of liver-specific genes. Our results showed that ALF does not influence the stem cell characteristics and cell activities of ADMSCs. Intriguingly, the expression levels of several liver-specific genes in ALF-derived ADMSCs are higher than in normal ADMSCs. In conclusion, our findings indicate that the stem cell characteristics and cell activities of ADMSCs were not altered by ALF and these cells can serve as a new source for regenerative medicine.

  11. 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

    on hepatic progenitor cells have focused on their origin and phenotypic characterization, recent attention has focused on the influence of the hepatic microenvironment on their activation and proliferation. This microenvironment comprises the extracellular matrix, epithelial and non-epithelial resident liver......, including hepatocytes, cholangiocytes and stromal cells. However, if the regenerative capacity of mature cells is impaired by liver-damaging agents, hepatic progenitor cells are activated and expand into the liver parenchyma. Upon transit amplification, the progenitor cells may generate new hepatocytes...... and biliary cells to restore liver homeostasis. In recent years, hepatic progenitor cells have been the subject of increasing interest due to their therapeutic potential in numerous liver diseases as alternative or supportive/complementary tools to liver transplantation. While the first investigations...

  12. Liver Cancer: Molecular Characterization, Clonal Evolution and Cancer Stem Cells

    OpenAIRE

    Castelli, Germana; Pelosi, Elvira; Testa, Ugo

    2017-01-01

    Liver cancer is the second most common cause of cancer-related death. The major forms of primary liver cancer are hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). Both these tumors develop against a background of cirrhotic liver, non-alcoholic fatty liver disease, chronic liver damage and fibrosis. HCC is a heterogeneous disease which usually develops within liver cirrhosis related to various etiologies: hepatitis B virus (HBV) infection (frequent in Asia and Africa)...

  13. In utero transplantation of fetal liver stem cells in humans.

    Science.gov (United States)

    Touraine, J L

    1991-01-01

    Following 15 years experience in postnatal fetal liver transplantation (FLT), we have developed a new therapeutical method, namely the in utero transplantation of stem cells from the human fetal liver. This early transplant takes advantage of the immunological tolerance that exists in young fetal recipients. The three fetuses that we treated were 28, 26, and 12 weeks of age (weeks after fecundation). The first two patients had immunodeficiencies, the third one had thalassemia major. Donor cells were obtained from 7- to 10-week-old fetuses, with conditions approved by the National Committee for Bioethics. Donors and recipients were not matched. The fetal cells were infused through the umbilical vein of the first two patients and injected intraperitoneally into the third one, under ultrasonic visualization. The first patient, born in 1988, has evidence of engraftment and reconstitution of cell-mediated immunity: initially 10% than 26% of lymphocytes of donor origin (with distinct phenotype), T cell responses to tetanus toxoid and candida antigens. This child, who had bare lymphocyte syndrome, has no clinical manifestation of the disease and lives normally at home. The second child was born in 1989 and it is too early for a thorough evaluation of the immunological effects of the transplant, although donor cell engraftment has been proven (Y chromosome in this female patient). The third patient has also evidence of donor cell take (Y chromosome in a female patient) but the effect on thalassemia has not yet been fully analyzed (donor hemoglobin present in small quantity). In all three cases, no side effect of any kind developed in the mother nor in the fetus.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. 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.

  15. 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

  16. Transplantation of mouse fetal liver cells for analyzing the function of hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Gudmundsson, Kristbjorn Orri; Stull, Steven W; Keller, Jonathan R

    2012-01-01

    Hematopoietic stem cells are defined by their ability to self-renew and differentiate through progenitor cell stages into all types of mature blood cells. Gene-targeting studies in mice have demonstrated that many genes are essential for the generation and function of hematopoietic stem and progenitor cells. For definitively analyzing the function of these cells, transplantation studies have to be performed. In this chapter, we describe methods to isolate and transplant fetal liver cells as well as how to analyze donor cell reconstitution. This protocol is tailored toward mouse models where embryonic lethality precludes analysis of adult hematopoiesis or where it is suspected that the function of fetal liver hematopoietic stem and progenitor cells is compromised.

  17. 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.

  18. [Influence of exogenous putrescine on the function of liver and apoptosis of liver cells in rats].

    Science.gov (United States)

    Zhou, Yueping; Rong, Xinzhou; Fan, Guicheng; Liu, Sirong; Wei, Yaming

    2014-02-01

    To explore the influence of exogenous putrescine on the function of liver and apoptosis of liver cells in rats. Ninety healthy clean SD rats were divided into control group (C, n = 10, intraperitoneally injected with 2 mL normal saline), low dosage putrescine group (LP, n = 40), and high dosage putrescine group (HP, n = 40) according to the random number table. Rats in the latter two groups were intraperitoneally injected with approximately 2 mL putrescine (2.5 or 5.0 g/L) with the dosage of 25 or 50 µg/g. Ten rats from group C at post injection hour (PIH) 24 and 10 rats from each of the latter two groups at PIH 24, 48, 72, 96 were sacrificed. Heart blood was obtained for determination of serum contents of ALT and AST. Liver was harvested for gross observation and histomorphological observation with HE staining. Apoptosis was shown with in situ end labeling, and apoptosis index (AI) was calculated. Data among the three groups and those at different time points within one group were processed with one-way analysis of variance or Welch test; LSD or Dunnett's T3 test was used for paired comparison; factorial design analysis of variance of two factors was applied for data between group LP and group HP. (1) No obvious abnormality was observed at gross observation of liver of rats in each group. Liver tissue of rats in group C was normal. Light edema was observed occasionally in liver of rats in groups LP and HP, but necrotic cells were not seen. (2) Content of ALT at PIH 24, 48, 96 and content of AST at PIH 72 and 96 in group LP were respectively (38 ± 10), (45 ± 6), (34 ± 4), (207 ± 18), (196 ± 19) U/L, and content of ALT at PIH 72 and 96 and content of AST at PIH 24, 72, 96 in group HP were respectively (38 ± 6), (48 ± 5), (213 ± 43), (209 ± 40), (230 ± 29) U/L. They were significantly higher than those of rats in group C [(29 ± 5), (163 ± 42) U/L, with P values all below 0.01]. There were statistically significant differences between group LP and group

  19. 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...

  20. Fetal liver cell transplantation : role and nature of the fetal haemopoietic stem cell

    NARCIS (Netherlands)

    B. Löwenberg (Bob)

    1975-01-01

    textabstractFetal liver cell transplantation deserves intensified interest because, according to previous experimental evidence, it may represent a useful approach to reduce or avoid severe Graft-versus-Host (GvH) reactions following treatment with allogeneic haemopoietic cell grafts. The

  1. 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...

  2. Memory T Cells Are Significantly Increased in Rejecting Liver Allografts of Rhesus Monkeys.

    Science.gov (United States)

    Kim, Hwajung; Kim, Hyeyoung; Lee, Sun-Kyung; Jin, Xue-Li; Kim, Tae Jin; Park, Chanho; Lee, Jae-Il; Kim, Hyo-Sin; Hong, Suk Kyun; Yoon, Kyung Chul; Ahn, Sung Woo; Lee, Kyoung-Bun; Yi, Nam-Joon; Yang, Jaeseok; Lee, Kwang-Woong; Hawthorne, Wayne J; Suh, Kyung-Suk

    2017-11-18

    The Rhesus monkey (RM) is an excellent preclinical model in kidney, heart and islet transplantation that has provided the basis for new immunosuppressive protocols for clinical studies. However, there remain relatively few liver transplantation (LT) models in nonhuman primates. In this study, we analyzed the immune cell populations of PBMCs and secondary lymphoid organs along with livers of normal rhesus monkeys and compared them to those of rejecting liver transplanted recipient's following withdrawal of immunosuppression. We undertook five allogeneic ABO compatible orthotopic LT in monkeys using five normal donor monkey livers. We collected tissues including lymph nodes, spleens, blood, recipient livers and performed flow cytometric analysis using isolated immune cells. We found that CD4 or CD8 naïve T cells were normally seen at low levels and memory T cells were seen at high levels in the liver rather than lymphoid organs or PBMC. However, regulatory cells such as CD4+FoxP-3(+)T cells and CD8+CD28- cells remained in high numbers in the liver, but not in lymph node or PBMC. The comparison of CD4/8 T sub-populations in normal and rejected livers and the various tissues showed that naïve cells were dramatically decreased in spleen, lymph node and PBMC of rejected liver transplanted monkeys, but rather memory CD4/8 T cells were increased in all tissues and PBMC. The normal liver has large numbers of CD4 Tregs, CD8+CD28- and myeloid-derived suppressor cells, which are known immunosuppressive cells occurring at much higher levels than those seen in lymph node or peripheral blood. Memory T cells are dramatically increased in rejecting liver allografts of rhesus monkeys compared to those seen in normal RM tissues. This article is protected by copyright. All rights reserved. © 2017 by the American Association for the Study of Liver Diseases.

  3. CWP232228 targets liver cancer stem cells through Wnt/β-catenin signaling: a novel therapeutic approach for liver cancer treatment

    National Research Council Canada - National Science Library

    Kim, Ji-Young; Lee, Hwa-Yong; Park, Kwan-Kyu; Choi, Yang-Kyu; Nam, Jeong-Seok; Hong, In-Sun

    2016-01-01

    Liver cancer stem cells (CSCs) are resistant to conventional chemotherapy and radiation, which may destroy tumor masses, but not all liver CSCs contribute to tumor initiation, metastasis, and relapse...

  4. Recombinant Wild-Type and Edmonston Strain Measles Viruses Bearing Heterologous H Proteins: Role of H Protein in Cell Fusion and Host Cell Specificity

    OpenAIRE

    Takeuchi, Kaoru; Takeda, Makoto; Miyajima, Naoko; Kobune, Fumio; Tanabayashi, Kiyoshi; Tashiro, Masato

    2002-01-01

    Wild-type measles virus (MV) isolated from B95a cells has a restricted host cell specificity and hardly replicates in Vero cells, whereas the laboratory strain Edmonston (Ed) replicates in a variety of cell types including Vero cells. To investigate the role of H protein in the differential MV host cell specificity and cell fusion activity, H proteins of wild-type MV (IC-B) and Ed were coexpressed with the F protein in Vero cells. Cell-cell fusion occurred in Vero cells when Ed H protein, but...

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

    Energy Technology Data Exchange (ETDEWEB)

    Ahsan, Muhammad H.; Gill, Amy F.; Alvarez, Xavier; Lackner, Andrew A.; Veazey, Ronald S., E-mail: rveazey@tulane.edu

    2013-11-15

    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.

  6. Targeting of naproxen covalently linked to HSA to sinusoidal cell types of the liver

    NARCIS (Netherlands)

    Melgert, BN; Lebbe, C; Wartna, E; Molema, G; Poelstra, K; Albrecht, C; Reichen, J; Meijer, DKF; Wisse, E; Knook, DL; Balabaud, C

    1997-01-01

    We have coupled the anti-inflammatory drug naproxen (Nap) covalently to human serum albumin (HSA) to deliver this drug selectively to non parenchymal cell types of the liver during endotoxin induced hepatic inflammation. Liver endothelial cells and Kupffer cells play an important role in the

  7. Side Population Cells Derived from Adult Human Liver Generate Hepatocyte-like Cells In Vitro

    Science.gov (United States)

    HUSSAIN, SUNNY ZAHEED; STROM, STEPHEN C.; KIRBY, MARTHA R.; BURNS, SEAN; LANGEMEIJER, SASKIA; UEDA, TAKAHIRO; HSIEH, MATTHEW; TISDALE, JOHN F.

    2009-01-01

    We sought to determine whether hepatic side population (SP) cells derived from adult human liver possess the potential of a novel candidate hepatic stem cell. Human cadaveric donor liver was subjected to collagenase perfusion and hepatocytes were separated from nonparenchymal cells by differential centrifugation. SP cells were isolated from the nonparenchymal portion after Hoechst 33342 staining. Since CD45 is a panleukocyte antigen, CD45-negative SP cells were separated from the vast majority of CD45-positive SP cells (90%), and hepatic growth medium was used to culture both groups. Both CD45-negative and CD45-positive hepatic SP cells generated colonies in the hepatic growth medium in 2–3 weeks. The colonies yielded large cells morphologically consistent with human hepatocytes, demonstrating granule-rich cytoplasm, dense, often double nuclei, and intracellular lipofuscin pigment. The cultured cells from both sources were positive for markers of human hepatocytes: HepPar, cytokeratin 8 (CK8), and human albumin. Reverse transcriptase–polymerase chain reaction (RT-PCR) performed on both groups demonstrated positivity for additional liver markers including human albumin, CK18, α-1 anti-trypsin, and the human cytochrome P450 enzyme CYP2B6. Double immunostaining (CD45 and HepPar) and RT-PCR confirmed that the hepatocyte-like cells derived from the CD45-negative SP cells acquired HepPar positivity but had no detectable CD45 antigen expression. In contrast, the cultured cells derived from the CD45-positive SP cells also acquired HepPar positivity, but only a minimal fraction expressed the CD45 antigen. We conclude that hepatic SP cells derived from the nonparenchymal portion of human liver are a potential source of human hepatocytes irrespective of their CD45 status, and further animal studies will be required to assess their regenerative potential. PMID:16187169

  8. 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

  9. 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

  10. 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.

  11. Multipotent mesenchymal stromal cells in liver cancer: implications for tumor biology and therapy.

    Science.gov (United States)

    Hernanda, Pratika Y; Pedroza-Gonzalez, Alexander; Sprengers, Dave; Peppelenbosch, Maikel P; Pan, Qiuwei

    2014-12-01

    Remodeling of tumor microenvironment is a hallmark in the pathogenesis of liver cancer. Being a pivotal part of tumor stroma, multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells (MSCs), are recruited and enriched in liver tumors. Owing to their tumor tropism, MSCs are now emerging as vehicles for anticancer drug/gene delivery against liver cancer. However, the exact impact of MSCs on liver cancer remains elusive, as a variety of effects of these cells that have been reported included a plethora of tumor-promoting effects and anti-oncogenic properties. This review aims to dissect the mechanistic insight regarding this observed discrepancy in different experimental settings of liver cancer. Furthermore, we call for caution using MSCs to treat liver cancer or even premalignant liver diseases, before conclusive evidence for safety and efficacy having been obtained. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Different Levels of Immunogenicity of Two Strains of Fowlpox Virus as Recombinant Vaccine Vectors Eliciting T-Cell Responses in Heterologous Prime-Boost Vaccination Strategies

    OpenAIRE

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

    2006-01-01

    The FP9 strain of Fowlpox virus 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 recom...

  13. Molecular Recognition of Human Liver Cancer Cells Using DNA Aptamers Generated via Cell-SELEX.

    Directory of Open Access Journals (Sweden)

    Jiehua Xu

    Full Text Available Most clinical cases of liver cancer cannot be diagnosed until they have evolved to an advanced stage, thus resulting in high mortality. It is well recognized that the implementation of early detection methods and the development of targeted therapies for liver cancer are essential to reducing the high mortality rates associated with this disease. To achieve these goals, molecular probes capable of recognizing liver cancer cell-specific targets are needed. Here we describe a panel of aptamers able to distinguish hepatocarcinoma from normal liver cells. The aptamers, which were selected by cell-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment, have Kd values in the range of 64-349 nM toward the target human hepatoma cell HepG2, and also recognize ovarian cancer cells and lung adenocarcinoma. The proteinase treatment experiment indicated that all aptamers could recognize target HepG2 cells through surface proteins. This outcome suggested that these aptamers could be used as potential probes for further research in cancer studies, such as developing early detection assays, targeted therapies, and imaging agents, as well as for the investigation of common membrane proteins in these distinguishable cancers.

  14. Novel protocol including liver biopsy to identify and treat CD8+ T-cell predominant acute hepatitis and liver failure.

    Science.gov (United States)

    McKenzie, Rebecca B; Berquist, William E; Nadeau, Kari C; Louie, Christine Y; Chen, Sharon F; Sibley, Richard K; Glader, Bertil E; Wong, Wendy B; Hofmann, Lawrence V; Esquivel, Carlos O; Cox, Kenneth L

    2014-08-01

    In the majority of children with ALF, the etiology is unknown and liver transplantation is often needed for survival. A patient case prompted us to consider that immune dysregulation may be the cause of indeterminate acute hepatitis and liver failure in children. Our study includes nine pediatric patients treated under a multidisciplinary clinical protocol to identify and treat immune-mediated acute liver injury. Patients with evidence of inflammation and no active infection on biopsy received treatment with intravenous immune globulin and methylprednisolone. Seven patients had at least one positive immune marker before or after treatment. All patients had a CD8+ T-cell predominant liver injury that completely or partially responded to immune therapy. Five of the nine patients recovered liver function and did not require liver transplantation. Three of these patients subsequently developed bone marrow failure and were treated with either immunosuppression or stem cell transplant. This series highlights the importance of this tissue-based approach to diagnosis and treatment that may improve transplant-free survival. Further research is necessary to better characterize the immune injury and to predict the subset of patients at risk for bone marrow failure who may benefit from earlier and stronger immunosuppressive therapy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Dynamics of Proliferative and Quiescent Stem Cells in Liver Homeostasis and Injury.

    Science.gov (United States)

    Cao, Wanlu; Chen, Kan; Bolkestein, Michiel; Yin, Yuebang; Verstegen, Monique M A; Bijvelds, Marcel J C; Wang, Wenshi; Tuysuz, Nesrin; Ten Berge, Derk; Sprengers, Dave; Metselaar, Herold J; van der Laan, Luc J W; Kwekkeboom, Jaap; Smits, Ron; Peppelenbosch, Maikel P; Pan, Qiuwei

    2017-10-01

    Adult liver stem cells are usually maintained in a quiescent/slow-cycling state. However, a proliferative population, marked by leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), was recently identified as an important liver stem cell population. We aimed to investigate the dynamics and functions of proliferative and quiescent stem cells in healthy and injured livers. We studied LGR5-positive stem cells using diphtheria toxin receptor and green fluorescent protein (GFP) knock-in mice. In these mice, LGR5-positive cells specifically coexpress diphtheria toxin receptor and the GFP reporter. Lineage-tracing experiments were performed in mice in which LGR5-positive stem cells and their daughter cells expressed a yellow fluorescent protein/mTmG reporter. Slow-cycling stem cells were investigated using GFP-based, Tet-on controlled transgenic mice. We studied the dynamics of both stem cell populations during liver homeostasis and injury induced by carbon tetrachloride. Stem cells were isolated from mouse liver and organoid formation assays were performed. We analyzed hepatocyte and cholangiocyte lineage differentiation in cultured organoids. We did not detect LGR5-expressing stem cells in livers of mice at any stage of a lifespan, but only following liver injury induced by carbon tetrachloride. In the liver stem cell niche, where the proliferating LGR5 + cells are located, we identified a quiescent/slow-cycling cell population, called label-retaining cells (LRCs). These cells were present in the homeostatic liver, capable of retaining the GFP label over 1 year, and expressed a panel of progenitor/stem cell markers. Isolated single LRCs were capable of forming organoids that could be carried in culture, expanded for months, and differentiated into hepatocyte and cholangiocyte lineages in vitro, demonstrating their bona fide stem cell properties. More interestingly, LRCs responded to liver injury and gave rise to LGR5-expressing stem cells, as well as

  16. Evaluation of non-reciprocal heterologous immunity between unrelated viruses.

    Science.gov (United States)

    Che, Jenny W; Selin, Liisa K; Welsh, Raymond M

    2015-08-01

    Heterologous immunity refers to the phenomenon whereby a history of an immune response against one pathogen can provide a level of immunity to a second unrelated pathogen. Previous investigations have shown that heterologous immunity is not necessarily reciprocal, such as in the case of vaccinia virus (VACV). Replication of VACV is reduced in mice immune to a variety of pathogens, while VACV fails to induce immunity to several of the same pathogens, including lymphocytic choriomeningitis virus (LCMV). Here we examine the lack of reciprocity of heterologous immunity between VACV and LCMV and find that they induce qualitatively different memory CD8 T cells. However, depending on the repertoire of an individual host, VACV can provide protection against LCMV simply by experimentally amplifying the quantity of T cells cross-reactive with the two viruses. Thus, one cause for lack of reciprocity is differences in the frequencies of cross-reactive T cells in immune hosts. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Fluorescent cell-traceable dexamethasone-loaded liposomes for the treatment of inflammatory liver diseases.

    Science.gov (United States)

    Bartneck, Matthias; Scheyda, Katharina M; Warzecha, Klaudia T; Rizzo, Larissa Y; Hittatiya, Kanishka; Luedde, Tom; Storm, Gert; Trautwein, Christian; Lammers, Twan; Tacke, Frank

    2015-01-01

    Liposomes are routinely used carrier materials for delivering drug molecules to pathological sites. Besides in tumors and inflammatory sites, liposomes also strongly accumulate in liver and spleen. The potential of using liposomes to treat acute and chronic liver disorders, however, has not yet been evaluated. We here explored the therapeutic potential of dexamethasone (Dex)-loaded liposomes for inflammatory liver diseases, using experimental models of acute and chronic liver injury in mice. Fluorescently labeled liposomes predominantly accumulated in hepatic phagocytes, but also in T cells. Importantly, Dex-loaded liposomes reduced T cells in blood and liver, more effectively than free Dex, and endorsed the anti-inflammatory polarization of hepatic macrophages. In experimental chronic liver damage, Dex-loaded liposomes significantly reduced liver injury and liver fibrosis. In immune-mediated acute hepatitis Dex-loaded liposomes, but not free Dex, significantly reduced disease severity. T cells, not macrophages, were significantly depleted by Dex liposomes in liver disease models in vivo, as further supported by mechanistic cell death in vitro studies. Our data indicate that Dex liposomes may be an interesting treatment option for liver diseases, in particular for immune-mediated hepatitis. The depletion of T cells might represent the major mechanism of action of Dex liposomes, rather than their macrophage-polarizing activities. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. 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...

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

    Science.gov (United States)

    Chen, Xiaojun; Yang, Xiaowei; Li, Yong; Zhu, Jifeng; Zhou, Sha; Xu, Zhipeng; He, Lei; Xue, Xue; Zhang, Weiwei; Dong, Xiaoxiao; Wu, Henry; Li, Carrie J; Hsu, Hsiang-Ting; Kong, Wenjun; Liu, Feng; Tripathi, Prem B; Yu, Michelle S; Chang, Jason; Zhou, Liang; Su, Chuan

    2014-05-01

    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.

  20. Inhibition of hepatic cells pyroptosis attenuates CLP-induced acute liver injury.

    Science.gov (United States)

    Chen, Yuan-Li; Xu, Guo; Liang, Xiao; Wei, Juan; Luo, Jing; Chen, Guan-Nan; Yan, Xiao-Di; Wen, Xue-Ping; Zhong, Ming; Lv, Xin

    2016-01-01

    Pyroptosis is a programmed cell death associated with caspase-1 and accompanied by the secretion of a large number of pro-inflammatory cytokines. In the acute stage of sepsis, the release of several pro-inflammatory cytokines aggravates hepatic cell death, and acute liver injury is aggravated with the progress of the disease, resulting in acute liver failure with a very high mortality rate. The present study investigated the effect of inhibiting hepatic cell pyroptosis on the septic acute liver injury. Septic acute liver injury mice model was established by cecal ligation and puncture (CLP model). The liver tissues were assessed for inflammatory infiltration by HE, serum concentrations of ALT, AST, IL-1β, and IL-18 were examined by ELISA, hepatic cell pyroptosis was determined by flow cytometry, and expressions of caspase-1 and NLRP3 were assessed by Western blot. CLP-induced acute liver injury was distinct at 24 h post-operation, with the highest hepatic cell pyroptosis rate. The pyroptosis rate and liver injury indexes were positively correlated. Western blot showed that the expressions of pyroptosis-related proteins, caspase-1, and NLRP3, were increased. Normal mouse hepatic cells were cultured in vitro and LPS+ATP introduced to establish the cell model of septic acute liver injury. The expressions of caspase-1, NLRP3, IL-1β, and IL-18 in LPS+ATP group were significantly higher than the control group by Western blot and ELISA. The inhibitors of NLRP3 (Glyburide) and caspase-1 (AC-YVAD-CMK) alone or in combination were used to pre-treat the hepatic cells, which revealed that the pyroptosis rate was decreased and the cell damage alleviated. The in vivo assay in rats showed that post inhibitor treatment, the 10-days survival was significantly improved and the liver damage reduced. Therefore, inhibiting the hepatic cell pyroptosis could alleviate CLP-induced acute liver injury, providing a novel treatment target for septic acute liver injury.

  1. Side Population Cells From an Immortalized Human Liver Epithelial Cell Line Exhibit Hepatic Stem-Like Cell Properties.

    Science.gov (United States)

    Tokiwa, Takayoshi; Yamazaki, Taisuke; Enosawa, Shin

    2012-01-01

    The existence of hepatic stem cells in human livers is controversial. We investigated whether the side population (SP) cells derived from an immortalized human liver epithelial cell line THLE-5b possess the properties of hepatic stem-like cells. SP cells derived from THLE-5b were isolated using flow cytometry and were assayed for the expression of phenotypic markers by reverse transcription polymerase chain reaction and immunostaining. THLE-5b SP cells retained the capacity to generate both SP and non-SP cells, showed a capacity for self-renewal, and were more efficient in colony formation than non-SP cells. Neither the SP nor the non-SP cells formed tumors when transplanted into athymic nude mice or severe combined immunodeficient mice. The expression level of stem cell-associated markers such as an ATP-binding cassette membrane transporter, epithelial cell adhesion molecule, c-kit, Thy-1, and octomer binding transcription factor 4 was higher in SP cells than in non-SP cells. When cultivated as rotation-mediated aggregates, the expression of liver-specific genes including tryptophan oxygenase and CYP3A4 was up-regulated in SP cells, suggesting that THLE-5b SP cells have the ability to differentiate into a hepatocyte phenotype. One of the clonal cell lines derived from the SP cells expressed stem cell-associated markers. These results indicate that SP cells derived from THLE-5b possess hepatic stem-like cell properties and suggest that THLE-5b can be used as a model of normal human liver progenitor or stem cell line.

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

    Science.gov (United States)

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

    2009-04-03

    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 alpha-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.

  3. CD52-Negative NK Cells Are Abundant in the Liver and Less Susceptible to Alemtuzumab Treatment.

    Science.gov (United States)

    Hotta, Ryuichi; Ohira, Masahiro; Matsuura, Toshiharu; Muraoka, Izumi; Tryphonopoulos, Panagiotis; Fan, Ji; Tekin, Akin; Selvaggi, Gennaro; Levi, David; Ruiz, Phillip; Ricordi, Camillo; Vianna, Rodrigo; Ohdan, Hideki; Waldmann, Herman; Tzakis, Andreas G; Nishida, Seigo

    2016-01-01

    T-cell depleting strategies have become an integral part of immunosuppressive regimens in organ transplantation. Alemtuzumab is a humanized monoclonal antibody against CD52, a cell-surface antigen on several immune cells. It has been suggested that lymphocyte depletion increases the risk of serious infections. However, this has not been observed with short-term alemtuzumab treatment in an organ transplant setting. For induction therapy using alemtuzumab following liver transplantation, we found that T- and B-cell numbers declined rapidly after alemtuzumab therapy; however, the natural killer (NK) cell number was sustained. NK cells are important effectors of innate immunity. Since the effects of alemtuzumab on NK cell functions, especially those of liver NK cells, are unknown, this study aimed to investigate this in detail. To assess the effect of alemtuzumab on NK cells, samples were obtained from 7 organ donors and examined by flow cytometry using Annexin V and propidium iodide. Phenotypical and functional differences within subsets of NK cells with different levels of CD52 expression were determined by flow cytometry and in vitro cytotoxicity assays. CD52 expression on NK cells was lower than that on other lymphocyte subsets. The liver contained a large number of CD52- NK cells compared with the peripheral blood. In vitro treatment of liver-derived NK cells with alemtuzumab did not result in cell death. In contrast, co-incubation with alemtuzumab induced cell death in peripheral blood mononuclear cells and non-NK cells in the liver. Furthermore, CD52- liver NK cells were more cytotoxic and produced more IFN-γ than CD52+ NK cells after cytokine activation. The liver contains a large number of CD52- NK cells. These cells are refractory to alemtuzumab and have robust activity. These findings indicate that CD52- NK cells persist and could protect against infection after alemtuzumab-based lymphocyte depletion.

  4. CD52-Negative NK Cells Are Abundant in the Liver and Less Susceptible to Alemtuzumab Treatment.

    Directory of Open Access Journals (Sweden)

    Ryuichi Hotta

    Full Text Available T-cell depleting strategies have become an integral part of immunosuppressive regimens in organ transplantation. Alemtuzumab is a humanized monoclonal antibody against CD52, a cell-surface antigen on several immune cells. It has been suggested that lymphocyte depletion increases the risk of serious infections. However, this has not been observed with short-term alemtuzumab treatment in an organ transplant setting. For induction therapy using alemtuzumab following liver transplantation, we found that T- and B-cell numbers declined rapidly after alemtuzumab therapy; however, the natural killer (NK cell number was sustained. NK cells are important effectors of innate immunity. Since the effects of alemtuzumab on NK cell functions, especially those of liver NK cells, are unknown, this study aimed to investigate this in detail.To assess the effect of alemtuzumab on NK cells, samples were obtained from 7 organ donors and examined by flow cytometry using Annexin V and propidium iodide. Phenotypical and functional differences within subsets of NK cells with different levels of CD52 expression were determined by flow cytometry and in vitro cytotoxicity assays.CD52 expression on NK cells was lower than that on other lymphocyte subsets. The liver contained a large number of CD52- NK cells compared with the peripheral blood. In vitro treatment of liver-derived NK cells with alemtuzumab did not result in cell death. In contrast, co-incubation with alemtuzumab induced cell death in peripheral blood mononuclear cells and non-NK cells in the liver. Furthermore, CD52- liver NK cells were more cytotoxic and produced more IFN-γ than CD52+ NK cells after cytokine activation.The liver contains a large number of CD52- NK cells. These cells are refractory to alemtuzumab and have robust activity. These findings indicate that CD52- NK cells persist and could protect against infection after alemtuzumab-based lymphocyte depletion.

  5. Advances in Liver Regeneration: Revisiting Hepatic Stem/Progenitor Cells and Their Origin

    Directory of Open Access Journals (Sweden)

    Ali-Reza Sadri

    2016-01-01

    Full Text Available The liver has evolved to become a highly plastic organ with extraordinary regenerative capabilities. What drives liver regeneration is still being debated. Adult liver stem/progenitor cells have been characterized and used to produce functional hepatocytes and biliary cells in vitro. However, in vivo, numerous studies have questioned whether hepatic progenitor cells have a significant role in liver regeneration. Mature hepatocytes have recently been shown to be more plastic than previously believed and give rise to new hepatocytes after acute and chronic injury. In this review, we discuss current knowledge in the field of liver regeneration and the importance of the serotonin pathway as a clinical target for patients with liver dysfunction.

  6. The CD8 T-cell response during tolerance induction in liver transplantation

    Science.gov (United States)

    Wong, Yik Chun; McCaughan, Geoffrey W; Bowen, David G; Bertolino, Patrick

    2016-01-01

    Both experimental and clinical studies have shown that the liver possesses unique tolerogenic properties. Liver allografts can be spontaneously accepted across complete major histocompatibility mismatch in some animal models. In addition, some liver transplant patients can be successfully withdrawn from immunosuppressive medications, developing ‘operational tolerance'. Multiple mechanisms have been shown to be involved in inducing and maintaining alloimmune tolerance associated with liver transplantation. Here, we focus on CD8 T-cell tolerance in this setting. We first discuss how alloreactive cytotoxic T-cell responses are generated against allografts, before reviewing how the liver parenchyma, donor passenger leucocytes and the host immune system function together to attenuate alloreactive CD8 T-cell responses to promote the long-term survival of liver transplants. PMID:27867515

  7. 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.

  8. [Damage effect of Polygonum multiflorum fractions on human normal liver cells L02 and liver cancer cells HepG2].

    Science.gov (United States)

    Zhang, Ruichen; Zhang, Chao; Sun, Zhenxiao; Deng, Qiaohong

    2012-06-01

    To investigate the damage effect of different fractions from Polygonum multiflorum on normal human liver and liver cancer cells, in order to seek for fractions that can obviously kill cancer cells but have less impact on normal liver cells, and make a preliminary study on different mechanism of the two kinds of cells. P. multiflorum water-eluted fraction (RW), 50% ethanol-eluted fraction (R50) and 95% ethanol-eluted fraction (R95) were successively obtained from 70% ethanol extracts of P. multiflorum, after being eluted by water, 50% ethanol and 95% ethanol and then absorbed by AB-8 macroporous resin. Normal human liver L02 cells and liver cancer HepG2 cells were incubated with cell supernatants from different fractions and cells. MTT method and inverted microscope were adopted to observe the impact of L02 on growth of HepG2 cells, screening fractions with damage effect and detect their doses and time effect. Giemsa stain showed changes in cell nucleus after administration and flow cytometry analysis was used to detect cycle and apoptosis of L02 cells. MTT method and inverted microscope showed that R50 had significant growth inhibition effects on L02 and HepG2 cells. According to giemsa stain and flow cytometry analysis, R50 showed different effect on inducing the two cells: there are much more apoptotic HepG2 cells than apoptotic L02 cells in each time phase (the proportion of the apoptosis cells in HepG2 group were 83.62%, 60.52% and 74.49%, and ID2 31.02%, 20.57% and 25.32% after treated with R50 for 24, 48, 72 h. Both cells showed less than 5% of apoptotic cells in the negative control group in each time phase). However, there is no significant impact on cycle of both cells. R50 from P. multiflorum extracts had different damage effects on human liver L02 cells and liver cancer HepG2 cells, which was caused by different degree of induction on apoptosis of the two cells in nature.

  9. 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.

  10. Concise review: cell therapies for hereditary metabolic liver diseases-concepts, clinical results, and future developments.

    Science.gov (United States)

    Cantz, Tobias; Sharma, Amar Deep; Ott, Michael

    2015-04-01

    The concept of cell-based therapies for inherited metabolic liver diseases has been introduced for now more than 40 years in animal experiments, but controlled clinical data in humans are still not available. In the era of dynamic developments in stem cell science, the "right" cell for transplantation is considered as an important key for successful treatment. Do we aim to transplant mature hepatocytes or do we consider the liver as a stem/progenitor-driven organ and replenish the diseased liver with genetically normal stem/progenitor cells? Although conflicting results from cell tracing and transplantation experiments have recently emerged about the existence and role of stem/progenitor cells in the liver, their overall contribution to parenchymal cell homeostasis and tissue repair is limited. Accordingly, engraftment and repopulation efficacies of extrahepatic and liver-derived stem/progenitor cell types are considered to be lower compared to mature hepatocytes. On the basis of these results, we will discuss the current clinical cell transplantation programs for inherited metabolic liver diseases and future developments in liver cell therapy. © 2014 AlphaMed Press.

  11. CD13 is a therapeutic target in human liver cancer stem cells

    National Research Council Canada - National Science Library

    Haraguchi, Naotsugu; Ishii, Hideshi; Mimori, Koshi; Tanaka, Fumiaki; Ohkuma, Masahisa; Kim, Ho Min; Akita, Hirofumi; Takiuchi, Daisuke; Hatano, Hisanori; Nagano, Hiroaki; Barnard, Graham F; Doki, Yuichiro; Mori, Masaki

    2010-01-01

    .... Here, we have demonstrated that CD13 is a marker for semiquiescent CSCs in human liver cancer cell lines and clinical samples and that targeting these cells might provide a way to treat this disease. CD13...

  12. 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.

  13. TAT-MTS-MCM fusion proteins reduce MMA levels and improve mitochondrial activity and liver function in MCM-deficient cells.

    Science.gov (United States)

    Erlich-Hadad, Tal; Hadad, Rita; Feldman, Anat; Greif, Hagar; Lictenstein, Michal; Lorberboum-Galski, Haya

    2018-03-01

    Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl-CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT-MTS-Protein approach for replacing a number of mitochondrial-mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT-MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear-encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9-engineered HepG2 MUT (-/-) liver cell line. Therefore, we suggest using this TAT-MTS-Protein approach for the treatment of MMA. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  14. 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.

  15. Role of hepatocytes and bile duct cells in preservation-reperfusion injury of liver grafts.

    Science.gov (United States)

    Kukan, M; Haddad, P S

    2001-05-01

    In liver transplantation, it is currently hypothesized that nonparenchymal cell damage and/or activation is the major cause of preservation-related graft injury. Because parenchymal cells (hepatocytes) appear morphologically well preserved even after extended cold preservation, their injury after warm reperfusion is ascribed to the consequences of nonparenchymal cell damage and/or activation. However, accumulating evidence over the past decade indicated that the current hypothesis cannot fully explain preservation-related liver graft injury. We review data obtained in animal and human liver transplantation and isolated perfused animal livers, as well as isolated cell models to highlight growing evidence of the importance of hepatocyte disturbances in the pathogenesis of normal and fatty graft injury. Particular attention is given to preservation time-dependent decreases in high-energy adenine nucleotide levels in liver cells, a circumstance that (1) sensitizes hepatocytes to various stimuli and insults, (2) correlates well with graft function after liver transplantation, and (3) may also underlie the preservation time-dependent increase in endothelial cell damage. We also review damage to bile duct cells, which is increasingly being recognized as important in the long-lasting phase of reperfusion injury. The role of hydrophobic bile salts in that context is particularly assessed. Finally, a number of avenues aimed at preserving hepatocyte and bile duct cell integrity are discussed in the context of liver transplantation therapy as a complement to reducing nonparenchymal cell damage and/or activation.

  16. 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.

  17. 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.

  18. Mechanisms of xenogeneic baboon platelet aggregation and phagocytosis by porcine liver sinusoidal endothelial cells.

    Directory of Open Access Journals (Sweden)

    Qiang Peng

    Full Text Available Baboons receiving xenogeneic livers from wild type and transgenic pigs survive less than 10 days. One of the major issues is the early development of profound thrombocytopenia that results in fatal hemorrhage. Histological examination of xenotransplanted livers has shown baboon platelet activation, phagocytosis and sequestration within the sinusoids. In order to study the mechanisms of platelet consumption in liver xenotransplantation, we have developed an in vitro system to examine the interaction between pig endothelial cells with baboon platelets and to thereby identify molecular mechanisms and therapies.Fresh pig hepatocytes, liver sinusoidal and aortic endothelial cells were isolated by collagenase digestion of livers and processing of aortae from GTKO and Gal+ MGH-miniature swine. These primary cell cultures were then tested for the differential ability to induce baboon or pig platelet aggregation. Phagocytosis was evaluated by direct observation of CFSE labeled-platelets, which are incubated with endothelial cells under confocal light microscopy. Aurintricarboxylic acid (GpIb antagonist blocking interactions with von Willebrand factor/vWF, eptifibatide (Gp IIb/IIIa antagonist, and anti-Mac-1 Ab (anti-α(Mβ(2 integrin Ab were tested for the ability to inhibit phagocytosis.None of the pig cells induced aggregation or phagocytosis of porcine platelets. However, pig hepatocytes, liver sinusoidal and aortic endothelial cells (GTKO and Gal+ all induced moderate aggregation of baboon platelets. Importantly, pig liver sinusoidal endothelial cells efficiently phagocytosed baboon platelets, while pig aortic endothelial cells and hepatocytes had minimal effects on platelet numbers. Anti-MAC-1 Ab, aurintricarboxylic acid or eptifibatide, significantly decreased baboon platelet phagocytosis by pig liver endothelial cells (P<0.01.Although pig hepatocytes and aortic endothelial cells directly caused aggregation of baboon platelets, only pig liver

  19. Comparative Pathogenicity of Liver Homogenate and Cell Culture Propagated Hydropericardium Syndrome Virus in Broiler Birds

    Directory of Open Access Journals (Sweden)

    M. D. Ahmad, S. Zaman1, M. H. Mushtaq*, A. A. Anjum1 and M. Akram1

    2011-10-01

    Full Text Available Comparative pathogenicity of liver homogenate and cell culture propagated agents of hydropericardium syndrome was studied in broiler birds. In Experiment I, 25-day-old while in experiment II, broiler birds at different ages were inoculated through different routes. In Experiment I, liver homogenate caused 64% mortality through intramuscular route and 33.33% mortality through oral route. The cell culture propagated HPS virus caused 60 and 13.33% mortality in broiler birds through intramuscular and oral routes, respectively. In Experiment II, none of the day-old-chicks died when challenged with liver homogenate and cell culture propagated HPS virus through S/C and oral route. The liver homogenate and cell culture propagated HPS virus caused higher mortality in different age groups of broiler birds through s/c route compared to oral route. The values of hemoglobin (Hb and packed cell volume (PCV showed highly significant (P<0.05 reduction indicating anemia. The values of Hb and PCV of the broiler birds inoculated with infectious liver homogenate were significantly lower as compared to birds inoculated with cell culture propagated HPS virus. The results indicated that the liver homogenate is more pathogenic than cell culture propagated HPS virus. These changes may be due to adoptability of the original FAdVs (fowl adenovirus after continued passages in the culture of chicken embryo liver cells. Importance of this study in vaccine production is also discussed.

  20. [The hepatic differentiation of adult and fetal liver stromal cells in vitro].

    Science.gov (United States)

    Kholodenko, I V; Kholodenko, R V; Manukyan, G V; Yarygin, K N

    2016-11-01

    The liver has a marked capacity for regeneration. In most cases the liver regeneration is determined by hepatocytes. The regenerative capacity of hepatocytes is significantly reduced in acute or chronic damage. In particular, repair mechanisms are not activated in patients with alcoholic cirrhosis. Organ transplantation or advanced methods of regenerative medicine can help such patients. The promising results were obtained in clinical trials involving patients with various forms of liver disease who received transplantation of autologous bone marrow stem cells. However, to improve the effectiveness of such treatment it is necessary to search for more optimal sources of progenitor cells, as well as to evaluate the possibility of using descendants of these cells differentiated in vitro. In this study we isolated stromal cells from the liver biopsies of three patients with alcoholic cirrhosis, conducted their morphological and phenotypic analysis, and evaluated the hepatic potential of these cells in vitro. The stromal cells isolated from fetal liver were used for comparison. The results of this can serve as a basis for the development of a new method for the treatment of end-stage liver disease. The stromal cells isolated from the liver biopsies for a long time proliferate in a culture and this which makes it possible to expand them to large amounts for subsequent differentiation into hepatocyte-like cells and autologous transplantation.

  1. Liver progenitor cells develop cholangiocyte-type epithelial polarity in three-dimensional culture.

    OpenAIRE

    Tanimizu, Naoki; Miyajima, Atsushi; Mostov, Keith E

    2007-01-01

    Cholangiocytes are cellular components of the bile duct system of the liver, which originate from hepatoblasts during embryonic liver development. Although several transcription factors and signaling molecules have been implicated in bile duct development, its molecular mechanism has not been studied in detail. Here, we applied a three-dimensional (3D) culture technique to a liver progenitor cell line, HPPL, to establish an in vitro culture system in which HPPL acquire differentiated cholangi...

  2. Cross-fostering to prevent maternal cell transfer did not prevent vaccine-associated enhanced respiratory disease that occurred following heterologous influenza challenge of pigs vaccinated in the presence of maternal immunity.

    Science.gov (United States)

    Loving, Crystal L; Brockmeier, Susan L; Vincent, Amy L; Gauger, Phillip C; Zanella, Eraldo L; Lager, Kelly M; Kehrli, Marcus E

    2014-09-01

    Whole inactivated virus (WIV) vaccines for influenza A virus (IAV) provide limited cross-protection to diverse antigenic strains that are circulating or may emerge in a population. Maternal vaccination is used to protect neonatal animals from disease through passive transfer of immunity. It is desirable to vaccinate at a young age to induce active immunity that provides protection against infection before maternal immunity wanes. However, maternal-derived immunity (MDI; antibody or cells) can interfere with vaccine priming. Previous work indicates that vaccine-associated enhanced respiratory disease (VAERD) occurs in pigs following heterologous IAV challenge if pigs were previously vaccinated with WIV vaccine in the presence of matched MDI. However, the component of MDI (antibody or cells) that is required for the mispriming of piglet immunity has not been determined. While antibody from colostrum is absorbed into piglet circulation regardless of the sow from which it receives colostrum, transfer of maternal cells requires colostrum from the biological dam. We used cross-fostering (CF) as a tool to determine if maternal cells are required for the mispriming of piglet immunity upon WIV vaccination in the presence of MDI. Piglets vaccinated in the presence of MDI, regardless of CF, displayed characteristics of VAERD following heterologous challenge. MDI alone (no piglet vaccination) did not provide cross-protection against the antigenic variant. However, it did not induce VAERD. WIV vaccination provided complete protection against homologous challenge when delivered to piglets without MDI. Vaccination in the presence of MDI inhibited an increase in hemagglutination inhibiting (HI) antibody titers to vaccine antigen, but did not alter development of total immunoglobulin levels to vaccine virus. Taken together, the cellular component of MDI did not contribute to the mispriming of piglet immunity to WIV vaccine, but maternal-derived antibody (MDA) alone was sufficient

  3. Glucocorticoids Have Opposing Effects on Liver Fibrosis in Hepatic Stellate and Immune Cells.

    Science.gov (United States)

    Kim, Kang Ho; Lee, Jae Man; Zhou, Ying; Harpavat, Sanjiv; Moore, David D

    2016-08-01

    Liver fibrosis is a reversible wound-healing process that is protective in the short term, but prolonged fibrotic responses lead to excessive accumulation of extracellular matrix components that suppresses hepatocyte regeneration, resulting in permanent liver damage. Upon liver damage, nonparenchymal cells including immune cells and hepatic stellate cells (HSCs) have crucial roles in the progression and regression of liver fibrosis. Here, we report differential roles of the glucocorticoid receptor (GR), acting in immune cells and HSCs, in liver fibrosis. In the carbon tetrachloride hepatotoxin-induced fibrosis model, both steroidal and nonsteroidal GR ligands suppressed expression of fibrotic genes and decreased extracellular matrix deposition but also inhibited immune cell infiltration and exacerbated liver injury. These counteracting effects of GR ligands were dissociated in mice with conditional GR knockout in immune cells (GR(LysM)) or HSC (GR(hGFAP)): the impacts of dexamethasone on immune cell infiltration and liver injury were totally blunted in GR(LysM) mice, whereas the suppression of fibrotic gene expression was diminished in GR(hGFAP) mice. The effect of GR activation in HSC was further confirmed in the LX-2 HSC cell line, in which antifibrotic effects were mediated by GR ligand inhibition of Sma and mad-related protein 3 (SMAD3) expression. We conclude that GR has differential roles in immune cells and HSCs to modulate liver injury and liver fibrosis. Specific activation of HSC-GR without alteration of GR activity in immune cells provides a potential therapeutic approach to treatment of hepatic fibrosis.

  4. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

    Science.gov (United States)

    Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

    2016-01-01

    Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications. PMID:27403430

  5. Regeneration and Cell Recruitment in an Improved Heterotopic Auxiliary Partial Liver Transplantation Model in the Rat.

    Science.gov (United States)

    Ono, Yoshihiro; Pérez-Gutiérrez, Angelica; Yovchev, Mladen I; Matsubara, Kentaro; Yokota, Shinichiro; Guzman-Lepe, Jorge; Handa, Kan; Collin de l'Hortet, Alexandra; Thomson, Angus W; Geller, David A; Yagi, Hiroshi; Oertel, Michael; Soto-Gutierrez, Alejandro

    2017-01-01

    Auxiliary partial liver transplantation (APLT) in humans is a therapeutic modality used especially to treat liver failure in children or congenital metabolic disease. Animal models of APLT have helped to explore therapeutic options. Though many groups have suggested improvements, standardizing the surgical procedure has been challenging. Additionally, the question of whether graft livers are reconstituted by recipient-derived cells after transplantation has been controversial. The aim of this study was to improve experimental APLT in rats and to assess cell recruitment in the liver grafts. To inhibit recipient liver regeneration and to promote graft regeneration, we treated recipients with retrorsine and added arterial anastomosis. Using green fluorescence protein transgenic rats as recipients, we examined liver resident cell recruitment within graft livers by immunofluorescence costaining. In the improved APLT model, we achieved well-regenerated grafts that could maintain regeneration for at least 4 weeks. Regarding the cell recruitment, there was no evidence of recipient-derived hepatocyte, cholangiocyte, or hepatic stellate cell recruitment into the graft. Macrophages/monocytes, however, were consistently recruited into the graft and increased over time, which might be related to inflammatory responses. Very few endothelial cells showed colocalization of markers. We have successfully established an improved rat APLT model with arterial anastomosis as a standard technique. Using this model, we have characterized cell recruitment into the regenerating grafts.

  6. A Case of Giant Cell Hepatitis Recurring after Liver Transplantation and Treated with Ribavirin

    Directory of Open Access Journals (Sweden)

    Ziad Hassoun

    2000-01-01

    Full Text Available A patient who underwent orthotopic liver transplantation for giant cell hepatitis with cirrhosis and in whom giant cell hepatitis recurred twice after orthotopic liver transplantation is reported. He was treated with ribavirin with an excellent result. The literature on this subject is reviewed. This observation clearly confirms the efficacy of ribavirin for the treatment of giant cell hepatitis, thus providing evidence for its viral origin.

  7. 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

    BACKGROUND: Although CD4 T cells are crucial for defense against M.tb, it is still not clear whether the optimal response against M.tb in fact involves both CD4 and CD8 T cells. To test this, we used a new vaccine strategy that generated a strong balanced T cell response consisting of both CD4......-alpha(+), whereas most of the CD8 T cells expressed IFN-gamma(+) and TNF-alpha(+) and possessed strong cytotoxic potential. The heterologous prime boost protocol also gave an increase in protective efficacy against M.tb challenge compared to H4/CAF01 and Ad-H4. Both the H4 specific CD4 and CD8 T cells were...... on the priming of CD4 and CD8 cells and in terms of the protective capacity of the vaccine, and therefore represent an interesting new vaccine strategy against M.tb. However, CD4 and CD8 T cells respond very differently to live M.tb challenge, in a manner which supports the consensus that CD4 T cells do play...

  8. Respiratory Influenza Virus Infection Induces Memory-like Liver NK Cells in Mice.

    Science.gov (United States)

    Li, Tingting; Wang, Jian; Wang, Yanshi; Chen, Yongyan; Wei, Haiming; Sun, Rui; Tian, Zhigang

    2017-02-01

    Although NK cells are classified as innate immune cells, recent studies have demonstrated the transformation of NK cells into long-lived memory cells that contribute to secondary immune responses in certain mouse models. However, whether NK cells mount an Ag-specific memory response to acute influenza virus infection has not yet been examined. Here, we show that, consistent with previous studies, lung NK cells play an important role in controlling viral proliferation after primary influenza virus infection. However, although lung NK cells display a memory phenotype at the late stage of infection, these cells do not protect mice against secondary influenza virus infection. Interestingly, liver NK cells from influenza virus-infected mice possess a memory phenotype and protect mice against secondary influenza virus infection. Memory-like liver NK cells display a CD49a+DX5- phenotype, and the adoptive transfer of purified liver CD49a+DX5- NK cells into naive mice followed by viral infection results in protective immunity and decreased viral titer. Moreover, we demonstrate that primary inactivated influenza virus induces memory NK cells residing in the liver of Rag1-/- mice. Collectively, these data suggest that liver CD49a+DX5- NK cells remember encountered Ag from influenza virus after primary infection and are more protective upon subsequent infection. Copyright © 2017 by The American Association of Immunologists, Inc.

  9. Claudin-2 Promotes Breast Cancer Liver Metastasis by Facilitating Tumor Cell Interactions with Hepatocytes

    Science.gov (United States)

    Tabariès, Sébastien; Dupuy, Fanny; Dong, Zhifeng; Monast, Anie; Annis, Matthew G.; Spicer, Jonathan; Ferri, Lorenzo E.; Omeroglu, Atilla; Basik, Mark; Amir, Eitan; Clemons, Mark

    2012-01-01

    We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes. PMID:22645303

  10. Organoids from adult liver and pancreas: Stem cell biology and biomedical utility.

    Science.gov (United States)

    Hindley, Christopher J; Cordero-Espinoza, Lucía; Huch, Meritxell

    2016-12-15

    The liver and pancreas are critical organs maintaining whole body metabolism. Historically, the expansion of adult-derived cells from these organs in vitro has proven challenging and this in turn has hampered studies of liver and pancreas stem cell biology, as well as being a roadblock to disease modelling and cell replacement therapies for pathologies in these organs. Recently, defined culture conditions have been described which allow the in vitro culture and manipulation of adult-derived liver and pancreatic material. Here we review these systems and assess their physiological relevance, as well as their potential utility in biomedicine. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. A Primary Human Liver Cell Culture Model for Hemorrhagic Fever Viruses.

    Science.gov (United States)

    Djavani, Mahmoud

    2018-01-01

    Viral hemorrhagic fevers affect liver functions such as important metabolic processes and the replacement of new blood cells, coagulation factors, and growth factors. Typically, multi-organ diseases such as viral hemorrhagic fevers are studied in an organism, but it is also possible to derive information about the molecular events involved in disease processes by focusing on liver cell culture. Here we describe a multi-cell culture system that is capable of replicating the arenavirus LCMV-WE, a virus that can cause hemorrhagic fever in primates, as a model for liver infection by a hemorrhagic fever virus.

  12. 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,

  13. 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...

  14. 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...

  15. 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.

  16. 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.

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    Afiana Rohmani

    2015-12-01

    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.

  19. 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.

  20. Heterologous Infection During Chagas' Disease

    Science.gov (United States)

    Sibona, G. J.; Condat, C. A.; Cossi Isasi, S.

    2007-05-01

    Human populations are often infected with more than one parasite strain. This is frequently the case with ChagasŠ disease, which is endemic to large regions of Latin America. In the present work we study the dynamics of the heterologous infection for this disease, using a model for the interaction between the trypanosoma cruzi parasite and the immune system. We find the dependence of the nature of the post-acute stage on the parameters characterizing the inoculated infectious strains.

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

    DEFF Research Database (Denmark)

    Wewer, U M; Albrechtsen, R

    1992-01-01

    . In normal adult liver the perisinusoidal spaces contained only minimal amounts of immunoreactive laminin. In 86% of patients dying from cancer with liver metastasis, however, a distinct increase in the amount of perisinusoidal laminin could be demonstrated. The perisinusoidal space also contained laminin...... in cancer patients without liver metastasis. In 3 cases of leukaemia sinusoids were laminin negative. In cirrhosis and chronic passive congestion there was, as expected, laminin immunoreactivity in the perisinusoidal space. The results obtained using polyclonal antibodies against laminin were confirmed...... that this perisinusoidal laminin may directly on indirectly mediate tumour cell metastasis to the liver....

  2. Molecular Imaging of Stem Cell Transplantation for Liver Diseases: Monitoring, Clinical Translation, and Theranostics

    Directory of Open Access Journals (Sweden)

    Ping Wang

    2016-01-01

    Full Text Available Stem cell transplantation has been investigated to rescue experimental liver failure and is promising to offer an alternative therapy to liver transplantation for liver diseases treatment. Several clinical studies in this field have been carried out, but the therapeutic benefit of this treatment is still controversial. A major obstacle to developing stem cell therapies in clinic is being able to visualize the cells in vivo. Imaging modalities allow optimization of delivery, detecting cell survival and functionality by in vivo monitoring these transplanted graft cells. Moreover, theranostic imaging is a brand new field that utilizes nanometer-scale materials to glean diagnostic insight for simultaneous treatment, which is very promising to improve stem cell-based therapy for treatment of liver diseases. The aim of this review was to summarize the various imaging tools that have been explored with advanced molecular imaging probes. We also outline some recent progress of preclinical and clinical studies of liver stem cells transplantation. Finally, we discuss theranostic imaging for stem cells transplantation for liver dysfunction and future opportunities afforded by theranostic imaging.

  3. Measurement of bioimpedance and cell viability during ischemia-reperfusion in the rat liver.

    Science.gov (United States)

    Ahn, H; Shin, H; Yun, S; Kim, J; Choi, J

    2005-01-01

    During liver resection and liver transplant, liver is damaged by ischemia-reperfusion injury. Until now, there is no approved method to measure or predict the extent of liver injury during the operation. This is the preliminary study to make the real time monitoring system by quantification of bioimpedance and ischemiareperfusion reperfusion injury in liver. Sprague-Dawley rats were subjected to different periods of 70% partial hepatic ischemia (30, 60, 90 and 120minutes ischemia) and reperfusion. We measured changes of liver tissue bioimpedance (120Hz-100KHz) every five minutes. Cell viability was assessed by metabolic capacity of fatty acid (palmitic acid metabolic rate), ATP content and histological examination (H/E and TUNEL stain) at every 30 minutes interval during ischemia.

  4. Liver Wellness

    Science.gov (United States)

    ... Fatty liver disease is the build up of fat in liver cells. It can cause the liver to swell and can lead to cirrhosis. Prevention: Ways to prevent fatty liver disease are: • Eating a healthy diet • Maintaining a healthy weight • Exercising regularly • Limiting the ...

  5. 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.

  6. CCR1 antagonism attenuates T cell trafficking to omentum and liver in obesity-associated cancer.

    Science.gov (United States)

    Conroy, Melissa J; Galvin, Karen C; Kavanagh, Maria E; Mongan, Ann Marie; Doyle, Suzanne L; Gilmartin, Niamh; O'Farrelly, Cliona; Reynolds, John V; Lysaght, Joanne

    2016-07-01

    Obesity is a global health problem presenting serious risk of disease fuelled by chronic inflammation, including type 2 diabetes mellitus, cardiovascular disease, liver disease and cancer. Visceral fat, in particular the omentum and liver of obese individuals are sites of excessive inflammation. We propose that chemokine-mediated trafficking of pro-inflammatory cells to the omentum and liver contributes to local and subsequent systemic inflammation. Oesophagogastric adenocarcinoma (OAC) is an exemplar model of obesity and inflammation driven cancer. We have demonstrated that T cells actively migrate to the secreted factors from the omentum and liver of OAC patients and that both CD4(+) and CD8(+) T cells bearing the chemokine receptor CCR5 are significantly more prevalent in these tissues compared to matched blood. The CCR5 ligand and inflammatory chemokine MIP-1α is also secreted at significantly higher concentrations in the omentum and liver of our OAC patient cohort compared to matched serum. Furthermore, we report that MIP-1α receptor antagonism can significantly reduce T cell migration to the secreted factors from OAC omentum and liver. These novel data suggest that chemokine receptor antagonism may have therapeutic potential to reduce inflammatory T cell infiltration to the omentum and liver and in doing so, may ameliorate pathological inflammation in obesity and obesity-associated cancer.

  7. 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)

  8. Targeting naproxen to non-parenchymal liver cells protects against endotoxin induced liver damage

    NARCIS (Netherlands)

    Lebbe, C; Reichen, J; Wartna, E.S; Sägesser, H; Poelstra, Klaas; Meijer, D.K F

    1997-01-01

    Non-steroidal anti-inflammatory drugs (NSAID's) could be of value in the treatment of liver disease; however, their use in this situation is limited by renal side effects. Therefore, we explored whether naproxen covalently bound to human serum albumin (NAP-HSA) was able to reduce toxicity in an

  9. Umbilical cord-derived mesenchymal stem cells alleviate liver fibrosis in rats

    Science.gov (United States)

    Chai, Ning-Li; Zhang, Xiao-Bin; Chen, Si-Wen; Fan, Ke-Xing; Linghu, En-Qiang

    2016-01-01

    AIM: To evaluate the efficacy of umbilical cord-derived mesenchymal stem cells (UC-MSCs) transplantation in the treatment of liver fibrosis. METHODS: Cultured human UC-MSCs were isolated and transfused into rats with liver fibrosis induced by dimethylnitrosamine (DMN). The effects of UC-MSCs transfusion on liver fibrosis were then evaluated by histopathology; serum interleukin (IL)-4 and IL-10 levels were also measured. Furthermore, Kupffer cells (KCs) in fibrotic livers were isolated and cultured to analyze their phenotype. Moreover, UC-MSCs were co-cultured with KCs in vitro to assess the effects of UC-MSCs on KCs’ phenotype, and IL-4 and IL-10 levels were measured in cell culture supernatants. Finally, UC-MSCs and KCs were cultured in the presence of IL-4 antibodies to block the effects of this cytokine, followed by phenotypical analysis of KCs. RESULTS: UC-MSCs transfused into rats were recruited by the injured liver and alleviated liver fibrosis, increasing serum IL-4 and IL-10 levels. Interestingly, UC-MSCs promoted mobilization of KCs not only in fibrotic livers, but also in vitro. Co-culture of UC-MSCs with KCs resulted in increased production of IL-4 and IL-10. The addition of IL-4 antibodies into the co-culture system resulted in decreased KC mobilization. CONCLUSION: UC-MSCs could increase IL-4 and promote mobilization of KCs both in vitro and in vivo, subsequently alleviating the liver fibrosis induced by DMN. PMID:27468195

  10. Role of Stem Cells Transplantation in Tissue Regeneration After Acute or Chronic Acetaminophen Induced Liver Injury.

    Science.gov (United States)

    Katselis, Charalampos; Apostolou, Konstantinos; Feretis, Themistoklis; Papanikolaou, Ioannis G; Zografos, George C; Toutouzas, Konstantinos; Papalois, Apostolos

    2016-01-01

    Acetaminophen-induced liver injury (APAP) is recognized as a frequent etiologic factor responsible for hepatic damage in the developed world. Management remains still elusive as treatment options are limited and their results are inconclusive. Consequently new strategies are explored at the experimental level. Mesenchymal stem cells (MSCs) present a promising modality as they can promote liver regeneration (LG) and compensate acute liver injury (ALI). Our research was focused on articles related to drug-induced liver injury, mechanisms of liver regeneration (LG) after Acute Liver Injury (ALI) and recent experimental protocols of Mesenchymal Stem Cells (MSCs) transplantation after chemical insult. All these studies are cited on Pubmed and MedLine. This review has three distinct sections. First recent developments in ALI pathogenesis are presented. The second section covers cellular pathways and histological findings relevant to liver regeneration. The final chapter analyzes MSCs transplantation protocols after ALI and interrelation between liver regeneration and hepatic differentiation of MSCs. Adipose tissue stem cells (ADSCs) and (MSCs) transplantation represents a promising modality in severe ALI management although many aspects remain to be clarified.

  11. Sustainable heterologous production of terpene hydrocarbons in cyanobacteria.

    Science.gov (United States)

    Formighieri, Cinzia; Melis, Anastasios

    2016-12-01

    Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial application. However, the slow catalytic activity of terpene synthases (k cat = 4 s-1 or slower) makes them noncompetitive for the pool of available substrate, thereby limiting the rate and yield of product generation. Work in this paper applied transformation technologies in Synechocystis for the heterologous production of β-phellandrene (monoterpene) hydrocarbons. Conditions were defined whereby expression of the β-phellandrene synthase (PHLS), as a CpcB·PHLS fusion protein with the β-subunit of phycocyanin, accounted for up to 20 % of total cellular protein. Moreover, CpcB·PHLS was heterologously co-expressed with enzymes of the mevalonic acid (MVA) pathway and geranyl-diphosphate synthase, increasing carbon flux toward the terpenoid biosynthetic pathway and enhancing substrate availability. These improvements enabled yields of 10 mg of β-phellandrene per g of dry cell weight generated in the course of a 48-h incubation period, or the equivalent of 1 % β-phellandrene:biomass (w:w) carbon-partitioning ratio. The work helped to identify prerequisites for the efficient heterologous production of terpene hydrocarbons in cyanobacteria: (i) requirement for overexpression of the heterologous terpene synthase, so as to compensate for the slow catalytic turnover of the enzyme, and (ii) enhanced endogenous carbon partitioning toward the terpenoid biosynthetic pathway, e.g., upon heterologous co-expression of the MVA pathway, thereby supplementing the native metabolic flux toward the universal isopentenyl-diphosphate and dimethylallyl-diphosphate terpenoid precursors. The two prerequisites are shown to be critical determinants of yield in the photosynthetic CO2 to terpene hydrocarbons conversion process.

  12. Hepatic NK cell-mediated hypersensitivity to ConA-induced liver injury in mouse liver expressing hepatitis C virus polyprotein.

    Science.gov (United States)

    Fu, Qiuxia; Yan, Shaoduo; Wang, Licui; Duan, Xiangguo; Wang, Lei; Wang, Yue; Wu, Tao; Wang, Xiaohui; An, Jie; Zhang, Yulong; Zhou, Qianqian; Zhan, Linsheng

    2017-08-08

    The role of hepatic NK cells in the pathogenesis of HCV-associated hepatic failure is incompletely understood. In this study, we investigated the effect of HCV on ConA-induced immunological hepatic injury and the influence of HCV on hepatic NK cell activation in the liver after ConA administration. An immunocompetent HCV mouse model that encodes the entire viral polyprotein in a liver-specific manner based on hydrodynamic injection and φC31o integrase was used to study the role of hepatic NK cells. Interestingly, the frequency of hepatic NK cells was reduced in HCV mice, whereas the levels of other intrahepatic lymphocytes remained unaltered. Next, we investigated whether the reduction in NK cells within HCV mouse livers might elicit an effect on immune-mediated liver injury. HCV mice were subjected to acute liver injury models upon ConA administration. We observed that HCV mice developed more severe ConA-induced immune-mediated hepatitis, which was dependent on the accumulated intrahepatic NK cells. Our results indicated that after the administration of ConA, NK cells not only mediated liver injury through the production of immunoregulatory cytokines (IFN-γ, TNF-α and perforin) with direct antiviral activity, but they also killed target cells directly through the TRAIL/DR5 and NKG2D/NKG2D ligand signaling pathway in HCV mice. Our findings suggest a critical role for NK cells in oversensitive liver injury during chronic HCV infection.

  13. From hepatocytes to stem and progenitor cells for liver regenerative medicine: advances and clinical perspectives.

    Science.gov (United States)

    Sokal, E M

    2011-04-01

    The parenchymal liver cell is a unique fully functional metabolic unit that can be used for liver regenerative medicine to restore function of the diseased organ; the aim of the procedure is to prevent progression of end-stage disease. The alternative, orthotopic liver transplantation, is highly intrusive, irreversible and limited by general organ shortage. Mature liver cell - hepatocyte - transplantation has been shown to have short- to medium-term efficacy for correction of miscellaneous inborn errors of metabolism. However, although proof of concept has been established, the procedure has not yet achieved full success, due to limited durability of functional benefit. Hepatocyte procurement is also restricted by organ shortage, and their storage is difficult due to poor tolerance of cryopreservation. Alternative cell sources are therefore needed for development and wider accessibility of cell-based liver regenerative medicine. Besides safety, the main challenge for these alternative cells is to acquire similar levels of functionality once implanted into the target organ. In this respect, liver derived progenitor cells may have some advantages over stem cells derived from other tissues. © 2011 Blackwell Publishing Ltd.

  14. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

    National Research Council Canada - National Science Library

    Skipper, Anthony; Sims, Jennifer N; Yedjou, Clement G; Tchounwou, Paul B

    2016-01-01

    ... 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...

  15. 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.)

  16. IL-7 licenses activation of human liver intrasinusoidal mucosal-associated invariant T cells.

    Science.gov (United States)

    Tang, Xin-Zi; Jo, Juandy; Tan, Anthony T; Sandalova, Elena; Chia, Adeline; Tan, Kai Chah; Lee, Kang Hoe; Gehring, Adam J; De Libero, Gennaro; Bertoletti, Antonio

    2013-04-01

    Human mucosal-associated invariant T (MAIT) cells are a T cell population characterized by the expression of a semi-invariant TCR capable of recognizing bacterial products in the context of MR1. MAIT cells are enriched in the human liver, which is constantly exposed to bacterial products from the intestine. Whether this specific parenchymal localization influences their function remains unknown. We analyzed MAIT cells resident in the vascular bed of livers and showed that they represented the majority of T cells expressing NK markers and the dominant IL-17A(+) T cell subset in the human liver sinusoids. In comparison with MAIT cells purified from peripheral blood, intrasinusoidal MAIT cells expressed markers of T cell activation; however, TCR-mediated cytokine production was equally suppressed in both circulating and intrasinusoidal MAIT cells. MAIT cells also expressed high levels of IL-7R, and we showed that IL-7, a cytokine produced by hepatocytes during inflammation, regulated TCR-mediated activation of MAIT cells, licensing them to dramatically increase Th1 cytokines and IL-17A production. Our quantitative and functional data indicate that MAIT cells are a specialized cell population highly adapted to exert their immune functions in the vascular network of the liver.

  17. Biochemical and Cytological Aspects of Liver Cell Function During Infection

    Science.gov (United States)

    1981-01-01

    with Na4 according to the principles of bioelec- trical balance and osmosis . The movement of water, therefore, helps maintain constant intracellular...centrifugation of liver homogenates on sucrose gradients show characteristically broad patterns with a mean density of 1.21. These patterns are affected only...Plasma membranes isolated from livers of control or infected rats have a similar density in alkaline sucrose , but differ significantly in their enzyme

  18. 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.

  19. 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.

  20. Long term culture of genome-stable bipotent progenitor cells from adult human liver

    OpenAIRE

    Huch, Meritxell; Gehart, Helmuth; van, Boxtel Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique MA; Ellis, Ewa; van, Wenum Martien; Fuchs, Sabine A; de, Ligt Joep; van, de Wetering Marc; Sasaki, Nobuo; Boers, Susanne J; Kemperman, Hans; de, Jonge Jeroen

    2014-01-01

    Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The e...

  1. 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.

  2. Primary cultures of endothelial cells of the rat liver: a model for ultrastructural and functional studies.

    Science.gov (United States)

    de Leeuw, A M; Barelds, R J; de Zanger, R; Knook, D L

    1982-01-01

    A new isolation and purification procedure for endothelial cells of the rat liver and the conditions for large scale survival of these cells in maintenance culture are reported. Cells isolated by this new method and cultured with homologous rat serum on a collagen matrix show the restoration of several ultrastructural characteristics typical of rat liver endothelial cells in situ, including the broad cytoplasmic extensions that contain the sieve plates. These fenestrated cytoplasmic projections, which cover the liver sinusoids in vivo, are well preserved and are reformed in a manner reminiscent of the situation in situ. Reformation of specific membrane receptors is indicated by the reappearance of the capacity to take up horseradish peroxidase by adsorptive endocytosis, a characteristic that is lost during the cell isolation procedure. From the results obtained in this study, maintenance culture of rat liver endothelial cells seems to be a promising system for studying the regulation of pore size of the fenestrated sieve plates by alcohol and certain hormones, for studying the interaction of endothelial cells with other liver cells and tumor cells, and for studying the mechanisms of adsorptive endocytosis.

  3. Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation

    Directory of Open Access Journals (Sweden)

    Hoda El-Kehdy

    2016-01-01

    Full Text Available In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL-MSCs. After isolation from 11-12 gestational weeks’ human fetal livers, FL-MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblastic differentiation potential. Thereafter, we explored their hepatocytic differentiation potential using the hepatogenic protocol applied for adult human liver mesenchymal cells. FL-MSCs differentiated in this way displayed significant features of hepatocyte-like cells as demonstrated in vitro by the upregulated expression of specific hepatocytic markers and the induction of metabolic functions including CYP3A4 activity, indocyanine green uptake/release, and glucose 6-phosphatase activity. Following transplantation, naive and differentiated FL-MSC were engrafted into the hepatic parenchyma of newborn immunodeficient mice and differentiated in situ. Hence, FL-MSCs appeared to be interesting candidates to investigate the liver development at the mesenchymal compartment level. Standardization of their isolation, expansion, and differentiation may also support their use for liver cell-based therapy development.

  4. 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.

  5. 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.

  6. 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.

  7. [H-3]thymidine incorporation into whole liver as an alternative to [H-3]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

    NARCIS (Netherlands)

    de Jong, KP; Brinker, M; van Veen, M; Daemen, T; Scherphof, GL; Slooff, MJH

    1999-01-01

    OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [H-3]thymidine incorporation into liver cell DNA. We hypothesized that [H-3]thymidine incorporation into whole liver tissue parallels [H-3]thymidine incorporation into

  8. Combined use of fluorescent dyes and flow cytometry to quantify the physiological state of Pichia pastoris during the production of heterologous proteins in high-cell-density fed-batch cultures.

    Science.gov (United States)

    Hyka, Petr; Züllig, Thomas; Ruth, Claudia; Looser, Verena; Meier, Christian; Klein, Joachim; Melzoch, Karel; Meyer, Hans-Peter; Glieder, Anton; Kovar, Karin

    2010-07-01

    Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut(+) (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.

  9. Toxicity Effect of Silver Nanoparticles on Mice Liver Primary Cell Culture and HepG2 Cell Line

    Science.gov (United States)

    Faedmaleki, Firouz; H Shirazi, Farshad; Salarian, Amir-Ahmad; Ahmadi Ashtiani, Hamidreza; Rastegar, Hossein

    2014-01-01

    Nano-silver (AgNP) has biological properties which are significant for consumer products, food technology, textiles and medical applications (e.g. wound care products, implantable medical devices, in diagnosis, drug delivery, and imaging). For their antibacterial activity, silver nanoparticles are largely used in various commercially available products. Thus, the use of nano-silver is becoming more and more widespread in medicine. In this study we investigated the cytotoxic effects of AgNPs on liver primary cells of mice, as well as the human liver HepG2 cell. Cell viability was examined with MTT assay after HepG2 cells exposure to AgNPs at 1, 2, 3, 4, 5, 7.5, 10 ppm compared to mice primary liver cells at 1, 10, 50, 100, 150, 200, 400 ppm for 24h. AgNPs caused a concentration-dependent decrease of cell viability in both cells. IC50 value of 2.764 ppm (µg/mL) was calculated in HepG2 cell line and IC50 value of 121.7 ppm (µg/mL) was calculated in primary liver cells of mice. The results of this experiment indicated that silver nanoparticles had cytotoxic effects on HepG2 cell line and primary liver cells of mice. The results illustrated that nano-silver had 44 times stronger inhibitory effect on the growth of cancerous cells (HepG2 cell line) compared to the normal cells (primary liver cells of mice). which might further justify AgNPs as a cytotoxic agents and a potential anticancer candidate which needs further studies in this regard. PMID:24734076

  10. 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

  11. 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.

  12. Local proliferation and extrahepatic recruitment of liver macrophages (Kupffer cells) in partial-body irradiated rats

    Energy Technology Data Exchange (ETDEWEB)

    Bouwens, L.; Knook, D.L.; Wisse, E.

    1986-06-01

    The relative significance of local proliferation and extrahepatic recruitment of Kupffer cells was investigated by partial-body irradiation before the induction of macrophage hyperplasia by zymosan. There was no difference in growth of the Kupffer cells population between nonirradiated rats and rats irradiated with the liver shielded, whereas irradiation of the liver with the rest of the body (bone marrow) shielded resulted in strong inhibition of growth (-61%). Splenectomy combined with bone marrow irradiation inhibited growth to a lesser extent as compared to liver irradiation (-38%). Monocyte and other leukocyte numbers were strongly reduced in peripheral blood and their accumulation in the liver was completely prevented by bone marrow irradiation. Our results demonstrate that local proliferation of resident Kupffer cells represents the predominant source for their increased number during hyperplasia.

  13. Infection of five human liver cell lines by dengue-2 virus.

    Science.gov (United States)

    Lin, Y L; Liu, C C; Lei, H Y; Yeh, T M; Lin, Y S; Chen, R M; Liu, H S

    2000-04-01

    Elevated serum transaminase levels of dengue patients indicate the possible impact of dengue virus infection on liver function. To elucidate the action of dengue virus infection in liver cells, an in vitro cell line system was established that mimicked the liver status of diverse clinical patients. Briefly, four hepatoma cell lines (HA22T, Huh7, Hep3B, and PLC) and one nonmalignant hepatocyte cell line (Chang liver) were included, representing various levels of tumorigenicity and differentiation. Our data showed that in these five cell lines, dengue-2 virus attached to each cell type equally well; however, this virus had higher replication rates and levels of virion production in differentiated Huh7, PLC, Hep3B, and Chang liver cells. Likewise, a lower replication rate was observed in the de-differentiated HA22T cells. Differentiation-related factors seem to play an important role in dengue virus replication. Further study showed that sodium butyrate (NaB, a differentiation inducer) treatment enhanced dengue virus replication in HA22T cells. Moreover, we found that the severity of morphologic aberration and the increase in aspartate aminotransferase (AST) levels correlated with the virus replication rate in the four infected hepatoma cells. In conclusion, we showed that dengue virus can infect diverse liver cells with differing replication efficiency, which causes cytopathic effects (CPEs) of diverse severity. Among the CPEs, the increased AST levels correlated with the clinical results from 24 dengue fever patients, who showed increased AST levels at the onset of fever. In summary, we find that dengue-2 virus replicates actively and causes severe CPEs in differentiated hepatoma cells. Factors related to differentiation as well as tumorigenicity seem to play critical roles, though the mechanisms of action remain unclear. Copyright 2000 Wiley-Liss, Inc.

  14. Animal model for liver cell banking from non-heart beating donors after prolonged ischaemia time.

    Science.gov (United States)

    Porretti, L; Gatti, S; Gramignoli, R; Colombo, F; Lopa, R; Cattaneo, A; Scalamogna, M; Colombo, G; Rossi, G; Bonino, F; Rebulla, P; Prati, D

    2006-12-01

    Although there is a growing interest on the use of non-heart beating donors to enlarge the liver donor pool, livers with prolonged warm ischaemia time are not currently considered for organ transplantation. We hypothesised that these organs may represent a source of hepatocytes for cell transplantation and/or use in bioartificial liver devices. Thus, we investigated if prolonged ischaemia could influence the recovery and viability of functional hepatocytes dissociated from rat livers. Hepatocytes were isolated from the liver within 15 min after death (t=15 min) and after 4, 8 and 12h of ischaemia. Cells were either maintained in culture or cryopreserved. In all products, we evaluated cell recovery and viability, hepatocyte markers and cellular functions, including albumin and urea production. The number of cells per gram of tissue was similar at 15 min, 4 and 8h, while it was significantly decreased at 12h. About 0.2 x 10(6) viable cells expressing hepatocyte markers and producing albumin and urea were isolated up to 8h of ischaemia per gram of tissue. Recovery of viable and functional hepatocytes seems possible after prolonged ischaemia time. These data warrant the evaluation of hepatocyte isolation from human livers of non-heart beating donors.

  15. Effects of cadmium on cell proliferation, apoptosis, and proto-oncogene expression in zebrafish liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying Ying; Zhu, Jin Yong; Chan, King Ming, E-mail: kingchan@cuhk.edu.hk

    2014-12-15

    Highlights: • Cd stimulated ZFL cell proliferation with decreasing apoptotic cell numbers. • Cd down regulated p53 and RAD51. • Cd up regulated immediate early cancer genes of GADD45 and growth factors. • Cd promoted tumorigenic effects in ZFL cells. - Abstract: Cadmium (Cd) is one of the major transitional metal that has toxic effects in aquatic organisms and their associated ecosystem; however, its hepatic toxicity and carcinogenicity are not very well characterized. We used a zebrafish liver (ZFL) cell line as a model to investigate the mechanism of Cd-induced toxicity on hepatocytes. Our results showed that Cd can be effectively accumulated in ZFL cells in our exposure experiments. Cell cytotoxicity assays and flow cytometer measurements revealed that Cd{sup 2+} stimulated ZFL cell proliferation with decreasing apoptotic cell numbers indicating potentially tumorigenic effects of Cd in ZFL cells. Gene expression profiles also indicated that Cd downregulated oncogenes p53 and rad51 and upregulated immediate response oncogenes, growth arrest and DNA damage-inducible (gadd45) genes, and growth factors. We also found dramatic changes in the gene expression of c-jun and igf1rb at different exposure time points, supporting the notion that potentially tumorigenic of Cd-is involved in the activation of immediate early genes or genes related to apoptosis in cancer promotion.

  16. 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.

  17. 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.

  18. Label-free quantitative proteomics of CD133-positive liver cancer stem cells

    Directory of Open Access Journals (Sweden)

    Tsai Sheng-Ta

    2012-11-01

    Full Text Available Abstract Background CD133-positive liver cancer stem cells, which are characterized by their resistance to conventional chemotherapy and their tumor initiation ability at limited dilutions, have been recognized as a critical target in liver cancer therapeutics. In the current work, we developed a label-free quantitative method to investigate the proteome of CD133-positive liver cancer stem cells for the purpose of identifying unique biomarkers that can be utilized for targeting liver cancer stem cells. Label-free quantitation was performed in combination with ID-based Elution time Alignment by Linear regression Quantitation (IDEAL-Q and MaxQuant. Results Initially, IDEAL-Q analysis revealed that 151 proteins were differentially expressed in the CD133-positive hepatoma cells when compared with CD133-negative cells. We then analyzed these 151 differentially expressed proteins by MaxQuant software and identified 10 significantly up-regulated proteins. The results were further validated by RT-PCR, western blot, flow cytometry or immunofluorescent staining which revealed that prominin-1, annexin A1, annexin A3, transgelin, creatine kinase B, vimentin, and EpCAM were indeed highly expressed in the CD133-positive hepatoma cells. Conclusions These findings confirmed that mass spectrometry-based label-free quantitative proteomics can be used to gain insights into liver cancer stem cells.

  19. 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.

  20. Liver Graft versus Host Disease after Allogeneic Peripheral Stem Cell Transplantation: Update on Etiopathogenesis and Diagnosis

    Directory of Open Access Journals (Sweden)

    Mihăilă R.-G.

    2016-06-01

    Full Text Available Graft versus host disease (GVHD is the main complication of allogeneic hematopoietic cell transplantation and is more frequent after peripheral stem cell transplants. Graft versus leukemia or lymphoma component of them is beneficial to eradicate residual tumor mass after previous treatment and conditioning regimen. A severe GVHD may endanger the patient's life. The most important liver manifestations of GVHD are increased serum alkaline phosphatase and bilirubin values. The last allows to estimate the GVHD severity. Sometimes, an increase of aminotransferases can mimic an acute hepatitis. Donor-derived hematopoietic cells appeared to turn in mesenchymal liver cells. Activated CD4(+ T cells, humoral and complement activation, a large number of cytokines and cytokine receptors are involved in GVHD development. Correct and early recognition of GVHD and its differentiation from the other liver diseases are essential for the medical practice.

  1. Liver-Derived Systemic Factors Drive β Cell Hyperplasia in Insulin-Resistant States

    Directory of Open Access Journals (Sweden)

    Abdelfattah El Ouaamari

    2013-02-01

    Full Text Available Integrative organ crosstalk regulates key aspects of energy homeostasis, and its dysregulation may underlie metabolic disorders such as obesity and diabetes. To test the hypothesis that crosstalk 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, as well as in vitro islet culture approaches, we demonstrate that humoral, nonneural, non-cell-autonomous factor(s induces 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 factor(s in insulin-resistant states.

  2. 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.

  3. Chlamydia pneumoniae replicates in Kupffer cells in mouse model of liver infection

    Institute of Scientific and Technical Information of China (English)

    Antonella Marangoni; Manuela Donati; Francesca Cavrini; Rita Aldini; Silvia Accardo; Vittorio Sambri; Marco Montagnani; Roberto Cevenini

    2006-01-01

    AIM: To develop an animal model of liver infection with Chlamydia pneumoniae (C.pneumoniae) in intraperitoneally infected mice for studying the presence of chlamydiae in Kupffer cells and hepatocytes.METHODS: A total of 80 BALB/c mice were inoculated intraperitoneally with C. pneumoniae and sacrificed at various time points after infection. Chlamydiae were looked for in liver homogenates as well as in Kupffer cells and hepatocytes separated by liver perfusion with collagenase. C. pneumoniae was detected by both isolation in LLC-MK2 cells and fluorescence in situ hybridization (FISH). The releasing of TNFA-α by C. pneumoniae in vitro stimulated Kupffer cells was studied by enzymelinked immunosorbent assay.RESULTS: C. pneumoniae isolation from liver homogenates reached a plateau on d 7 after infection when 6 of 10 animals were positive, then decreased, and became negative by d 20. C. pneumoniae isolation from separated Kupffer cells reached a plateau on d 7 when 5 of 10 animals were positive, and became negative by d 20.The detection of C. pneumoniae in separated Kupffer cells by FISH, confirmed the results obtained by culture.Isolated hepatocytes were always negative. Stimulation of Kupffer cells by alive C. pneumoniae elicited high TNF-α levels.CONCLUSION: A productive infection by C. pneumoniae may take place in Kupffer cells and C. pneumoniae induces a local pro-inflammatory activity. C. pneumoniae is therefore, able to act as antigenic stimulus when localized in the liver. One could speculate that C. pneumoniae infection, involving cells of the innate immunity such as Kupffer cells, could also trigger pathological immune reactions involving the liver, as observed in human patients with primary biliary cirrhosis.

  4. Liver cancer - hepatocellular carcinoma

    Science.gov (United States)

    Primary liver cell carcinoma; Tumor - liver; Cancer - liver; Hepatoma ... Hepatocellular carcinoma accounts for most liver cancers. This type of cancer occurs more often in men than women. It is usually diagnosed in people age 50 or ...

  5. 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 ...

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

    Indian Academy of Sciences (India)

    2010-12-06

    Dec 6, 2010 ... 1995). Studies have shown that histidine and its catabolites have important physiological activities (Wu 2009), there- fore this study is concentrated on the relevance between his- tidine metabolism and liver regeneration. Biosynthesis of histidine consists of three steps: first, phosphoribosyl py- rophosphate ...

  7. Technique of Hepatectomy for Primary Liver Cell Carcinoma in a ...

    African Journals Online (AJOL)

    The experience in managing a 36 year-old Nigerian male with a histopathologically confirmed hepatocellular carcinoma confined to the left lobe by surgical resection of the liver, using the finger-fracture technique is presented. The literature review on the aetiopathogenesis of this disease and the role and technique of ...

  8. Cell volume changes affect gluconeogenesis in the perfused liver of ...

    Indian Academy of Sciences (India)

    Unknown

    Janssens P A and Waterman J 1988 Hormonal regulation of gluconeogenesis and glycogenolysis in carp (Cyprinus carpio) liver pieces cultured in vitro; Comp. Biochem. Physiol. A91. 451–455. Knox D, Walton M J and Cowey C B 1980 Distribution of enzy- mes of glycolysis and gluconeogenesis in fish tissues; Mar. Biol.

  9. Levels of hepatic Th17 cells and regulatory T cells upregulated by hepatic stellate cells in advanced HBV-related liver fibrosis.

    Science.gov (United States)

    Li, Xiaoyan; Su, Yujie; Hua, Xuefeng; Xie, Chan; Liu, Jing; Huang, Yuehua; Zhou, Liang; Zhang, Min; Li, Xu; Gao, Zhiliang

    2017-04-11

    Liver fibrosis which mainly occurs upon chronic hepatitis virus infection potentially leads to portal hypertension, hepatic failure and hepatocellular carcinoma. However, the immune status of Th17 and Treg cells in liver fibrosis is controversial and the exact mechanisms remain largely elusive. Liver tissues and peripheral blood were obtained simultaneously from 32 hepatitis B virus infected patients undergoing surgery for hepatocellular carcinoma at the medical center of Sun Yat-sen University. Liver tissues at least 3 cm away from the tumor site were used for the analyses. Levels of Th17 cells and regulatory T cells were detected by flow cytometry analysis and immunohistochemistry. In vitro experiment, we adopted magnetic cell sorting to investigate how hepatic stellate cells regulate the levels of Th17 cells and regulatory T cells. We found that hepatic Th17 cells and regulatory T cells were increased in patients with advanced stage HBV-related liver fibrosis. Hepatic stellate cells upregulated the levels of Th17 cells and regulatory T cells via PGE2/EP2 and EP4 pathway. We found that the increased levels of Th17 cells and regulatory T cells were upregulated by hepatic stellate cells. These results may provide insight into the role of hepatic stellate cells and Th17 cells and regulatory T cells in the persistence of fibrosis and into the occurrence of hepatocellular carcinoma following cirrhosis.

  10. 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.

  11. Cancer stem cells in primary liver cancers: pathological concepts and imaging findings.

    Science.gov (United States)

    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.

  12. 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.

  13. Neoplastic transformation of rat liver epithelial cells is enhanced by non-transferrin-bound iron

    Directory of Open Access Journals (Sweden)

    Messner Donald J

    2008-02-01

    Full Text Available Abstract Background Iron overload is associated with liver toxicity, cirrhosis, and hepatocellular carcinoma in humans. While most iron circulates in blood as transferrin-bound iron, non-transferrin-bound iron (NTBI also becomes elevated and contributes to toxicity in the setting of iron overload. The mechanism for iron-related carcinogenesis is not well understood, in part due to a shortage of suitable experimental models. The primary aim of this study was to investigate NTBI-related hepatic carcinogenesis using T51B rat liver epithelial cells, a non-neoplastic cell line previously developed for carcinogenicity and tumor promotion studies. Methods T51B cells were loaded with iron by repeated addition of ferric ammonium citrate (FAC to the culture medium. Iron internalization was documented by chemical assay, ferritin induction, and loss of calcein fluorescence. Proliferative effects were determined by cell count, toxicity was determined by MTT assay, and neoplastic transformation was assessed by measuring colony formation in soft agar. Cyclin levels were measured by western blot. Results T51B cells readily internalized NTBI given as FAC. Within 1 week of treatment at 200 μM, there were significant but well-tolerated toxic effects including a decrease in cell proliferation (30% decrease, p Conclusion These results establish NTBI as a tumor promoter in T51B rat liver epithelial cells. Changes in cyclin proteins suggest cell cycle disregulation contributes to tumor promotion by NTBI in this liver cell model.

  14. Phases I-II Matched Case-Control Study of Human Fetal Liver Cell Transplantation for Treatment of Chronic Liver Disease

    National Research Council Canada - National Science Library

    Pietrosi, Giada; Vizzini, Giovanni; Gerlach, Jorg; Chinnici, Cinzia; Luca, Angelo; Amico, Giandomenico; D'amato, Monica; Conaldi, Pier Giulio; Petri, Sergio Li; Spada, Marco; Tuzzolino, Fabio; Alio, Luigi; Schmelzer, Eva; Gridelli, Bruno

    2015-01-01

    Fetal hepatocytes have a high regenerative capacity. The aim of the study was to assess treatment safety and clinical efficacy of human fetal liver cell transplantation through splenic artery infusion...

  15. 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.

    Science.gov (United States)

    Rodríguez, 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

    2008-04-01

    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 serotype 35 coding for CS (rAd35.CS). The current study was designed to answer the question whether such an effect can be extended to liver-stage antigens (LSA) of Plasmodium falciparum such as LSA-1. Studies with mice have demonstrated that the LSA-1 protein induces strong antibody response but a weak T-cell immunity. We first identified T-cell epitopes in LSA-1 by use of intracellular gamma interferon (IFN-gamma) staining and confirmed these epitopes by means of enzyme-linked immunospot assay and pentamer staining. We show that a single immunization with rAd35.LSA-1 induced a strong antigen-specific IFN-gamma CD8(+) T-cell response but no measurable antibody response. In contrast, vaccinations with the adjuvanted recombinant LSA-1 protein induced remarkably low cellular responses but strong antibody responses. Finally, both priming and boosting of the adjuvanted protein by rAd35 resulted in enhanced T-cell responses without impairing the level of antibody responses induced by the protein immunizations alone. Furthermore, the incorporation of rAd35 in the vaccination schedule led to a skewing of LSA-1-specific antibody responses toward a Th1-type immune response. Our results show the ability of rAd35 to induce potent T-cell immunity in combination with protein in a prime-boost schedule without impairing the B-cell response.

  16. GMP-grade human fetal liver-derived mesenchymal stem cells for clinical transplantation.

    Science.gov (United States)

    Larijani, Bagher; Aghayan, Hamid-Reza; Goodarzi, Parisa; Arjmand, Babak

    2015-01-01

    Stem cell therapy seems a promising avenue in regenerative medicine. Within various stem cells, mesenchymal stem cells have progressively used for cellular therapy. Because of the age-related decreasing in the frequency and differentiating capacity of adult MSCs, fetal tissues such as fetal liver, lung, pancreas, spleen, etc. have been introduced as an alternative source of MSCs for cellular therapy. On the other hand, using stem cells as advanced therapy medicinal products, must be performed in compliance with cGMP as a quality assurance system to ensure the safety, quality, and identity of cell products during translation from the basic stem cell sciences into clinical cell transplantation. In this chapter the authors have demonstrated the manufacturing of GMP-grade human fetal liver-derived mesenchymal stem cells.

  17. Intraportal mesenchymal stem cell transplantation prevents acute liver failure through promoting cell proliferation and inhibiting apoptosis.

    Science.gov (United States)

    Sang, Jian-Feng; Shi, Xiao-Lei; Han, Bin; Huang, Tao; Huang, Xu; Ren, Hao-Zhen; Ding, Yi-Tao

    2016-12-01

    Transplantation of mesenchymal stem cells (MSCs) has been regarded as a potential treatment for acute liver failure (ALF), but the optimal route was unknown. The present study aimed to explore the most effective MSCs transplantation route in a swine ALF model. The swine ALF model induced by intravenous injection of D-Gal was treated by the transplantation of swine MSCs through four routes including intraportal injection (InP group), hepatic intra-arterial injection (AH group), peripheral intravenous injection (PV group) and intrahepatic injection (IH group). The living conditions and survival time were recorded. Blood samples before and after MSCs transplantation were collected for the analysis of hepatic function. The histology of liver injury was interpreted and scored in terminal samples. Hepatic apoptosis was detected by TUNEL assay. Apoptosis and proliferation related protein expressions including cleaved caspase-3, survivin, AKT, phospho-AKT (Ser473), ERK and phospho-ERK (Tyr204) were analyzed by Western blotting. The average survival time of each group was 10.7+/-1.6 days (InP), 6.0+/-0.9 days (AH), 4.7+/-1.4 days (PV), 4.3+/-0.8 days (IH), respectively, when compared with the average survival time of 3.8+/-0.8 days in the D-Gal group. The survival rates between the InP group and D-Gal group revealed a statistically significant difference (Pliver damage was the worst in the D-Gal group, while less injury in the InP group. Histopathological scores revealed a significant decrease in the InP group (3.17+/-1.04, Pliver function, inhibit apoptosis and prolong the survival time of swine with ALF. The transplanted MSCs may participate in liver regeneration via promoting cell proliferation and suppressing apoptosis during the initial stage of ALF.

  18. [Induction by mycotoxins of somatic mosaicism in Drosophila and DNA repair in mammalian liver cell cultures].

    Science.gov (United States)

    Belitskiĭ, G A; Khovanova, E M; Budunova, I V; Sharunich, E G

    1983-07-01

    The genotoxic activity of four mycotoxins has been studied. High level of somatic mutagenesis in imaginal discs of Drosophila melanogaster larvae and DNA repair synthesis in human embryo and adult rat liver cell cultures were inducible only by highly carcinogenic aflatoxin B1. Patulin, a weak direct-action carcinogenic substance, slightly elevated the mutagenesis in somatic cells of Drosophila but did not induce DNA repair synthesis in liver cell cultures. Citrinin that did not exhibit any carcinogenic properties when used alone and stachybotrotoxin with non-reported carcinogenic activity appeared inactive in the test-systems applied. The possibilities of rapid recognition of carcinogenic mycotoxins by detecting their genotoxic properties are discussed.

  19. 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.

  20. Berbamine inhibits the growth of liver cancer cells and cancer initiating cells by targeting Ca2+/Calmodulin-dependent protein kinase II

    OpenAIRE

    Meng, Zhipeng; Li, Tao; Ma, Xiaoxiao; Wang, Xiaoqiong; Van Ness, Carl; Gan, Yichao; Zhou, Hong; Tang, Jinfen; Lou, Guiyu; Wang, Yafan; Wu, Jun; Yen, Yun; Xu, Rongzhen; Huang, Wendong

    2013-01-01

    Liver cancer is the third leading cause of cancer deaths worldwide but no effective treatment toward liver cancer is available so far. Therefore, there is an unmet medical need to identify novel therapies to efficiently treat liver cancer and improve the prognosis of this disease. Here we report that berbamine (BBM) and one of its derivatives, bbd24, potently suppressed liver cancer cell proliferation and induced cancer cell death by targeting Ca2+/calmodulin-dependent protein kinase II (CAMK...

  1. Cell Spheroids with Enhanced Aggressiveness to Mimic Human Liver Cancer In Vitro and In Vivo

    OpenAIRE

    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

    2017-01-01

    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-1?) and ERK signal activation were detected in the cell spheroids. To diminish core necros...

  2. Rodent animal models for surrogate analysis of cell therapy in acute liver failure

    Directory of Open Access Journals (Sweden)

    Bruno eChrist

    2012-04-01

    Full Text Available Without therapeutic intervention acute liver failure is the consequence of a progredient destruction of the liver parenchyma due to metabolic exhaustion of the hepatocytes. Perivenous hepatocytes are responsible for the detoxification of noxes via the cytochrome P450 enzyme system. Liver transplantation is the only remaining therapeutic option in the end-stage of the disease. Assuming that metabolic capacity could be provided by healthy hepatocytes and thus substitute for the genuine parenchymal cells hepatocyte transplantation since quite some time is considered to be an alternative to whole liver transplantaton. While this hypothesis achieved proof-of-concept in animal trials clinical breakthrough is still awaiting success, the reasons of which are ongoing matter of debate. In recent times mesenchymal stem cells came into focus as a transplantable cell source to treat ALF. Interestingly, as demonstrated in various rodent animal models their mode of action is rather based on trophic support of hepatocytes remaining in the damaged host parenchyma rather than substitution of tissue loss. Mechanistically, either direct or indirect paracrine effects from the transplanted cells acting pro-proliferative, anti-apoptotic and anti-inflammatory seem to trigger the regenerative response of the residual healthy hepatocytes in the otherwise lethally injured liver parenchyma. Thus, allogeneic mesenchymal stem cells may be the best choice for the treatment of ALF taking advantage of their short-term benefit to sustain the critical phase of the acute insult avoiding long-term immunosuppression.

  3. Liver Enzymes and Trace Elements in the Acute Phase of Sickle Cell ...

    African Journals Online (AJOL)

    BACKGROUND: Trace elements are required for the performance of numerous functions of immune cells. It is not clear whether levels of trace elements are elevated and whether there is a relationship between the levels of liver enzymes and trace elements in patients with sickle cell anaemia in crisis. OBJECTIVE: To ...

  4. Small cell variant of renal oncocytoma with liver metastases: a case report.

    Science.gov (United States)

    Trivedi, Priti P; Kriplani, Divya; Gami, Amisha; Shah, Manoj J; Shah, Pankaj M

    2013-12-01

    Renal oncocytoma is a renal neoplasm considered to be benign. A small cell variant comprising predominantly of oncoblasts is rare. Metastases from a renal oncocytoma are extremely rare. A case of small cell variant of renal oncocytoma with liver metastases is described.

  5. 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

  6. Murine liver-resident group 1 innate lymphoid cells regulate optimal priming of anti-viral CD8+ T cells.

    Science.gov (United States)

    Krueger, Peter D; Narayanan, Sowmya; Surette, Fionna A; Brown, Michael G; Sung, Sun-Sang J; Hahn, Young S

    2017-01-01

    The liver contains 2 transcriptionally distinct group 1 ILC subsets: CD49a+ ILC1s and CD49b+ NK cells. However, little is known about how group 1 ILCs contribute to hepatic immune responses. Therefore, we characterized murine liver-resident group 1 ILCs and found that CD49a+ ILC1s express high levels of the inhibitory receptor NKG2A and localize near DCs in perivascular spaces surrounding the portal triads. Upon hepatic viral infection, NKG2A signaling in group 1 ILCs, especially in CD49a+ ILC1s, inhibits CXCL9 expression required for robust accumulation of IFN-γ+CD49b+ NK cells. As a consequence, NKG2A-/- mice showed increased numbers of IFN-γ-producing NK cells that preferentially activate liver CD103+ DCs, leading to the sustained proliferation of adoptively transferred, virus-specific CD8+ T cells. Collectively, these data suggest that group 1 ILCs play a role in maintaining the liver as a tolerogenic site by limiting the recruitment of peripheral NK cells during the early phase of viral infection. Furthermore, our findings implicate that the inhibition of NKG2A signaling on group 1 ILCs may be a novel vaccine strategy to induce robust CD8+ T cell responses against persistent liver pathogens. © Society for Leukocyte Biology.

  7. Successful Transplantation of Reduced Sized Rat Alcoholic Fatty Livers Made Possible by Mobilization of Host Stem Cells

    Science.gov (United States)

    Hisada, Masayuki; Ota, Yoshihiro; Zhang, Xiuying; Cameron, Andrew M; Gao, Bin; Montgomery, Robert A; Williams, George Melville; Sun, Zhaoli

    2015-01-01

    Livers from Lewis rats fed with 7% alcohol for 5 weeks were used for transplantation. Reduced sized (50%) livers or whole livers were transplanted into normal DA recipients, which, in this strain combination, survive indefinitely when the donor has not been fed alcohol. However, none of the rats survived a whole fatty liver transplant while six of seven recipients of reduced sized alcoholic liver grafts survived long term. SDF-1 and HGF were significantly increased in reduced size liver grafts compared to whole liver grafts. Lineage-negative Thy-1+CXCR4+CD133+ stem cells were significantly increased in the peripheral blood and in allografts after reduced size fatty liver transplantation. In contrast, there were meager increases in cells reactive with anti Thy-1, CXCR4 and CD133 in peripheral blood and allografts in whole alcoholic liver recipients. The provision of plerixafor, a stem cell mobilizer, salvaged 5 of 10 whole fatty liver grafts. Conversely, blocking SDF-1 activity with neutralizing antibodies diminished stem cell recruitment and four of five reduced sized fatty liver recipients died. Thus chemokine insuficiency was associated with transplant failure of whole grafts which was overcome by the increased regenerative requirements promoted by the small grafts and mediated by SDF-1 resulting in stem cell influx. PMID:22994609

  8. MiR-525-3p Enhances the Migration and Invasion of Liver Cancer Cells by Downregulating ZNF395

    Science.gov (United States)

    Pang, Fei; Zha, Ruopeng; Zhao, Yingjun; Wang, Qifeng; Chen, Di; Zhang, Zhenfeng; Chen, Taoyang; Yao, Ming; Gu, Jianren; He, Xianghuo

    2014-01-01

    Liver cancer is one of leading causes of cancer-related deaths. A deeper mechanistic understanding of liver cancer could lead to the development of more effective therapeutic strategies. In our previous work, we screened 646 miRNAs and identified 11 that regulate liver cancer cell migration. The current study shows that miR-525-3p is frequently up-regulated in liver cancer tissues, and enhanced expression of miR-525-3p can promote liver cancer cell migration and invasion. Zinc finger protein 395 (ZNF395) is the direct functional target gene for miR-525-3p, and it is frequently down-regulated in liver cancer tissues. High expression of ZNF395 can significantly inhibit while knockdown of ZNF395 expression can markedly enhance the migration and invasion of liver cancer cells, suggesting that ZNF395 suppresses metastasis in liver cancer. Down-regulation of ZNF395 can mediate miR-525-3p induced liver cancer cell migration and invasion. In conclusion, miR-525-3p promotes liver cancer cell migration and invasion by directly targeting ZNF395, and the fact that miR-525-3p and ZNF395 both play important roles in liver cancer progression makes them potential therapeutic targets. PMID:24599008

  9. Berbamine inhibits the growth of liver cancer cells and cancer-initiating cells by targeting Ca²⁺/calmodulin-dependent protein kinase II.

    Science.gov (United States)

    Meng, Zhipeng; Li, Tao; Ma, Xiaoxiao; Wang, Xiaoqiong; Van Ness, Carl; Gan, Yichao; Zhou, Hong; Tang, Jinfen; Lou, Guiyu; Wang, Yafan; Wu, Jun; Yen, Yun; Xu, Rongzhen; Huang, Wendong

    2013-10-01

    Liver cancer is the third leading cause of cancer deaths worldwide but no effective treatment toward liver cancer is available so far. Therefore, there is an unmet medical need to identify novel therapies to efficiently treat liver cancer and improve the prognosis of this disease. Here, we report that berbamine and one of its derivatives, bbd24, potently suppressed liver cancer cell proliferation and induced cancer cell death by targeting Ca(2+)/calmodulin-dependent protein kinase II (CAMKII). Furthermore, berbamine inhibited the in vivo tumorigenicity of liver cancer cells in NOD/SCID mice and downregulated the self-renewal abilities of liver cancer-initiating cells. Chemical inhibition or short hairpin RNA-mediated knockdown of CAMKII recapitulated the effects of berbamine, whereas overexpression of CAMKII promoted cancer cell proliferation and increased the resistance of liver cancer cells to berbamine treatments. Western blot analyses of human liver cancer specimens showed that CAMKII was hyperphosphorylated in liver tumors compared with the paired peritumor tissues, which supports a role of CAMKII in promoting human liver cancer progression and the potential clinical use of berbamine for liver cancer therapies. Our data suggest that berbamine and its derivatives are promising agents to suppress liver cancer growth by targeting CAMKII. Mol Cancer Ther; 12(10); 2067-77. ©2013 AACR.

  10. 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.

  11. 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

    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......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...... 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....

  12. Novel roles of DC-SIGNR in colon cancer cell adhesion, migration, invasion, and liver metastasis.

    Science.gov (United States)

    Na, Heya; Liu, Xiaoli; Li, Xiaomeng; Zhang, Xinsheng; Wang, Yu; Wang, Zhaohui; Yuan, Menglang; Zhang, Yu; Ren, Shuangyi; Zuo, Yunfei

    2017-01-21

    Tumor metastasis is an essential cause of the poor prognosis of colon cancer. DC-SIGNR is a C-type lectin that is frequently found on human liver sinusoidal endothelial cells. LSECtin, which is a homologue of DC-SIGNR, has been demonstrated to participate in colon cancer liver metastasis. Due to the similarities in the expression pattern and structure of the two proteins, we speculated that DC-SIGNR could also be involved in this process. Colon cancer cells were treated with the DC-SIGNR protein or control IgG, after which cell migration, invasion, and morphology were assayed. Xenograft mouse models were used to determine the role of DC-SIGNR in colon cancer liver metastasis in vivo. In addition, a human gene expression array was used to detect differential gene expression in colon cancer cells stimulated with the DC-SIGNR protein. The serum level of DC-SIGNR was examined in colon cancer patients by ELISA, and the significance of DC-SIGNR was determined. In our research, we investigated whether DC-SIGNR promotes colon cancer cell adhesion, migration, and invasion. Knocking down mouse DC-SIGNR decreased the liver metastatic potency of colon cancer cells and increased survival time. Expressing human DC-SIGNR enhanced colon cancer liver metastasis. Furthermore, DC-SIGNR conferred metastatic capability on cancer cells by upregulating various metallothionein isoforms. To validate the above results, we also found that the serum DC-SIGNR level was statistically higher in colon cancer patients with liver metastasis compared with those without metastasis. These results imply that DC-SIGNR may promote colon carcinoma hepatic metastasis and could serve as a promising therapeutic target for anticancer treatment.

  13. Therapeutic PEG-ceramide nanomicelles synergize with salinomycin to target both liver cancer cells and cancer stem cells.

    Science.gov (United States)

    Wang, Meiping; Xie, Fangyuan; Wen, Xikai; Chen, Han; Zhang, Hai; Liu, Junjie; Zhang, He; Zou, Hao; Yu, Yuan; Chen, Yan; Sun, Zhiguo; Wang, Xinxia; Zhang, Guoqing; Yin, Chuan; Sun, Duxin; Gao, Jie; Jiang, Beige; Zhong, Yanqiang; Lu, Ying

    2017-05-01

    Salinomycin (SAL)-loaded PEG-ceramide nanomicelles (SCM) were prepared to target both liver cancer cells and cancer stem cells. The synergistic ratio of SAL/PEG-ceramide was evaluated to prepare SCM, and the antitumor activity of SCM was examined both in vitro and in vivo. SAL/PEG-ceramide molar ratio of 1:4 was chosen as the synergistic ratio, and SCM showed superior cytotoxic effect and increased apoptosis-inducing activity in both liver cancer cells and cancer stem cells. In vivo, SCM showed the best tumor inhibitory effect with a safety profile. Thus, PEG-ceramide nanomicelles could serve as an effective and safe therapeutic drug carrier to deliver SAL into liver cancer, opening up the avenue of using PEG-ceramide as therapeutic drug carriers.

  14. [Effects of extract of Polygonum multiflorum on cell cycle arrest and apoptosis of human liver cell line L02].

    Science.gov (United States)

    Zhang, Rui-Chen; Liu, Bin; Sun, Zhen-Xiao; Xu, Dong-Yan

    2010-06-01

    To analyze the chemical constituents of Polygonum multiflorum extract which may cause human liver cell damage and to explore the mechanism. Raw and processed Polygonum multiflorum were extracted by 70% ethanol, then raw and processed Polygonum multiflorum water-eluted material (RW and PW), 50% ethanol-eluted material (R50 and P50) and 95% ethanol-eluted material (R95 and P95) were obtained by absorbing through AB-8 macroporous resin, followed by water, 50% ethanol and 95% ethanol elution in order. The water extracts of raw and processed Polygonum multiflorum (RWE or PWE) were obtained by boiling them in water as usual. Normal human liver L02 cells were treated by different concentrations of eluted Polygonum multiflorum materials for different time, and the cell growth inhibition of each group was determined by methylthiazolyldiphenyl-tetrazolium bromide method. The chemical constituents which had a significant cytotoxicity to L02 cells were analyzed by high-performance liquid chromatography (HPLC). Morphological changes of L02 cells were observed by Giemsa staining and cell cycle distribution was observed by flow cytometry. It was found that 95% ethanol-eluted extracts of raw and processed Polygonum multiflorum showed significant growth inhibition on normal human liver L02 cells, while the other components showed no significant inhibition on cell growth. HPLC analysis showed that the main component in 95% ethanol-eluted extract of raw and processed Polygonum multiflorum was emodin at content of (18.53+/-2.96)% and (10.28+/-1.34)% respectively. Cell cycle analysis showed that 95% ethanol-eluted material of Polygonum multiflorum and emodin had a similar significant effect of S phase arrest and all could induce L02 cell apoptosis. The main part of Polygonum multiflorum causing liver cell damage is the 95% ethanol-eluted extract, and emodin is one of the important chemical constituents leading to liver cell damage.

  15. PAMAM-pullulan conjugates as targeted gene carriers for liver cell.

    Science.gov (United States)

    Askarian, Saeedeh; Abnous, Khalil; Ayatollahi, Sara; Farzad, Sara Amel; Oskuee, Reza Kazemi; Ramezani, Mohammad

    2017-02-10

    Targeted nano-carriers are highly needed to promote nucleic acid delivery into the specific cell for therapeutic approaches. Pullulan as a linear carbohydrate has an intrinsic liver targeting property interacting with asialoglycoprotein receptor (ASGPR) found on liver cells. In the present study, we developed polyamidoamine (PAMAM)-pullulan conjugates and investigated their targeting activity in delivering gene into liver cells. The particle size, zeta potential, buffering capacity and ethidium bromide exclusion assays of the conjugates were evaluated. The cytotoxicity and transfection efficiency of new derivatives were assessed following in vitro transfection of HepG2 (receptor positive) and N2A (receptor negative) cell lines. Size of conjugated polymers ranged between 118 and 184 nanometers and their cytotoxicity were similar to PAMAM. Among six produced nanocarriers, G4PU4 and G5PU4 enhanced transfection efficiency in HepG2 cells compared to unmodified PAMAM. Therefore, the PAMAM-pullulan derivatives seem to improve delivery of nucleic acids into the liver cells expressing asialoglycoprotein receptor with minimal transfection in non-targeted cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Deregulation of Regulatory T Cells in Acute-on-Chronic Liver Failure: A Rat Model.

    Science.gov (United States)

    Ni, Shunlan; Li, Shanshan; Yang, Naibin; Tang, Xinyue; Zhang, Shengguo; Hu, Danping; Lu, Mingqin

    2017-01-01

    Aims. Acute-on-chronic liver failure (ACLF) and acute liver failure (ALF) are similar in many respects during their acute exacerbation; however, ACLF generally has a poorer prognosis. We aimed to investigate the role and dynamic changes of regulatory T cell (Treg) and T helper 17 (Th17) cell proportions during ACLF progress. Methods. All rats were classified into two groups randomly: ACLF group and ALF group (control group). The rat model of ACLF was preestablished by intraperitoneal injection of carbon tetrachloride for 2 months. Then acute liver injury was induced by combined D-galactosamine and lipopolysaccharide. Six time points were examined before or after acute induction. Liver samples were performed with hematoxylin-eosin and Masson staining; circulatory Treg and Th17 cell frequencies were determined using flow cytometry assays; serum levels of alanine aminotransferase, aspartate aminotransferase, interleukin-10 (IL-10), and interferon-γ (IFN-γ) were examined. Results. In group ACLF, both Th17 cell proportion and IFN-γ level presented upgrade firstly and then descend latter tendency; the trends of Treg cell proportion and IL-10 level were observed to gradually decrease and became stable. Conclusion. The Treg cells played an important role in the immunologic mechanism during the process of ACLF. And the function of Treg cells in ACLF was defective.

  17. Level of oxidative stress in the red blood cells of patients with liver cirrhosis.

    Science.gov (United States)

    Geetha, A; Lakshmi Priya, M D; Jeyachristy, S Annie; Surendran, R

    2007-09-01

    Liver cirrhosis is associated with gastrointestinal haemorrhage and oesophageal variceal bleeding. Altered platelet functions has been reported to be a cause of bleeding complication. We carried out this study to find out the level of oxidative stress in the red blood cells of patients with liver cirrhosis. Fifty patients admitted with the complication of liver cirrhosis (with bleeding complications, n=30 and without bleeding complications, n=20) were included in the study. Age and sex matched normal healthy volunteers (n=45) served as controls. The levels of oxyhaemoglobin and methaemoglobin were assayed in the red blood cells. Oxidative stress markers such as lipid peroxides, lipid hydroperoxides and nitric oxide were determined along with enzymatic antioxidants. Membrane bound adenosine triphosphatases, cytosolic glucose-6-phosphate dehydrogenase and NADHmethaemoglobin reductase were also measured. The levels of cholesterol and total phospholipids were assessed in red blood cell membrane. The osmotic fragility of red blood cells was monitored using different concentrations of sodium chloride. The level of methaemoglobin was significantly higher (P blood cells of liver cirrhotic patients with bleeding complication compared to that of non bleeding patients. The activity level of NADH-methaemoglobin reductase was significantly lower (Penzymatic antioxidants were low except of glutathione peroxidase. The activity levels of adenosine triphosphatases were also found to be significantly lower (Pblood cells was also altered in patients. In cirrhotic condition red blood cells are subjected to severe oxidative stress with significant alterations in the membrane properties.

  18. Anticarcinogenic effects of glycoalkaloids from potatoes against human cervical, liver, lymphoma, and stomach cancer cells.

    Science.gov (United States)

    Friedman, Mendel; Lee, Kap-Rang; Kim, Hyun-Jeong; Lee, In-Seon; Kozukue, Nobuyuke

    2005-07-27

    Methods were devised for the isolation of large amounts of pure alpha-chaconine and alpha-solanine from Dejima potatoes and for the extraction and analysis of total glycoalkaloids from five fresh potato varieties (Dejima, Jowon, Sumi, Toya, and Vora Valley). These compounds were then evaluated in experiments using a tetrazolium microculture (MTT) assay to assess the anticarcinogenic effects of (a) the isolated pure glycoalkaloids separately, (b) artificial mixtures of the two glycoalkaloids, and (c) the total glycoalkaloids isolated from each of the five potato varieties. All samples tested reduced the numbers of the following human cell lines: cervical (HeLa), liver (HepG2), lymphoma (U937), stomach (AGS and KATO III) cancer cells and normal liver (Chang) cells. The results show that (a) the effects of the glycoalkaloids were concentration dependent in the range of 0.1-10 mug/mL (0.117-11.7 nmol/mL); (b) alpha-chaconine was more active than was alpha-solanine; (c) some mixtures exhibited synergistic effects, whereas other produced additive ones; (d) the different cancer cells varied in their susceptibilities to destruction; and (e) the destruction of normal liver cells was generally lower than that of cancer liver cells. The decreases in cell populations were also observed visually by reversed-phase microscopy. The results complement related observations on the anticarcinogenic potential of food ingredients.

  19. Label-free detection of liver cancer cells by aptamer-based microcantilever biosensor.

    Science.gov (United States)

    Chen, Xuejuan; Pan, Yangang; Liu, Huiqing; Bai, Xiaojing; Wang, Nan; Zhang, Bailin

    2016-05-15

    Liver cancer is one of the most common and highly malignant cancers in the world. There are no effective therapeutic options if an early liver cancer diagnosis is not achieved. In this work, detection of HepG2 cells by label-free microcantilever array aptasensor was developed. The sensing microcantilevers were functionalized by HepG2 cells-specific aptamers. Meanwhile, to eliminate the interferences induced by the environment, the reference microcantilevers were modified with 6-mercapto-1-hexanol self-assembled monolayers. The aptasensor exhibits high specificity over not only human liver normal cells, but also other cancer cells of breast, bladder, and cervix tumors. The linear relation ranges from 1×10(3) to 1×10(5)cells/mL, with a detection limit of 300 cells/mL (S/N=3). Our work provides a simple method for detection of liver cancer cells with advantages in terms of simplicity and stability. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Exploiting the unique regenerative capacity of the liver to underpin cell and gene therapy strategies for genetic and acquired liver disease.

    Science.gov (United States)

    Logan, Grant J; de Alencastro, Gustavo; Alexander, Ian E; Yeoh, George C

    2014-11-01

    The number of genetic or acquired diseases of the liver treatable by organ transplantation is ever-increasing as transplantation techniques improve placing additional demands on an already limited organ supply. While cell and gene therapies are distinctly different modalities, they offer a synergistic alternative to organ transplant due to distinct architectural and physiological properties of the liver. The hepatic blood supply and fenestrated endothelial system affords relatively facile accessibility for cell and/or gene delivery. More importantly, however, the remarkable capacity of hepatocytes to proliferate and repopulate the liver creates opportunities for new treatments based on emerging technologies. This review will summarise current understanding of liver regeneration, describe clinical and experimental cell and gene therapeutic modalities and discuss critical challenges to translate these new technologies to wider clinical utility. This article is part of a Directed Issue entitled: "Regenerative Medicine: the challenge of translation". Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. 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

  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. Autophagy-Modulated Human Bone Marrow-Derived Mesenchymal Stem Cells Accelerate Liver Restoration in Mouse Models of Acute Liver Failure.

    Science.gov (United States)

    Amiri, Fatemeh; Molaei, Sedigheh; Bahadori, Marzie; Nasiri, Fatemeh; Deyhim, Mohammad Reza; Jalili, Mohammad Ali; Nourani, Mohammad Reza; Habibi Roudkenar, Mehryar

    2016-07-01

    Mesenchymal stem cells (MSCs) have been recently received increasing attention for cell-based therapy, especially in regenerative medicine. However, the low survival rate of these cells restricts their therapeutic applications. It is hypothesized that autophagy might play an important role in cellular homeostasis and survival. This study aims to investigate the regenerative potentials of autophagy-modulated MSCs for the treatment of acute liver failure (ALF) in mice. ALF was induced in mice by intraperitoneal injection of 1.5 ml/kg carbon tetrachloride. Mice were intravenously infused with MSCs, which were suppressed in their autophagy pathway. Blood and liver samples were collected at different intervals (24, 48 and 72 h) after the transplantation of MSCs. Both the liver enzymes and tissue necrosis levels were evaluated using biochemical and histopathological assessments. The survival rate of the transplanted mice was also recorded during one week. Biochemical and pathological results indicated that 1.5 ml/kg carbon tetrachloride induces ALF in mice. A significant reduction of liver enzymes and necrosis score were observed in autophagy-modulated MSC-transplanted mice compared to sham (with no cell therapy) after 24 h. After 72 h, liver enzymes reached their normal levels in mice transplanted with autophagy-suppressed MSCs. Interestingly, normal histology without necrosis was also observed. Autophagy suppression in MSCs ameliorates their liver regeneration potentials due to paracrine effects and might be suggested as a new strategy for the improvement of cell therapy in ALF.

  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. The Human Amnion Epithelial Cell Secretome Decreases Hepatic Fibrosis in Mice with Chronic Liver Fibrosis

    Directory of Open Access Journals (Sweden)

    Majid Alhomrani

    2017-10-01

    Full Text Available Background: Hepatic stellate cells (HSCs are the primary collagen-secreting cells in the liver. While HSCs are the major cell type involved in the pathogenesis of liver fibrosis, hepatic macrophages also play an important role in mediating fibrogenesis and fibrosis resolution. Previously, we observed a reduction in HSC activation, proliferation, and collagen synthesis following exposure to human amnion epithelial cells (hAEC and hAEC-conditioned media (hAEC-CM. This suggested that specific factors secreted by hAEC might be effective in ameliorating liver fibrosis. hAEC-derived extracellular vesicles (hAEC-EVs, which are nanosized (40–100 nm membrane bound vesicles, may act as novel cell–cell communicators. Accordingly, we evaluated the efficacy of hAEC-EV in modulating liver fibrosis in a mouse model of chronic liver fibrosis and in human HSC.Methods: The hAEC-EVs were isolated and characterized. C57BL/6 mice with CCl4-induced liver fibrosis were administered hAEC-EV, hAEC-CM, or hAEC-EV depleted medium (hAEC-EVDM. LX2 cells, a human HSC line, and bone marrow-derived mouse macrophages were exposed to hAEC-EV, hAEC-CM, and hAEC-EVDM. Mass spectrometry was used to examine the proteome profile of each preparation.Results: The extent of liver fibrosis and number of activated HSCs were reduced significantly in CCl4-treated mice given hAEC-EVs, hAEC-CM, and hAEC EVDM compared to untreated controls. Hepatic macrophages were significantly decreased in all treatment groups, where a predominant M2 phenotype was observed. Human HSCs cultured with hAEC-EV and hAEC-CM displayed a significant reduction in collagen synthesis and hAEC-EV, hAEC-CM, and hAEC-EVDM altered macrophage polarization in bone marrow-derived mouse macrophages. Proteome analysis showed that 164 proteins were unique to hAEC-EV in comparison to hAEC-CM and hAEC-EVDM, and 51 proteins were co-identified components with the hAEC-EV fraction.Conclusion: This study provides novel data

  6. Transplantation of Thy1+Cells Accelerates Liver Regeneration by Enhancing the Growth of Small Hepatocyte-Like Progenitor Cells via IL17RB Signaling.

    Science.gov (United States)

    Ichinohe, Norihisa; Ishii, Masayuki; Tanimizu, Naoki; Kon, Junko; Yoshioka, Yusuke; Ochiya, Takahiro; Mizuguchi, Toru; Hirata, Koichi; Mitaka, Toshihiro

    2017-04-01

    Small hepatocyte-like progenitor cells (SHPCs) transiently form clusters in rat livers treated with retrorsine (Ret)/70% partial hepatectomy (PH). When Thy1 + cells isolated from d-galactosamine-treated rat livers were transplanted into the livers of Ret/PH-treated rats, the mass of the recipient liver transiently increased during the first 30 days after transplantation, suggesting that liver regeneration was enhanced. Here we addressed how Thy1 + cell transplantation stimulates liver regeneration. We found that the number and size of SHPC clusters increased in the liver at 14 days after transplantation. GeneChip analysis revealed that interleukin 17 receptor b (IL17rb) expression significantly increased in SHPCs from livers transplanted with Thy1 + cells. We subsequently searched for ligand-expressing cells and found that sinusoidal endothelial cells (SECs) and Kupffer cells expressed Il17b and Il25, respectively. Moreover, extracellular vesicles (EVs) separated from the conditioned medium of Thy1 + cell culture induced IL17b and IL25 expression in SECs and Kupffer cells, respectively. Furthermore, EVs enhanced IL17rb expression in small hepatocytes (SHs), which are hepatocytic progenitor cells; in culture, IL17B stimulated the growth of SHs. These results suggest that Thy1-EVs coordinate IL17RB signaling to enhance liver regeneration by targeting SECs, Kupffer cells, and SHPCs. Indeed, the administration of Thy1-EVs increased the number and size of SHPC clusters in Ret/PH-treated rat livers. Sixty days post-transplantation, most expanded SHPCs entered cellular senescence, and the enlarged liver returned to its normal size. In conclusion, Thy1 + cell transplantation enhanced liver regeneration by promoting the proliferation of intrinsic hepatic progenitor cells via IL17RB signaling. Stem Cells 2017;35:920-931. © 2016 AlphaMed Press.

  7. The potential of bone marrow stem cells to correct liver dysfunction in a mouse model of Wilson's disease.

    Science.gov (United States)

    Allen, Katrina J; Cheah, Daphne M Y; Lee, Xiao Ling; Pettigrew-Buck, Nicole E; Vadolas, Jim; Mercer, Julian F B; Ioannou, Panayiotis A; Williamson, Robert

    2004-01-01

    Metabolic liver diseases are excellent targets for correction using novel stem cell, hepatocyte, and gene therapies. In this study, the use of bone marrow stem cell transplantation to correct liver disease in the toxic milk (tx) mouse, a murine model for Wilson's disease, was evaluated. Preconditioning with sublethal irradiation, dietary copper loading, and the influence of cell transplantation sites were assessed. Recipient tx mice were sublethally irradiated (4 Gy) prior to transplantation with bone marrow stem cells harvested from normal congenic (DL) littermates. Of 46 transplanted tx mice, 11 demonstrated genotypic repopulation in the liver. Sublethal irradiation was found to be essential for donor cell engraftment and liver repopulation. Dietary copper loading did not improve cell engraftment and repopulation results. Both intravenously and intrasplenically transplanted cells produced similar repopulation successes. Direct evidence of functionality and disease correction following liver repopulation was observed in the 11 mice where liver copper levels were significantly reduced when compared with mice with no liver repopulation. The reversal of copper loading with bone marrow cells is similar to the level of correction seen when normal congenic liver cells are used. Transplantation of bone marrow cells partially corrects the metabolic phenotype in a mouse model for Wilson's disease.

  8. 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.

  9. Autophagy is Required for the Maintenance of Liver Progenitor Cell Functionality

    Directory of Open Access Journals (Sweden)

    Yiji Cheng

    2015-06-01

    Full Text Available Background: Liver progenitor cells (LPCs are bipotent stem cells existing in the adult liver, which could be activated upon massive liver injury and contribute to liver regeneration. However, mechanisms of maintenance of LPC functionality remain poorly understood. Previous studies found that autophagy was required for the self-renewal and differentiation of several tissue stem cells. Methods: The study compared the level of autophagic activity in LPCs and differentiated hepatocytes. Then, autophagic activity was inhibited in LPCs by lentivirus-mediated autophagy-related gene 5 or Beclin 1 knockdown. Clonogenic assay, cell viability assays, hepatic differentiation assay, and senescence analysis were conducted to assess the role of autophagy in regulating self-renewal, hepatic differentiation and senescence of LPCs. Results: We observed high autophagic activity in LPCs compared with differentiated hepatocytes. We found that inhibition of autophagy impaired the self-renewal, proliferation, and hepatic differentiation capability of LPCs under normal cultural condition, but had little impact on cell viability. Interestingly, while wild-type LPCs remained rarely affected by the toxin, etoposide, inhibition of autophagy induced the senescent phenotype of LPCs. Overexpression of Beclin 1 in Beclin 1-knockdown LPCs restored the functionality of stem cells. Conclusion: Our findings indicate that autophagy may function as a critical regulator of LPC functionality under both physiological and pathological condition.

  10. Small intestine follicular dendritic cell sarcoma with liver metastasis: A case report.

    Science.gov (United States)

    Chang, Yun-Chen; Chau, Ivy Yenwen; Yeh, Yi-Chen; Chau, Gar-Yang

    2017-08-01

    Follicular dendritic cell sarcoma (FDCS) is a rare neoplasia composed of spindle or oval cells with follicular dendritic cell differentiation, usually occurring in lymphoid tissue. In this report, we present a case of FDCS of the small intestine with liver metastasis. A 19-year-old female presented with recent onset of left upper abdominal pain. Abdominal computed tomography scan showed a large tumor mass in the liver lateral segment with compression to the pancreas upper part, and a smaller mass in the terminal ileum, respectively. High serum levels of amylase and lipase were noted. Resection of the tumors was performed. Microscopically, both tumors consisted of ovoid to spindle-shaped nuclei cells admixed with some lymphocytes arranged in fascicles, whorls, storiform arrays. Immunohistochemistry demonstrated that the tumor cells were positive for follicular dendritic cell markers, including CD21, CD23, and CD35. Epstein-Barr virus encoding small RNA (EBER; Inform EBER probe; Ventana Medical Systems, Tucson, AZ) in situ hybridization was negative. According to the clinicopathological features, diagnosis of FDCS of intestinal origin was made. Resection of tumors located in the liver and at the small intestine was performed. After the operation, patient received adjuvant vinblastin chemotherapy. There was no evidence of recurrence at 8-month follow-up. It was unusual for FDCS of intestinal origin with liver metastasis and expressing with high serum levels of pancreatic enzymes.

  11. [Hepatic intra-arterial infusion of BAK immune cells to treat metastatic liver cancer].

    Science.gov (United States)

    Ebina, Takusaburo

    2011-11-01

    Based on the "living with cancer" concept while maintaining a favorable QOL and avoiding side effects and drug resistance, we have developed a new immune cell treatment called BAK (BRM activated killer) therapy, primarily using CD56+ cells for a case of advanced progressive solid cancer. In the present case, we administered BAK cells by hepatic intra-arterial infusion to a patient who happened to be a surgeon and wished to undergo this therapy. The patient was a 52-year- old male surgeon who underwent surgery for rectal cancer in April 2007. Heavy particle radiotherapy was administered when liver metastases were identified in July 2008. Starting in December 2008, 10 billion BAK cells were administered each month by hepatic intra-arterial infusion via a catheter on a total of six different occasions. The 10 billion autologous lymphocytes were suspended in 200 mL of Ringer's solution and returned to the patient by hepatic intra-arterial infusion over a period of one hour. Interactions between the activated lymphocytes and liver cancer cells increased levels of serum α1AG, an inflammation marker, but these levels normalized following the sixth and final administration. Conventional drip-infusion BAK therapy was administered thereafter. Diagnostic imaging, including PET-CT and PET, confirmed a complete disappearance of liver metastases. This case suggests the effectiveness of hepatic intra-arterial infusion BAK cell therapy in treating liver cancer.

  12. CUDR promotes liver cancer stem cell growth through upregulating TERT and C-Myc

    Science.gov (United States)

    Pu, Hu; Zheng, Qidi; Li, Haiyan; Wu, Mengying; An, Jiahui; Gui, Xin; Li, Tianming; Lu, Dongdong

    2015-01-01

    Cancer up-regulated drug resistant (CUDR) is a novel non-coding RNA gene. Herein, we demonstrate excessive CUDR cooperates with excessive CyclinD1 or PTEN depletion to accelerate liver cancer stem cells growth and liver stem cell malignant transformation in vitro and in vivo. Mechanistically, we reveal the decrease of PTEN in cells may lead to increase binding capacity of CUDR to CyclinD1. Therefore, CUDR-CyclinD1 complex loads onto the long noncoding RNA H19 promoter region that may lead to reduce the DNA methylation on H19 promoter region and then to enhance the H19 expression. Strikingly, the overexpression of H19 increases the binding of TERT to TERC and reduces the interplay between TERT with TERRA, thus enhancing the cell telomerase activity and extending the telomere length. On the other hand, insulator CTCF recruits the CUDR-CyclinD1 complx to form the composite CUDR-CyclinD1-insulator CTCF complex which occupancied on the C-myc gene promoter region, increasing the outcome of oncogene C-myc. Ultimately, excessive TERT and C-myc lead to liver cancer stem cell and hepatocyte-like stem cell malignant proliferation. To understand the novel functions of long noncoding RNA CUDR will help in the development of new liver cancer therapeutic and diagnostic approaches. PMID:26513297

  13. Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

    OpenAIRE

    Huch Meritxell; Gehart Helmuth; van Boxtel Ruben; Hamer Karien; Blokzijl Francis; Verstegen Monique M. A.; Ellis Ewa; van Wenum Martien; Fuchs Sabine A.; de Ligt Joep; van de Wetering Marc; Sasaki Nobuo; Boers Susanne J.; Kemperman Hans; de Jonge Jeroen

    2015-01-01

    Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in?vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in?vitro and can be differentiated into functional hepatocytes in?vitro and in?vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human live...

  14. Human mesenchymal stromal cell lysates as a novel strategy to recover liver function.

    Science.gov (United States)

    Herencia, Carmen; Almadén, Yolanda; Martínez-Moreno, Julio M; Espejo, Isabel; Herrera, Concha; Pérez-Sánchez, Carlos; Guerrero, Fátima; Ciria, Rubén; Briceño, Francisco Javier; Ferrín, Gustavo; de la Mata, Manuel; Muñoz-Castañeda, Juan R

    2015-01-01

    It is unknown if the beneficial effects of mesenchymal stromal cells (MSC) transplantation into the liver are dependent on their anchorage and differentiation into hepatocytes or rather the result of the release of stem cell intracellular content with hepatoprotector properties. The effects of intact MSC transplantation were compared with the infusion of MSC lysates in an experimental rat model of acute liver failure. A more powerful hepatoprotective and antiapoptotic effect was obtained after infusion of MSC lysates than intact MSC. Changes in IL-6 levels and miRNAs might explain the beneficial effects of MSC lysates. Infusion of MSC lysates show a better hepatoprotective effect than the transplantation of intact MSC.

  15. 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.

  16. 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

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

    Science.gov (United States)

    Kegel, Victoria; Deharde, Daniela; Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

    2016-03-30

    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 Medicine(1). 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.

  18. Effect of Astragaloside II combined with 5-fluorouracil treatment on liver cell proliferation

    Directory of Open Access Journals (Sweden)

    Yan-Qing Chen

    2016-09-01

    Full Text Available Objective: To study the effect of astragaloside II combined with 5-fluorouracil treatment on liver cell proliferation and analyze the specific molecular mechanisms. Methods: Liver cancer cell lines HepG2 were cultured, cells of logphase growth were collected and treated with different conditions, blank control group were treated with DMEM without serum, 5-Fu treatment group were treated with 0.5, 1, 5 and 10 μmol/L 5-Fu, and 5-Fu combined with astragaloside II treatment group were treated with 10 μmol/L 5-Fu combined with 20, 40 and 80 μmol/L astragaloside II. 24, 48 and 72 h after treatment, MTS kit was used to determine the cell viability; 24 h after treatment, ELISA kit was used to determine the content of apoptosis protein in cells. Results: Compared with blank control group, 5-Fu treatment could reduce the survival rate of liver cancer cells in time-dependent and dose-dependent manner; compared with 10 μmol/L 5-Fu single drug treatment group, 10 μmol/L 5-Fu combined with astragaloside II treatment could reduce the survival rate of liver cancer cells in time-dependent and dosedependent manner; Fas, FasL, Bax, Caspase-3, Caspase-8 and Caspase-9 expression levels after 10 μmol/L 5-Fu treatment were significantly higher than those of blank control group, and Fas, FasL, Bax, Caspase-3, Caspase-8 and Caspase-9 expression levels after 10 μmol/L 5-Fu combined with 40 μmol/L astragaloside II treatment were significantly higher than those of 10 μmol/L 5-Fu treatment group. Conclusions: Astragaloside II combined with 5-fluorouracil treatment can enhance the inhibitory effect of 5-fluorouracil on liver cancer cell proliferation, and the apoptosis proteins mediating the effect are Fas/FasL and Bax.

  19. 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

  20. 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.

  1. Inactivation of kupffer cells by gadolinium chloride protects murine liver from radiation-induced apoptosis.

    Science.gov (United States)

    Du, Shi-Suo; Qiang, Min; Zeng, Zhao-Chong; Ke, Ai-Wu; Ji, Yuan; Zhang, Zheng-Yu; Zeng, Hai-Ying; Liu, Zhongshan

    2010-03-15

    To determine whether the inhibition of Kupffer cells before radiotherapy (RT) would protect hepatocytes from radiation-induced apoptosis. A single 30-Gy fraction was administered to the upper abdomen of Sprague-Dawley rats. The Kupffer cell inhibitor gadolinium chloride (GdCl3; 10 mg/kg body weight) was intravenously injected 24 h before RT. The rats were divided into four groups: group 1, sham RT plus saline (control group); group 2, sham RT plus GdCl3; group 3, RT plus saline; and group 4, RT plus GdCl3. Liver tissue was collected for measurement of apoptotic cytokine expression and evaluation of radiation-induced liver toxicity by analysis of liver enzyme activities, hepatocyte micronucleus formation, apoptosis, and histologic staining. The expression of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha was significantly attenuated in group 4 compared with group 3 at 2, 6, 24, and 48 h after injection (p <0.05). At early points after RT, the rats in group 4 exhibited significantly lower levels of liver enzyme activity, apoptotic response, and hepatocyte micronucleus formation compared with those in group 3. Selective inactivation of Kupffer cells with GdCl3 reduced radiation-induced cytokine production and protected the liver against acute radiation-induced damage. Copyright 2010 Elsevier Inc. All rights reserved.

  2. Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes.

    Science.gov (United States)

    Joshi, Meghnad; B Patil, Pradeep; He, Zhong; Holgersson, Jan; Olausson, Michael; Sumitran-Holgersson, Suchitra

    2012-07-01

    One important problem commonly encountered after hepatocyte transplantation is the low numbers of transplanted cells found in the graft. If hepatocyte transplantation is to be a viable therapeutic approach, significant liver parenchyma repopulation is required. Mesenchymal stromal cells (MSC) produce high levels of various growth factors, cytokines and metalloproteinases, and have immunomodulatory effects. We therefore hypothesized that co-transplantation of MSC with human fetal hepatocytes (hFH) could augment in vivo expansion after transplantation. We investigated the ability of human fetal liver MSC (hFLMSC) to augment expansion of phenotypically and functionally well-characterized hFH. Two million hFH (passage 6) were either transplanted alone or together (1:1 ratio) with green fluorescence protein-expressing hFLMSC into the spleen of C57BL/6 nude mice with retrorsine-induced liver injury. After 4 weeks, engraftment of cells was detected by fluorescence in situ hybridization using a human-specific DNA probe. Significantly higher numbers of cells expressing human cytokeratin (CK)8, CK18, CK19, Cysteine-rich MNNG HOS Transforming gene (c-Met), alpha-fetoprotein (AFP), human nuclear antigen, mitochondrial antigen, hepatocyte-specific antigen and albumin (ALB) were present in the livers of recipient animals co-transplanted with hFLMSC compared with those without. Furthermore, expression of human hepatocyte nuclear factor (HNF)-4α and HNF-1β, and cytochrome P450 (CYP) 3A7 mRNA was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) in these animals. In addition, significantly increased amounts of human ALB were detected. Importantly, hFLMSC did not transdifferentiate into hepatocytes. Our study reports the use of a novel strategy for enhanced liver repopulation and thereby advances this experimental procedure closer to clinical liver cell therapy.

  3. Polycomb group protein Ezh2 regulates hepatic progenitor cell proliferation and differentiation in murine embryonic liver.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Koike

    Full Text Available In embryonic liver, hepatic progenitor cells are actively proliferating and generate a fundamental cellular pool for establishing parenchymal components. However, the molecular basis for the expansion of the progenitors maintaining their immature state remains elusive. Polycomb group proteins regulate gene expression throughout the genome by modulating of chromatin structure and play crucial roles in development. Enhancer of zeste homolog 2 (Ezh2, a key component of polycomb group proteins, catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3, which trigger the gene suppression. In the present study, we investigated a role of Ezh2 in the regulation of the expanding hepatic progenitor population in vivo. We found that Ezh2 is highly expressed in the actively proliferating cells at the early developmental stage. Using a conditional knockout mouse model, we show that the deletion of the SET domain of Ezh2, which is responsible for catalytic induction of H3K27me3, results in significant reduction of the total liver size, absolute number of liver parenchymal cells, and hepatic progenitor cell population in size. A clonal colony assay in the hepatic progenitor cells directly isolated from in vivo fetal livers revealed that the bi-potent clonogenicity was significantly attenuated by the Ezh2 loss of function. Moreover, a marker expression based analysis and a global gene expression analysis showed that the knockout of Ezh2 inhibited differentiation to hepatocyte with reduced expression of a number of liver-function related genes. Taken together, our results indicate that Ezh2 is required for the hepatic progenitor expansion in vivo, which is essential for the functional maturation of embryonic liver, through its activity for catalyzing H3K27me3.

  4. 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

  5. A Preclinical Model of Chronic Alcohol Consumption Reveals Increased Metastatic Seeding of Colon Cancer Cells in the Liver.

    Science.gov (United States)

    Im, Hwi-Jin; Kim, Hyeong-Geug; Lee, Jin-Seok; Kim, Hyo-Seon; Cho, Jung-Hyo; Jo, Il-Joo; Park, Sung-Joo; Son, Chang-Gue

    2016-04-01

    Liver metastasis is the main cause of death from colorectal cancer. Alcohol consumption impacts liver function and is suggested to be an independent risk factor for liver metastasis of colorectal cancer, but no experimental evidence supporting this hypothesis has been demonstrated to date. In this study, we investigated the effect of alcohol intake on liver metastasis. We examined colon cancer cell spread from the spleen in mice provided with water (control group), alcohol for 4 weeks before tumor injection (prealcohol), alcohol for 3 weeks after tumor injection (postalcohol), or alcohol throughout the 7-week study (alcohol). Alcohol intake significantly increased hepatic metastatic burden in the prealcohol (2.4-fold, P liver (2.5-fold, P liver metastasis of colorectal cancer cells through alterations to the liver microenvironment and inactivation of immune surveillance. Cancer Res; 76(7); 1698-704. ©2016 AACR. ©2016 American Association for Cancer Research.

  6. p62/SQSTM1 by binding to vitamin D receptor inhibits hepatic stellate cell activity, fibrosis and liver cancer

    OpenAIRE

    Duran, Angeles; Hernandez, Eloy D.; Reina-Campos, Miguel; Castilla, Elias A.; Subramaniam, Shankar; Raghunandan, Sindhu; Roberts, Lewis R.; Kisseleva, Tatiana; Karin, Michael; Diaz-Meco, Maria T.; Moscat, Jorge

    2016-01-01

    Hepatic stellate cells (HSC) play critical roles in liver fibrosis and hepatocellular carcinoma (HCC). Vitamin D receptor (VDR) activation in HSC inhibits liver inflammation and fibrosis. We found that p62/SQSTM1, a protein upregulated in liver parenchymal cells but downregulated in HCC-associated HSC, negatively controls HSC activation. Total body or HSC-specific p62 ablation potentiates HSC and enhances inflammation, fibrosis and HCC progression. p62 directly interacts with VDR and RXR prom...

  7. 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......, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion...

  8. 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.

  9. Quantification of fenestrations in liver sinusoidal endothelial cells by atomic force microscopy.

    Science.gov (United States)

    Zapotoczny, Bartlomiej; Szafranska, Karolina; Kus, Edyta; Chlopicki, Stefan; Szymonski, Marek

    2017-10-01

    Liver sinusoidal endothelial cells present unique morphology characterized by the presence of transmembrane pores called fenestrations. The size and number of fenestrations in live cells change dynamically in response to variety of chemical and physical factors. Although scanning electron microscopy is a well-established method for investigation of fixed liver sinusoidal endothelial cells morphology, atomic force microscopy is the interesting alternative providing detailed 3D topographical information. Moreover, simple sample preparation, only by wet-fixation, minimizing sample preparation artifacts enable high-resolution atomic force microscopy-based measurements. In this work, we apply imaging methods based on atomic force microscopy, to describe characteristic features of glutaraldehyde-fixed primary murine liver sinusoidal endothelial cells, namely: mean fenestration diameter, porosity, and fenestrations frequency. We also investigate the effect of different tip apex radius on evaluation of single fenestration diameter. By quantitative description of fenestrations, we demonstrate that atomic force microscopy became a well competing tool for nondestructive quantitative investigation of the liver sinusoidal endothelial cell morphology. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

    Science.gov (United States)

    Huch, Meritxell; Gehart, Helmuth; van Boxtel, Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique M.A.; Ellis, Ewa; van Wenum, Martien; Fuchs, Sabine A.; de Ligt, Joep; van de Wetering, Marc; Sasaki, Nobuo; Boers, Susanne J.; Kemperman, Hans; de Jonge, Jeroen; Ijzermans, Jan N.M.; Nieuwenhuis, Edward E.S.; Hoekstra, Ruurdtje; Strom, Stephen; Vries, Robert R.G.; van der Laan, Luc J.W.; Cuppen, Edwin; Clevers, Hans

    2015-01-01

    Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. PMID:25533785

  11. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line.

    Science.gov (United States)

    Talbot, Neil C; Blomberg, Le Ann; Garrett, Wesley M; Caperna, Thomas J

    2010-10-01

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication, morphology, and function were lost if the cells were cultured without STO feeder cells. A method for the feeder-independent continuous culture of PICM-19 cells (FI-PICM-19) is presented. PICM-19 cells were maintained and grown without feeder cells on collagen I-coated tissue culture plastic for 26 passages (P26) with initial split ratios of 1:3 that diminished to split ratios of less than 1:2 after passage 16. Once plated, the FI-PICM-19 cells were overlaid with a 1:12 to 1:50 dilution of Matrigel or related extracellular matrix product. Growth of the cells was stimulated by daily refeedings with STO feeder-cell conditioned medium. The FI-PICM-19 cells grew to an approximate confluence of 50% prior to each passage at 2-wk intervals. Growth curve analysis showed their average cell number doubling time to be ~96 h. Morphologically, the feeder-independent cells closely resembled PICM-19 cells grown on feeder cells, and biliary canalicui were present at cell-to-cell junctions. However, no spontaneous multicellular ductules formed in the monolayers of FI-PICM-19 cells. Ultrastructural subcellular features of the FI-PICM-19 cells were similar to those of PICM-19 cells cultured on feeder cells. The FI-PICM-19 cells produced a spectrum of serum proteins and expressed many liver/hepatocyte-specific genes. Importantly, cytochrome P450 (EROD) activity, ammonia clearance, and urea production were maintained by the feeder-independent cells. This simple method for the propagation of the PICM-19 cell line without feeder cells should simplify the generation and selection of functional mutants within the population and enhances the cell line's potential for use in toxicological

  12. 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......-collidin (DDC) diet; and N-acetyl-paraaminophen (APAP). Reproducibly, oval cells showing reactivity for cytokeratins (CKs), muscle pyruvate kinase (MPK), the adenosine triphosphate-binding cassette transporter ABCG2/BCRP1 (ABCG2), alpha-fetoprotein (AFP), and delta-like protein 1/preadipocyte factor 1 (Dlk...

  13. 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.

  14. Research advances in the role of autophagy in liver cells and regulation of MAPK pathway

    Directory of Open Access Journals (Sweden)

    JIANG Na

    2016-05-01

    Full Text Available Autophagy is a highly conservative cellular process in eukaryotes that plays an important role in nutrition and energy metabolism in the liver. It can promote the autophagy of hepatocytes and protect the hepatocytes against the adverse external stimulation, but excessive autophagy can cause autophagic cell death of hepatocytes. Hepatic stellate cells (HSC play an important role in the development and progression of liver fibrosis. Autophagy can provide energy for their activation, but may also lead to their death. This article introduces the relationship of autophagy with hepatocytes/HSC and the mitogen-activated protein kinase pathway, and points out that in-depth studies on the relationship between autophagy and pathway regulation during liver fibrosis can provide new targets for developing antifibrotic drugs.

  15. Malignant melanocytic neoplasm of pancreas with liver metastasis: Is it malignant melanoma or clear cell sarcoma?

    Science.gov (United States)

    Kodiatte, Thomas Alex; George, Sam Varghese; Chacko, Raju Titus; Ramakrishna, Banumathi

    2017-01-01

    Malignant melanocytic neoplasm, usually seen in soft tissues, is rare in a visceral location and presents as a diagnostic dilemma. We present a case of pancreatic malignant melanocytic neoplasm with liver metastasis. A 58-year-old man presented with left upper abdominal swelling and loss of appetite. Imaging revealed a large mass arising from the pancreatic tail, and this was diagnosed as malignant neoplasm with melanocytic differentiation on biopsy with the possible differentials of malignant melanoma, clear cell sarcoma (CCS), and perivascular epithelioid cell neoplasm. The patient underwent distal pancreatectomy and splenectomy for the same. Follow-up imaging 6 months later showed a metastatic liver lesion, for which he also underwent a liver resection. BRAF mutational analysis was found to be negative. Both CCS and malignant melanoma have similar morphological features and melanocytic differentiation, but each harbors a distinct genetic background. Differentiation of both has diagnostic and therapeutic implications.

  16. Inhibition of sphingosine kinase 1 ameliorates acute liver failure by reducing high-mobility group box 1 cytoplasmic translocation in liver cells.

    Science.gov (United States)

    Lei, Yan-Chang; Yang, Ling-Ling; Li, Wen; Luo, Pan; Zheng, Pei-Fen

    2015-12-14

    To determine the therapeutic potential of sphingosine kinase 1 (Sphk1) inhibition and its underlying mechanism in a well-characterized mouse model of D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver failure (ALF). Balb/c mice were randomly assigned to different groups, with ALF induced by intraperitoneal injection of D-GaIN (600 mg/kg) and LPS (10 μg/kg). The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at different time points within one week were determined using a multi-parametric analyzer. Serum high-mobility group box 1 (HMGB1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-10, and sphingosine-1-phosphate were detected by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after acute liver injury induction were assessed by hematoxylin and eosin staining. HMGB1 expression in hepatocytes and cytoplasmic translocation were detected by immunohistochemistry. Expression of Sphk1 in liver tissue and peripheral blood mononuclear cells (PBMCs) was analyzed by Western blot. The expression of Sphk1 in liver tissue and PBMCs was upregulated in GalN/LPS-induced ALF. Upregulated Sphk1 expression in liver tissue was mainly caused by Kupffer cells, the resident macrophages of the liver. The survival rates of mice in the N,N-dimethylsphingosine (DMS, a specific inhibitor of SphK1) treatment group were significantly higher than that of the control group (P liver cells were significantly decreased in the DMS treatment group compared to the control group (43.72% ± 5.51% vs 3.57% ± 0.83%, χ(2) = 12.81, P liver cells, and so might be a potential therapeutic strategy for this disease.

  17. 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...

  18. Delivery of viral vectors to hepatic stellate cells in fibrotic livers using HVJ envelopes fused with targeted liposomes

    NARCIS (Netherlands)

    Adrian, Joanna E.; Kamps, Jan A. A. M.; Poelstra, Klaas; Scherphof, Gerrit L.; Meijer, Dirk K. F.; Kaneda, Yasufumi

    2007-01-01

    Hepatic stellate cells (HSC) are a major target for antifibrotic therapies in the liver and in particular gene delivery to these cells would be relevant. Previously, we demonstrated that mannose 6-phosphate human serum albumin (M6P-HSA) coupled liposomes accumulate in HSC in fibrotic livers. Here we

  19. 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.

  20. 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.

  1. 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 × 105 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.

  2. Liver Cirrhosis in a Patient with Sickle Cell Trait (Hb Sβ+ Thalassemia without Other Known Causes of Hepatic Disease

    Directory of Open Access Journals (Sweden)

    Luca Santi

    2009-09-01

    Full Text Available Liver involvement in patients with sickle cell anemia/trait includes a wide range of alterations, from mild liver function test abnormalities to cirrhosis and acute liver failure. Approximately 15–30% of patients with sickle cell anemia present cirrhosis at autopsy. The pathogenesis of cirrhosis is usually related to chronic hepatitis B or C infection or to iron overload resulting from the many transfusions received by these patients in their lifetime. Thus, cirrhosis has been described almost exclusively in patients with sickle cell anemia, while only mild liver abnormalities have been associated with the sickle cell trait. In the present case study, we describe a young Mediterranean man carrying a sickle cell trait (Hb Sβ+ thalassemia who developed liver cirrhosis being negative for hepatitis C and B viruses or for other causes of cirrhosis and not receiving chronic blood transfusions.

  3. Caffeine attenuates liver fibrosis via defective adhesion of hepatic stellate cells in cirrhotic model.

    Science.gov (United States)

    Shim, Sung Gon; Jun, Dae Won; Kim, Eun Kyung; Saeed, Waqar Khalid; Lee, Kang Nyeong; Lee, Hang Lak; Lee, Oh Young; Choi, Ho Soon; Yoon, Byung Chul

    2013-12-01

    Several epidemiological studies have shown that coffee intake attenuates the progression of liver fibrosis; however, the mechanism is unclear. We investigated the direct effects of caffeine on hepatic stellate cells (HSCs) and assessed whether caffeine attenuated intrahepatic fibrosis in rat model of liver cirrhosis. Human hepatic stellate cell line, an immortalized human HSCs line, was used in in vitro assay system. Cell migration and proliferation were assessed in presence of various caffeine concentrations (0, 1, 5, and 10 mmol), and levels of procollagen type Ic and α-smooth muscle actin (α-SMA) were measured by Western blot. Severity of liver inflammation and fibrosis were compared between thioacetamide-treated rats with and without caffeine supplementation. Caffeine increased HSCs apoptosis and intracellular F-actin and cyclic adenosine monophosphate expression. Caffeine also inhibited procollagen type Ic and α-SMA expression in a dose- and time-dependent manner. In rat model, caffeine decreased periportal inflammation, levels of inflammatory cells (1.4 ± 0.52 vs 2.6 ± 0.46, P caffeine. Caffeine attenuates the progression of liver fibrosis by inhibiting HSCs adhesion and activation. © 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  4. 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

  5. In vitro functionality of human fetal liver cells and clonal derivatives under proliferative conditions

    NARCIS (Netherlands)

    Deurholt, Tanja; ten Bloemendaal, Lysbeth; Chhatta, Aniska A.; van Wijk, Albert C. W. A.; Weijer, Kees; Seppen, Jurgen; Elferink, Ronald P. J. Oude; Chamuleau, Robert A. F. M.; Hoekstra, Ruurdtje

    2006-01-01

    Mature human hepatocytes are not suitable for large-scale in vitro applications that rely on hepatocyte function, due to their limited availability and insufficient proliferation capacity in vitro. In contrast, human fetal liver cells (HFLC) can be easily expanded in vitro. In this study we

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

    NARCIS (Netherlands)

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

    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 (less than 1 mg Cu/kg) for 10 wk. Copper-deficient rats were characterized by low copper concentrations in plasma and

  7. Effect of extracellular vesicles of human adipose tissue on insulin signaling in liver and muscle cells

    NARCIS (Netherlands)

    Kranendonk, Mariëtte E G; Visseren, Frank L J; van Herwaarden, Joost A; Nolte-'t Hoen, Esther N M; de Jager, Wilco; Wauben, Marca H M; Kalkhoven, Eric; Nolte - t Hoen, Esther

    2014-01-01

    OBJECTIVE: Insulin resistance (IR) is a key mechanism in obesity-induced cardiovascular disease. To unravel mechanisms whereby human adipose tissue (AT) contributes to systemic IR, the effect of human AT-extracellular vesicles (EVs) on insulin signaling in liver and muscle cells was determined.

  8. Cell volume regulation in the perfused liver of a freshwater air ...

    Indian Academy of Sciences (India)

    Madhu

    ; J. Biosci. 29 337–347. Graf J, Haddad P, Häussinger D and Lang F 1988 Cell volume regulation in liver; Renal Physiol. Biochem. 11 202–220. Grinstein S, Cohen S and Rothstein A 1984 Cytoplasmic pH regulation in thymic lymphocytes by ...

  9. Targeting dexamethasone to Kupffer cells : Effects on liver inflammation and fibrosis in rats

    NARCIS (Netherlands)

    Melgert, BN; Olinga, P; Van der Laan, JMS; Weert, B; Cho, J; Schuppan, D; Groothuis, GMM; Meijer, DKF; Poelstra, K

    2001-01-01

    Kupffer cells (KC) play an important role in the pathogenesis of inflammatory liver diseases leading to fibrosis. Anti-inflammatory drugs are only effective when administered at high doses that may cause side effects. Therefore, dexamethasone coupled to mannosylated albumin (Dexa(5)-Man(10)-HSA) was

  10. 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.

  11. [Protective effect of intraperitoneal transplantation of human liver-derived stem cells at different times against concanavalin A-induced acute liver injury in mice].

    Science.gov (United States)

    Bi, Y Z; Fan, Z; Chen, D F; Li, S S; Wang, Q Y; Gao, P F; Wang, Q Q; Duan, Z P; Chen, Y; Kong, L B; Wang, Y B; Hong, F

    2017-03-20

    Objective: To investigate the protective effect of intraperitoneal transplantation of human liver-derived stem cells at different times against concanavalin A (ConA)-induced acute liver injury in mice. Methods: A total of 88 male C57BL/6 mice were randomly divided into normal control group (group C), ConA model group (group M), and human liver-derived stem cells (HYX1)+ConA group (group E); according to the interval between phosphate buffer/HYX1 injection and ConA injection, Groups M and E were further divided into 3-hour groups (M1 and E1 groups), 6-hour groups (M2 and E2 groups), 12-hour groups (M3 and E3 groups), 24-hour groups (M4 and E4 groups), and 48-hour groups (M5 and E5 groups). The levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and total bilirubin (TBil) in peripheral blood were measured, liver tissue sections were used to observe pathological changes, and the Ishak score for liver inflammation was determined. The independent samples t-test was used for comparison between groups, and P 0.05). The pathological sections of liver tissue showed that compared with group M, group E had significant reductions in the degree of necrosis and Ishak score (both P transplantation of human liver-derived stem cells has a protective effect against ConA-induced acute liver injury in mice, and the injection at 6 and 12 hours in advance has the best protective effect.

  12. Multiple hormonal control of enzyme synthesis in liver and hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Kenney, F.T.; Lee, K.L.; Pomato, N.; Nickol, J.M.

    1978-01-01

    Synthesis of hepatic tyrosine aminotransferase is accelerated in vivo by either of the pancreatic hormones, insulin and glucagon as well as by glucocorticoids, and glucagon acts via the intracellular mediator, cyclic AMP. The mechanisms responsive to these hormones have also been retained in cultured hepatoma cells: in H-35 cells the responses appear to be essentially identical to those in liver, especially in that each inducer can act independently of the others. In this paper we describe recent analyses of the cellular mechanisms involved in this multiple hormonal control of synthesis of a single enzyme. These experiments have been done with rat liver in vivo, owing to a need for larger quantities of cellular components that can readily be obtained from cultured cells. As some of these results appear to be at variance in important respects with those of earlier analyses carried out in H-35 cells, we briefly review these earlier experiments as well.

  13. 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.

  14. 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.

  15. Cell transplantation after oxidative hepatic preconditioning with radiation and ischemia-reperfusion leads to extensive liver repopulation

    Science.gov (United States)

    Malhi, Harmeet; Gorla, Giridhar R.; Irani, Adil N.; Annamaneni, Pallavi; Gupta, Sanjeev

    2002-10-01

    The inability of transplanted cells to proliferate in the normal liver hampers cell therapy. We considered that oxidative hepatic DNA damage would impair the survival of native cells and promote proliferation in transplanted cells. Dipeptidyl peptidase-deficient F344 rats were preconditioned with whole liver radiation and warm ischemia-reperfusion followed by intrasplenic transplantation of syngeneic F344 rat hepatocytes. The preconditioning was well tolerated, although serum aminotransferase levels rose transiently and hepatic injury was observed histologically, along with decreased catalase activity and 8-hydroxy adducts of guanine, indicating oxidative DNA damage. Transplanted cells did not proliferate in the liver over 3 months in control animals and animals preconditioned with ischemia-reperfusion alone. Animals treated with radiation alone showed some transplanted cell proliferation. In contrast, the liver of animals preconditioned with radiation plus ischemia-reperfusion was replaced virtually completely over 3 months. Transplanted cells integrated in the liver parenchyma and liver architecture were preserved normally. These findings offer a paradigm for repopulating the liver with transplanted cells. Progressive loss of cells experiencing oxidative DNA damage after radiation and ischemia-reperfusion injury could be of significance for epithelial renewal in additional organs.

  16. Galactose-specific recognition system of mammalian liver: receptor distribution on the hepatocyte cell surface

    OpenAIRE

    1981-01-01

    An isolated perfused liver system was used to study the distribution of asialoglycoprotein (ASGP) binding sites on rat hepatocyte cell surfaces. The number of surface receptors was quantitated by monitoring clearance of 125I-labeled ligands from the perfusate medium under two conditions that blocked their internalization: low temperature (less than 5 degrees C) or brief formaldehyde fixation. The cell surface distribution of binding sites was visualized in the electron microscope with either ...

  17. Liver Transplant

    Science.gov (United States)

    ... The Progression of Liver Disease Diagnosing Liver Disease – Liver Biopsy and Liver Function Tests Clinical Trials Liver Transplant ... The Progression of Liver Disease Diagnosing Liver Disease: Liver Biopsy and Liver Function Tests Clinical Trials Liver Transplant ...

  18. 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.

  19. 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

  20. Role and regulation of autophagy and apoptosis by nitric oxide in hepatic stellate cells during acute liver failure.

    Science.gov (United States)

    Jin, Li; Gao, Heng; Wang, JiuPing; Yang, ShuJuan; Wang, Jing; Liu, JingFeng; Yang, Yuan; Yan, TaoTao; Chen, Tianyan; Zhao, Yingren; He, Yingli

    2017-11-01

    We previously found that hepatic stellate cell activation induced by autophagy maintains the liver architecture to prevent collapse during acute liver failure. Nitric oxide has shown to induce hepatic stellate cell apoptosis. Whether and how nitric oxide is involved in acute liver failure and autophagy remains unclear. Acute liver failure patients were recruited to investigate the correlation between plasma nitric oxide levels and clinical features. Liver tissues were collected from chronic hepatitis patients by biopsy and from acute liver failure patients who had undergone liver transplantation. The expression of nitric oxide synthases and hepatic stellate cell activation (alpha-SMA), and autophagic activity (LC3) were investigated by immunohistochemistry. Autophagy and apoptosis were investigated by immunoblot analysis, confocal microscopy, and flow cytometry in hepatic stellate cells treated with nitric oxide donors. Plasma nitric oxide level was significantly increased in patients with acute liver failure compared to those with cirrhosis (53.60±19.74 μM vs 19.40±9.03 μM, Z=-7.384, Pfailure. At least some Nitric oxide was produced by overexpression of inducible nitric oxide synthases and endothelial nitric oxide synthases, but not neuronal nitric oxide synthases in the liver tissue. In vivo observation revealed that autophagy was inhibited in hepatic stellate cells based on decreased LC3 immunostaining, and in vitro experiments demonstrated that Nitric oxide can inhibit autophagy. Moreover, nitric oxide promoted hepatic stellate cell apoptosis, which was rescued by an autophagy inducer. Increased nitric oxide synthases/ nitric oxide promotes apoptosis through autophagy inhibition in hepatic stellate cells during acute liver failure, providing a novel strategy for the treatment of patients with acute liver failure. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells

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    Canto Tomas

    2004-08-01

    Full Text Available Abstract Background RNA interference (RNAi in animals and post-transcriptional gene silencing (PTGS in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system. Results Here we demonstrate that both transient and constitutive expression of the Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein. Conclusion These results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi.

  2. Effects of environmental pollution on the liver parenchymal cells and Kupffer-melanomacrophagic cells of the frog Rana esculenta.

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    Fenoglio, Carla; Boncompagni, Eleonora; Fasola, Mauro; Gandini, Carlo; Comizzoli, Sergio; Milanesi, Gloria; Barni, Sergio

    2005-03-01

    In vertebrates, the biotransformation processes of xenobiotics are performed mainly by the liver which involves both hepatocytes and Kupffer-melanomacrophagic cells through enzymatic and nonenzymatic mechanisms. In this study, we investigated the liver of Rana esculenta adult frogs collected at two sample rice fields, one heavily polluted and one relatively unpolluted. Water pollution was determined by chemical analysis on tadpoles. The specific activities of some enzymes (glucose-6-phosphate dehydrogenase (G6PDH), acid and alkaline phosphatases (AcPase and AlkPase), succinic dehydrogenase (SDH), and catalase) were studied in the liver of adult frogs to identify the possible changes induced by contamination in the metabolic processes which depend on the function of the liver. The production of reactive oxygen species (ROS) were also evaluated through histochemical techniques. In the polluted samples, hepatocytes showed variations in the activity of G6PDH, AlkPase, and SDH and a moderate to intense ROS expression. Prominent changes were observed in Kupffer cells (KCs) and melanomacrophages (MMPs), both showing intense reactivity for AcPase and catalase and variations in melanin content and distribution. Results thus indicate a general adaptive response of liver parenchyma to environmental pollution. The possible role of both KCs and MMPs as scavengers of foreign substances is discussed.

  3. Observations of liver cancer cells in scanning probe acoustic microscope: a preliminary study

    Science.gov (United States)

    Chen, Xiaohui; Fang, Xiaoyue; Xi, Qing; Guo, Hua; Zhang, Ning; Ding, Mingyue

    2016-04-01

    Scanning probe acoustic microscope (SPAM) can be used to acquire the morphology image as well as the non-destructive internal structures acoustic image. However, the observations of the morphology image as well as the internal structures acoustic image of liver cancer cells in SPAM are few. In this paper, we cultured 4 different types of liver cancer cells on the silicon wafer and coverslip to observe their morphology images as well as acoustic images in SPAM, and made a preliminary study of the 8 types of cells specimens (hereinafter referred to as the silicon specimens and coverslips specimens). The experimental measurement results showed that some cellular pseudopodium were observed in the morphology images of the coverslip specimens while no such cellular pseupodium were appeared in the morphology images of the silicon specimens, which concluded that the living liver cancer cells were less likely to grow on the silicon wafer. SPAM provides a rapid and sensitive visual method for studying the morphology and internal structures of the cancer cells. The proposed method can be also used to obtain the morphology and internal information in both solid and soft material wafers, such as silicon and cells, with the resolution of nanometer scale.

  4.  IL-1β siRNA adenovirus benefits liver regeneration by improving mesenchymal stem cells survival after acute liver failure.

    Science.gov (United States)

    Ma, Hucheng; Shi, Xiaolei; Yuan, Xianwen; Ding, Yitao

    2016-01-01

      Uncontrolled hapatic inflammatory response is regarded as the primary pathological mechanism of acute liver failure and impairs the regeneration of hepatocytes and stem cell grafts. Interleukin-1 plays a key role for activating immune and inflammatory response. Recently, siRNA has made quite a few progresses in treating inflammatory response. To assess the effect of IL-1? siRNA adenovirus on MSC and the therapeutic effect of MSC combined with IL-1? siRNA adenovirus in ALF. We implanted MSC or/and IL-1? siRNA adenovirus via the tail vein, using CCl4-induced ALF in a mice model. Mice were sacrificed at different time points. Blood samples and liver tissues were collected. Hepatic injury, liver regeneration, cytokines (CXCL1, IL-1?, IL-10, IL-6, VEGF and HGF), animal survival and vital MSC were assessed after cell transplantation. MSC combined with IL-1? siRNA reduced the inflammatory levels and prevented liver failure. These animals administrated with MSC and IL-1? siRNA also exhibited improved liver regeneration and increased survival rates. Immunohistochemistry and fluorescence microscopy revealed the number of vital MSC in ALF + MSC + IL-1? siRNA group were significantly more than that in ALF + MSC group. IL-1? siRNA adenovirus could enhance MSC ability of tissue regeneration through increasing its survival rate. Accordingly, combination of IL-1? siRNA adenovirus and MSC had a synergistic effect on acute liver failure.

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

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    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.

  6. Immunization with radiation-attenuated Plasmodium berghei sporozoites induces liver cCD8alpha+DC that activate CD8+T cells against liver-stage malaria.

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    Ousman Jobe

    Full Text Available Immunization with radiation (gamma-attenuated Plasmodia sporozoites (gamma-spz confers sterile and long-lasting immunity against malaria liver-stage infection. In the P. berghei gamma-spz model, protection is linked to liver CD8+ T cells that express an effector/memory (T(EM phenotype, (CD44(hiCD45RB(loCD62L(lo, and produce IFN-gamma. However, neither the antigen presenting cells (APC that activate these CD8+ T(EM cells nor the site of their induction have been fully investigated. Because conventional (cCD8alpha+ DC (a subset of CD11c+ DC are considered the major inducers of CD8+ T cells, in this study we focused primarily on cCD8alpha+ DC from livers of mice immunized with Pb gamma-spz and asked whether the cCD8alpha+ DC might be involved in the activation of CD8+ T(EM cells. We demonstrate that multiple exposures of mice to Pb gamma-spz lead to a progressive and nearly concurrent accumulation in the liver but not the spleen of both the CD11c+NK1.1(- DC and CD8+ T(EM cells. Upon adoptive transfer, liver CD11c+NK1.1(- DC from Pb gamma-spz-immunized mice induced protective immunity against sporozoite challenge. Moreover, in an in vitro system, liver cCD8alpha(+ DC induced naïve CD8+ T cells to express the CD8+ T(EM phenotype and to secrete IFN-gamma. The in vitro induction of functional CD8+ T(EM cells by cCD8alpha+ DC was inhibited by anti-MHC class I and anti-IL-12 mAbs. These data suggest that liver cCD8alpha+ DC present liver-stage antigens to activate CD8+ T(EM cells, the pre-eminent effectors against pre-erythrocytic malaria. These results provide important implications towards a design of anti-malaria vaccines.

  7. Comparison of Liver Cell Models Using the Basel Phenotyping Cocktail.

    Science.gov (United States)

    Berger, Benjamin; Donzelli, Massimiliano; Maseneni, Swarna; Boess, Franziska; Roth, Adrian; Krähenbühl, Stephan; Haschke, Manuel

    2016-01-01

    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 suitability 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 four-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20 μM 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 four-fold (CYP3A4) for HepG2. In 3D-PHH at least a two-fold increase 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 assess 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.

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

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    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.

  9. A CD13-targeting peptide integrated protein inhibits human liver cancer growth by killing cancer stem cells and suppressing angiogenesis.

    Science.gov (United States)

    Zheng, Yan-Bo; Gong, Jian-Hua; Liu, Xiu-Jun; Li, Yi; Zhen, Yong-Su

    2017-05-01

    CD13 is a marker of angiogenic endothelial cells, and recently it is proved to be a biomarker of human liver cancer stem cells (CSCs). Herein, the therapeutic effects of NGR-LDP-AE, a fusion protein composed of CD13-targeting peptide NGR and antitumor antibiotic lidamycin, on human liver cancer and its mechanism were studied. Western blot and immunofluorescence assay demonstrated that CD13 (WM15 epitope) was expressed in both human liver cancer cell lines and vascular endothelial cells, while absent in normal liver cells. MTT assay showed that NGR-LDP-AE displayed potent cytotoxicity to cultured tumor cell lines with IC50 values at low nanomolar level. NGR-LDP-AE inhibited tumorsphere formation of liver cancer cells, and the IC50 values were much lower than that in MTT assay, indicating selectively killing of CSCs. In endothelial tube formation assay, NGR-LDP-AE at low cytotoxic dose significantly inhibited the formation of intact tube networks. Animal experiment demonstrated that NGR-LDP-AE inhibited the growth of human liver cancer xenograft. Immunohistochemical analysis showed that NGR-LDP-AE induced the down-regulation of CD13. In vitro experiment using cultured tumor cells also confirmed this result. NGR-LDP-AE activated both apoptotic and autophagic pathways in cultured tumor cells, while the induced autophagy protected cells from death. Conclusively, NGR-LDP-AE exerts its antitumor activity via killing liver CSCs and inhibiting angiogenesis. With one targeting motif, NGR-LDP-AE acts on both liver CSCs and angiogenic endothelial cells. It is a promising dual targeting fusion protein for liver cancer therapy, especially for advanced or relapsed cancers. © 2017 Wiley Periodicals, Inc.

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

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    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.

  11. Short-Chain Flavor Ester Synthesis in Organic Media by an E. coli Whole-Cell Biocatalyst Expressing a Newly Characterized Heterologous Lipase

    Science.gov (United States)

    Brault, Guillaume; Shareck, François; Hurtubise, Yves; Lépine, François; Doucet, Nicolas

    2014-01-01

    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. PMID:24670408

  12. Bone marrow derived stem cells for the treatment of end-stage liver disease.

    Science.gov (United States)

    Margini, Cristina; Vukotic, Ranka; Brodosi, Lucia; Bernardi, Mauro; Andreone, Pietro

    2014-07-21

    End-stage disease due to liver cirrhosis is an important cause of death worldwide. Cirrhosis results from progressive, extensive fibrosis and impaired hepatocyte regeneration. The only curative treatment is liver transplantation, but due to the several limitations of this procedure, the interest in alternative therapeutic strategies is increasing. In particular, the potential of bone marrow stem cell (BMSC) therapy in cirrhosis has been explored in different trials. In this article, we evaluate the results of 18 prospective clinical trials, and we provide a descriptive overview of recent advances in the research on hepatic regenerative medicine. The main message from the currently available data in the literature is that BMSC therapy is extremely promising in the context of liver cirrhosis. However, its application should be further explored in randomized, controlled trials with large cohorts and long follow-ups.

  13. Liver Cancer Cell of Origin, Molecular Class, and Effects on Patient Prognosis.

    Science.gov (United States)

    Sia, Daniela; Villanueva, Augusto; Friedman, Scott L; Llovet, Josep M

    2017-03-01

    Primary liver cancer is the second leading cause of cancer-related death worldwide and therefore a major public health challenge. We review hypotheses of the cell of origin of liver tumorigenesis and clarify the classes of liver cancer based on molecular features and how they affect patient prognosis. Primary liver cancer comprises hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (iCCA), and other rare tumors, notably fibrolamellar carcinoma and hepatoblastoma. The molecular and clinical features of HCC versus iCCA are distinct, but these conditions have overlapping risk factors and pathways of oncogenesis. A better understanding of the cell types originating liver cancer can aid in exploring molecular mechanisms of carcinogenesis and therapeutic options. Molecular studies have identified adult hepatocytes as the cell of origin. These cells have been proposed to transform directly into HCC cells (via a sequence of genetic alterations), to dedifferentiate into hepatocyte precursor cells (which then become HCC cells that express progenitor cell markers), or to transdifferentiate into biliary-like cells (which give rise to iCCA). Alternatively, progenitor cells also give rise to HCCs and iCCAs with markers of progenitor cells. Advances in genome profiling and next-generation sequencing have led to the classification of HCCs based on molecular features and assigned them to categories such as proliferation-progenitor, proliferation-transforming growth factor β, and Wnt-catenin β1. iCCAs have been assigned to categories of proliferation and inflammation. Overall, proliferation subclasses are associated with a more aggressive phenotype and poor outcome of patients, although more specific signatures have refined our prognostic abilities. Analyses of genetic alterations have identified those that might be targeted therapeutically, such as fusions in the FGFR2 gene and mutations in genes encoding isocitrate dehydrogenases (in approximately 60% of iCCAs) or

  14. Characterization of CD133+ parenchymal cells in the liver: Histology and culture

    Science.gov (United States)

    Yoshikawa, Seiichi; Zen, Yoh; Fujii, Takahiko; Sato, Yasunori; Ohta, Tetsuo; Aoyagi, Yutaka; Nakanuma, Yasuni

    2009-01-01

    AIM: To reveal the characteristics of CD133+ cells in the liver. METHODS: This study examined the histological characteristics of CD133+ cells in non-neoplastic and neoplastic liver tissues by immunostaining, and also analyzed the biological characteristics of CD133+ cells derived from human hepatocellular carcinoma (HCC) or cholangiocarcinoma cell lines. RESULTS: Immunostaining revealed constant expression of CD133 in non-neoplastic and neoplastic biliary epithelium, and these cells had the immunophenotype CD133+/CK19+/HepPar-1-. A small number of CD133+/CK19-/HepPar-1+ cells were also identified in HCC and combined hepatocellular and cholangiocarcinoma. In addition, small ductal structures, resembling the canal of Hering, partly surrounded by hepatocytes were positive for CD133. CD133 expression was observed in three HCC (HuH7, PLC5 and HepG2) and two cholangiocarcinoma cell lines (HuCCT1 and CCKS1). Fluorescence-activated cell sorting (FACS) revealed that CD133+ and CD133- cells derived from HuH7 and HuCCT1 cells similarly produced CD133+ and CD133- cells during subculture. To examine the relationship between CD133+ cells and the side population (SP) phenotype, FACS was performed using Hoechst 33342 and a monoclonal antibody against CD133. The ratios of CD133+/CD133- cells were almost identical in the SP and non-SP in HuH7. In addition, four different cellular populations (SP/CD133+, SP/CD133-, non-SP/CD133+, and non-SP/CD133-) could similarly produce CD133+ and CD133- cells during subculture. CONCLUSION: This study revealed that CD133 could be a biliary and progenitor cell marker in vivo. However, CD133 alone is not sufficient to detect tumor-initiating cells in cell lines. PMID:19842219

  15. Triple Staining Including FOXA2 Identifies Stem Cell Lineages Undergoing Hepatic and Biliary Differentiation in Cirrhotic Human Liver.

    Science.gov (United States)

    Rogler, Charles E; Bebawee, Remon; Matarlo, Joe; Locker, Joseph; Pattamanuch, Nicole; Gupta, Sanjeev; Rogler, Leslie E

    2017-01-01

    Recent investigations have reported many markers associated with human liver stem/progenitor cells, "oval cells," and identified "niches" in diseased livers where stem cells occur. However, there has remained a need to identify entire lineages of stem cells as they differentiate into bile ducts or hepatocytes. We have used combined immunohistochemical staining for a marker of hepatic commitment and specification (FOXA2 [Forkhead box A2]), hepatocyte maturation (Albumin and HepPar1), and features of bile ducts (CK19 [cytokeratin 19]) to identify lineages of stem cells differentiating toward the hepatocytic or bile ductular compartments of end-stage cirrhotic human liver. We identified large clusters of disorganized, FOXA2 expressing, oval cells in localized liver regions surrounded by fibrotic matrix, designated as "micro-niches." Specific FOXA2-positive cells within the micro-niches organize into primitive duct structures that support both hepatocytic and bile ductular differentiation enabling identification of entire lineages of cells forming the two types of structures. We also detected expression of hsa-miR-122 in primitive ductular reactions expected for hepatocytic differentiation and hsa-miR-23b cluster expression that drives liver cell fate decisions in cells undergoing lineage commitment. Our data establish the foundation for a mechanistic hypothesis on how stem cell lineages progress in specialized micro-niches in cirrhotic end-stage liver disease.

  16. INTRACELLULAR DISTRIBUTION OF ALKALINE PHOSPHATASE IN RAT LIVER CELLS

    Science.gov (United States)

    Emery, Arthur J.; Dounce, Alexander L.

    1955-01-01

    1. Cytochemical studies of the intracellular distribution of alkaline phosphatase in rat liver have been made, using a fractionation procedure recently developed in this laboratory (8) and a similar but modified method not described previously. Aqueous media were used in both cases. 2. The alkaline phosphatase was found to consist of two forms, one of which is strongly activated by magnesium and one of which is not sensitive to this metal. 3. The form of the enzyme that is not activated by magnesium occurs mainly in the nuclear fraction, where it seems to be rather firmly bound. Some of this form of the enzyme is also found in the microsomes, but very little if any occurs in the soluble supernatant fraction. 4. The form of alkaline phosphatase which is activated by magnesium occurs mainly in the soluble supernatant fraction, but what is believed are significant amounts also occur in nuclei. A significant portion of this form of the enzyme can be extracted from the isolated nuclei with cold, isotonic saline solution. Some activity of this form of the enzyme is also found in the microsomal fraction. 5. Mitochondria appear to contain relatively little alkaline phosphatase of either kind. 6. The concept of a porous nuclear membrane has been invoked to explain some of the results obtained in this work. It is postulated that part at least of the form of the enzyme that is activated by magnesium is free to diffuse back and forth through pores in the nuclear membrane, whereas this is considered not to be possible for the form of the enzyme that is insensitive to magnesium as a result of the firm binding of the latter to nuclear substance. PMID:13242596

  17. Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells.

    Science.gov (United States)

    Gesell, Andreas; Chávez, Maria Luisa Díaz; Kramell, Robert; Piotrowski, Markus; Macheroux, Peter; Kutchan, Toni M

    2011-06-01

    Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

  18. The nitric oxide donor S-nitrosoglutathione reduces apoptotic primary liver cell loss in a three-dimensional perfusion bioreactor culture model developed for liver support.

    Science.gov (United States)

    Prince, Jose M; Vodovotz, Yoram; Baun, Matthew J; Monga, Satdarshan Pal; Billiar, Timothy R; Gerlach, Jörg C

    2010-03-01

    Artificial extracorporeal support for hepatic failure has met with limited clinical success. In hepatocytes, nitric oxide (NO) functions as an antiapoptotic modulator in response to a variety of stresses. We hypothesized that NO administration would yield improved viability and hepatocellular restructuring in a four-compartment, hollow fiber-based bioreactor with integral oxygenation for dynamic three-dimensional perfusion of hepatic cells in bioartificial liver support systems. Isolated adult rat liver cells were placed in culture medium alone (control) or medium supplemented with various concentrations of an NO donor (S-nitrosoglutathione [GSNO]) in the bioreactors. Media samples were obtained from the cell perfusion circuit to monitor cellular response. After 24 and 72 h, histology biopsies were taken to investigate spontaneous restructuring of the cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to quantify apoptotic nuclei. Control bioreactors exhibited 47.9 +/- 2.9% (mean +/- standard error of the mean) apoptotic nuclei. In contrast, NO-treated bioreactors exhibited a biphasic response. Fewer apoptotic nuclei were seen in the 200 and 500 microM GSNO groups (14.4 +/- 0.4%). No effect was observed in the 10 microM GSNO group (47.3%), and increased TUNEL staining was observed in the 1000 microM GSNO group (82.6%). Media lactate dehydrogenase levels were lower in bioreactor groups treated with 200 or 500 microM GSNO (310 +/- 38 IU/L) compared with the control group (919 +/- 188 IU/L; p bioreactors at 24 h vs. 110 +/- 13 in controls; p = 0.851). Histologically, all of the bioreactor groups exhibited liver cell aggregates with some attached to the bioreactor capillaries. Increased numbers of cells in the aggregates and superior spontaneous restructuring of the cells were seen at 24 and 72 h in the bioreactor groups treated with either 200 or 500 microM GSNO compared with the control groups. Addition of an NO donor

  19. Liver Panel

    Science.gov (United States)

    ... Analysis Kidney Stone Risk Panel KRAS Mutation Lactate Lactate Dehydrogenase (LD) Lactoferrin Lactose Tolerance Tests LDL Cholesterol LDL ... GGT) – another enzyme found mainly in liver cells Lactate dehydrogenase (LD) – an enzyme released with cell damage; found ...

  20. Liver sinusoidal endothelial cells and acute non-oxidative hepatic injury induced by Pseudomonas aeruginosa pyocyanin.

    Science.gov (United States)

    Cheluvappa, Rajkumar; Cogger, Victoria C; Kwun, Sun Young; O'Reilly, Jennifer N; Le Couteur, David G; Hilmer, Sarah N

    2008-12-01

    The liver sinusoidal endothelial cell (LSEC) is damaged by many toxins, including oxidants and bacterial toxins. Any effect on LSECs of the Pseudomonas aeruginosa virulence factor, pyocyanin, may be relevant for systemic pseudomonal infections and liver transplantation. In this study, the effects of pyocyanin on in vivo rat livers and isolated LSECs were assessed using electron microscopy, immunohistochemistry and biochemistry. In particular, the effect on fenestrations, a crucial morphological aspect of LSECs was assessed. Pyocyanin treatment induced a dose-dependent reduction in fenestrations in isolated LSECs. In the intact liver, intraportal injection of pyocyanin (11.9 microM in blood) was associated with a reduction in endothelial porosity from 3.4 +/- 0.2% (n = 5) to 1.3 +/- 0.1% (n = 7) within 30 min. There were decreases in both diameter and frequency of fenestrations in the intact endothelium. There was also a decrease in endothelial thickness from 175.8 +/- 5.8 to 156.5 +/- 4.0 nm, an endothelial pathology finding previously unreported. Hepatocyte ultrastructure, liver function tests and immunohistochemical markers of oxidative stress (3-nitrotyrosine and malondialdehyde) were not affected. Pyocyanin induces significant ultrastructural changes in the LSEC in the absence of immunohistochemical evidence of oxidative stress or hepatocyte injury pointing to a novel mechanism for pyocyanin pathogenesis.

  1. Inhibitory effects of capsaicinoids on fatty acid desaturation in a rat liver cell line.

    Science.gov (United States)

    Nakano, N; Shirasaka, N; Masuoka, K; Murakami, T; Watanabe, T; Kobata, K; Shimizu, S; Yoshizumi, H

    2001-08-01

    The inhibitory effects of such vanillylamides as capsaicin and nine capsaicinoids on fatty acid desaturation in liver cells were investigated by using the cultured rat liver cell line, BRL-3A. When capsaicin was added to the medium, it had a relatively strong inhibitory effect on delta6 desaturation and clear inhibitory effects on delta5 and C24delta16 desaturation (delta16 desaturation of C24-polyunsaturated fatty acids). Capsaicinoids with side carbon chain lengths of C10:0 and C12:0 expressed the maximum inhibitory effects of the nine capsaicinoids on fatty acid desaturation in the BRL-3A cells. The inhibitory effects of the capsaicinoids were not correlated with their pungency.

  2. Anti-Erythropoietin Antibody Associated Pure Red Cell Aplasia Resolved after Liver Transplantation.

    Science.gov (United States)

    Hung, Annie K; Guy, Jennifer; Behler, Caroline M; Lee, Eugene E

    2015-01-01

    Patients undergoing antiviral therapy for chronic hepatitis C often develop anemia secondary to ribavirin and interferon. Recombinant erythropoietin has been used to improve anemia associated with antiviral therapy and to minimize dose reductions, which are associated with decreased rates of sustained virologic response. A rare potential side effect of recombinant erythropoietin is anti-erythropoietin antibody associated pure red cell aplasia. In chronic kidney disease patients with this entity, there have been good outcomes associated with renal transplant and subsequent immunosuppression. In this case, a chronic liver disease patient developed anti-erythropoietin associated pure red cell aplasia and recovered after liver transplantation and immunosuppression. It is unclear whether it is the transplanted organ, the subsequent immunosuppression, or the combination that contributed to the response. In conclusion, anti-erythropoietin associated pure red cell aplasia is a serious complication of erythropoietin therapy, but this entity should not be considered a contraindication for solid organ transplantation.

  3. Anti-Erythropoietin Antibody Associated Pure Red Cell Aplasia Resolved after Liver Transplantation

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    Annie K. Hung

    2015-01-01

    Full Text Available Patients undergoing antiviral therapy for chronic hepatitis C often develop anemia secondary to ribavirin and interferon. Recombinant erythropoietin has been used to improve anemia associated with antiviral therapy and to minimize dose reductions, which are associated with decreased rates of sustained virologic response. A rare potential side effect of recombinant erythropoietin is anti-erythropoietin antibody associated pure red cell aplasia. In chronic kidney disease patients with this entity, there have been good outcomes associated with renal transplant and subsequent immunosuppression. In this case, a chronic liver disease patient developed anti-erythropoietin associated pure red cell aplasia and recovered after liver transplantation and immunosuppression. It is unclear whether it is the transplanted organ, the subsequent immunosuppression, or the combination that contributed to the response. In conclusion, anti-erythropoietin associated pure red cell aplasia is a serious complication of erythropoietin therapy, but this entity should not be considered a contraindication for solid organ transplantation.

  4. Genomic instability in liver cells caused by an LPS-induced bystander-like effect.

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    Igor Kovalchuk

    Full Text Available Bacterial infection has been linked to carcinogenesis, however, there is lack of knowledge of molecular mechanisms that associate infection with the development of cancer. We analyzed possible effects of the consumption of heat-killed E. coli O157:H7 cells or its cellular components, DNA, RNA, protein or lipopolysaccharides (LPS on gene expression in naïve liver cells. Four week old mice were provided water supplemented with whole heat-killed bacteria or bacterial components for a two week period. One group of animals was sacrificed immediately, whereas another group was allowed to consume uncontaminated tap water for an additional two weeks, and liver samples were collected, post mortem. Liver cells responded to exposure of whole heat-killed bacteria and LPS with alteration in γH2AX levels and levels of proteins involved in proliferation, DNA methylation (MeCP2, DNMT1, DNMT3A and 3B or DNA repair (APE1 and KU70 as well as with changes in the expression of genes involved in stress response, cell cycle control and bile acid biosynthesis. Other bacterial components analysed in this study did not lead to any significant changes in the tested molecular parameters. This study suggests that lipopolysaccharides are a major component of Gram-negative bacteria that induce molecular changes within naïve cells of the host.

  5. Allicin induces p53-mediated autophagy in Hep G2 human liver cancer cells.

    Science.gov (United States)

    Chu, Yung-Lin; Ho, Chi-Tang; Chung, Jing-Gung; Rajasekaran, Raghu; Sheen, Lee-Yan

    2012-08-29

    Garlic has been used throughout history for both culinary and medicinal purpose. Allicin is a major component of crushed garlic. Although it is sensitive to heat and light and easily metabolized into various compounds such as diallyl disulfide, diallyl trisulfide, and diallyl sulfide, allicin is still a major bioactive compound of crushed garlic. The mortality of hepatocellular carcinoma is quite high and ranks among the top 10 cancer-related deaths in Taiwan. Although numerous studies have shown the cancer-preventive properties of garlic and its components, there is no study on the effect of allicin on the growth of human liver cancer cells. In this study, we focused on allicin-induced autophagic cell death in human liver cancer Hep G2 cells. Our results indicated that allicin induced p53-mediated autophagy and inhibited the viability of human hepatocellular carcinoma cell lines. Using Western blotting, we observed that allicin decreased the level of cytoplasmic p53, the PI3K/mTOR signaling pathway, and the level of Bcl-2 and increased the expression of AMPK/TSC2 and Beclin-1 signaling pathways in Hep G2 cells. In addition, the colocalization of LC3-II with MitoTracker-Red (labeling mitochondria), resulting in allicin-induced degradation of mitochondria, could be observed by confocal laser microscopy. In conclusion, allicin of garlic shows great potential as a novel chemopreventive agent for the prevention of liver cancer.

  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

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    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. Role of Cortico-Cancellous Heterologous Bone in Human Periodontal Ligament Stem Cell Xeno-Free Culture Studied by Synchrotron Radiation Phase-Contrast Microtomography.

    Science.gov (United States)

    Mazzoni, Serena; Mohammadi, Sara; Tromba, Giuliana; Diomede, Francesca; Piattelli, Adriano; Trubiani, Oriana; Giuliani, Alessandra

    2017-02-10

    This study was designed to quantitatively demonstrate via three-dimensional (3D) images, through the Synchrotron Radiation Phase-Contrast Microtomography (SR-PhC-MicroCT), the osteoinductive properties of a cortico-cancellous scaffold (Osteobiol Dual Block-DB) cultured with human Periodontal Ligament Stem Cells (hPDLSCs) in xeno-free media. In vitro cultures of hPDLSCs, obtained from alveolar crest and horizontal fibers of the periodontal ligament, were seeded onto DB scaffolds and cultured in xeno-free media for three weeks. 3D images were obtained by SR-PhC-microCT after one and three weeks from culture beginning. MicroCT data were successively processed with a phase-retrieval algorithm based on the Transport of Intensity Equation (TIE). The chosen experimental method, previously demonstratively applied for the 3D characterization of the same constructs in not xeno-free media, quantitatively monitored also in this case the early stages of bone formation in basal and differentiating conditions. Interestingly, it quantitatively showed in the xeno-free environment a significant acceleration of the mineralization process, regardless of the culture (basal/differentiating) medium. This work showed in 3D that the DB guides the osteogenic differentiation of hPDLSCs in xeno-free cultures, in agreement with 2D observations and functional studies previously performed by some of the authors. Indeed, here we fully proved in 3D that expanded hPDLSCs, using xeno-free media formulation, not only provide the basis for Good Manufacturing Practice (preserving the stem cells' morphological features and their ability to differentiate into mesenchymal lineage) but have to be considered, combined to DB scaffolds, as interesting candidates for potential clinical use in new custom made tissue-engineered constructs.

  9. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

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    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

  10. 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.

  11. Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes

    Science.gov (United States)

    Yovchev, Mladen I.; Xue, Yuhua; Shafritz, David A.; Locker, Joseph; Oertel, Michael

    2013-01-01

    Background & Aim Considerable progress has been made in developing anti-fibrotic agents and other strategies to treat liver fibrosis; however, significant long-term restoration of functional liver mass has not yet been achieved. Therefore, we investigated whether transplanted hepatic stem/progenitor cells can effectively repopulate the liver with advanced fibrosis/cirrhosis. Methods Stem/progenitor cells derived from fetal livers or mature hepatocytes from DPPIV+ F344 rats were transplanted into DPPIV− rats with thioacetamide (TAA)-induced fibrosis/cirrhosis; rats were sacrificed 1, 2, or 4 months later. Liver tissues were analyzed by histochemistry, hydroxyproline determination, RT-PCR, and immunohistochemistry. Results After chronic TAA administration, DPPIV− F344 rats exhibited progressive fibrosis, cirrhosis and severe hepatocyte damage. Besides stellate cell activation, increased numbers of stem/progenitor cells (Dlk-1+, AFP+, CD133+, Sox-9+, FoxJ1+) were observed. In conjunction with partial hepatectomy (PH), transplanted stem/progenitor cells engrafted, proliferated competitively compared to host hepatocytes, differentiated into hepatocytic and biliary epithelial cells, and generated new liver mass with extensive long-term liver repopulation (40.8 ± 10.3%). Remarkably, more than 20% liver repopulation was achieved in the absence of PH, associated with reduced fibrogenic activity (e.g., expression of α-SMA, PDGFRβ, desmin, vimentin, TIMP1) and fibrosis (reduced collagen). Furthermore, hepatocytes can also replace liver mass with advanced fibrosis/cirrhosis, but to a lesser extent than FLSPCs. Conclusions This study is a Proof of Principle demonstration that transplanted epithelial stem/progenitor cells can restore injured parenchyma in a liver environment with advanced fibrosis/cirrhosis and exhibit anti-fibrotic effects. PMID:23840008

  12. Modulation of RANTES expression by HCV core protein in liver derived cell lines

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    Rapicetta Maria

    2007-06-01

    Full Text Available Abstract Background Hepatitis C virus (HCV infection is associated with high percentage of chronicity which implies the ability of the virus to evade or modulate host cell immune system. Modulation of chemokines, such as RANTES may be part of the virus induced pathogenicity. We examined the effect of core and structural proteins of HCV on RANTES expression in two liver derived cell lines, HepG2 and Chang Liver (CHL. Methods HepG2 and Chang Liver (CHL cell lines were established and selected for constitutive expression of HCV core and structural genes. Flow cytometry and quantitative RT-PCR analysis were performed to examine the effect of HCV core protein on RANTES expression. Luciferase analysis after RANTES-Luc-promoter transfection of established cell lines was assayed by luminometer measurements (RLU of RANTES promoter activity. IRF-1 and IRF-7 expression was then examined by immunoblotting analysis. Results Results of flow cytometry and RT-PCR analysis indicated that RANTES is differentially regulated by HCV core protein in the two cell lines examined as its expression was inhibited in HepG2 cells, by a reduction of RANTES promoter activity. Conversely, RANTES protein and mRNA were induced by the core protein in CHL cells, through the induction of the promoter. Since HCV genome modulates IRF-1 and IRF-7 in replicon system and IRF-1, IRF-3 and IRF-7 have been reported to regulate RANTES promoter in various cell systems, analysis of the mechanism underlying RANTES modulation by the core protein revealed that IRF-1 expression was induced in HepG2 cells by the core protein, whereas in CHL cells it was expressed at a very low level that was not influenced by transfection with the core protein construct. This suggested that IRF-1 level may mediate the expression of RANTES in cell lines of liver origin. The effect of the core protein on RANTES promoter was countered by co-transfection with NF90, a double-stranded-RNA binding protein that activates

  13. Noncoding RNAs in liver cancer stem cells: The big impact of little things.

    Science.gov (United States)

    Lv, Hongwei; Lv, Guishuai; Han, Qin; Yang, Wen; Wang, Hongyang

    2018-04-01

    The high rate of recurrence and heterogeneity make hepatocellular carcinoma (HCC) as one of the most prevalent malignancy worldwide. Cancer stem cells (CSCs), also known as tumor-initiating cells, are responsible for cancer initiation, metastasis, drug resistance and relapse, accounting for the heterogeneous and hierarchical organizations of HCC. Therefore, CSCs are novel targets for more efficient liver cancer therapies. New data continues to mount on the functions of noncoding RNAs in multiple processes of liver CSCs, including malignant proliferation, self-renewal, tumorigenicity, and survival. Thus, it is highly critical to develop improved understanding of the mechanisms by which noncoding RNAs control CSCs functions for developing better diagnostic and therapeutic strategies. Herein, we will shed light on the regulatory roles of noncoding RNAs, especially microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), in liver CSCs acquisition and maintenance after presenting an overview about the characteristics of liver CSCs and their origins. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Postnatal hyperoxia exposure differentially affects hepatocytes and liver haemopoietic cells in newborn rats.

    Directory of Open Access Journals (Sweden)

    Guya Diletta Marconi

    Full Text Available Premature newborns are frequently exposed to hyperoxic conditions and experimental data indicate modulation of liver metabolism by hyperoxia in the first postnatal period. Conversely, nothing is known about possible modulation of growth factors and signaling molecules involved in other hyperoxic responses and no data are available about the effects of hyperoxia in postnatal liver haematopoiesis. The aim of the study was to analyse the effects of hyperoxia in the liver tissue (hepatocytes and haemopoietic cells and to investigate possible changes in the expression of Vascular Endothelial Growth Factor (VEGF, Matrix Metalloproteinase 9 (MMP-9, Hypoxia-Inducible Factor-1α (HIF-1α, endothelial Nitric Oxide Synthase (eNOS, and Nuclear Factor-kB (NF-kB. Experimental design of the study involved exposure of newborn rats to room air (controls, 60% O2 (moderate hyperoxia, or 95% O2 (severe hyperoxia for the first two postnatal weeks. Immunohistochemical and Western blot analyses were performed. Severe hyperoxia increased hepatocyte apoptosis and MMP-9 expression and decreased VEGF expression. Reduced content in reticular fibers was found in moderate and severe hyperoxia. Some other changes were specifically produced in hepatocytes by moderate hyperoxia, i.e., upregulation of HIF-1α and downregulation of eNOS and NF-kB. Postnatal severe hyperoxia exposure increased liver haemopoiesis and upregulated the expression of VEGF (both moderate and severe hyperoxia and eNOS (severe hyperoxia in haemopoietic cells. In conclusion, our study showed different effects of hyperoxia on hepatocytes and haemopoietic cells and differential involvement of the above factors. The involvement of VEGF and eNOS in the liver haemopoietic response to hyperoxia may be hypothesized.

  15. Targeting dexamethasone to Kupffer cells: effects on liver inflammation and fibrosis in rats.

    Science.gov (United States)

    Melgert, B N; Olinga, P; Van Der Laan, J M; Weert, B; Cho, J; Schuppan, D; Groothuis, G M; Meijer, D K; Poelstra, K

    2001-10-01

    Kupffer cells (KC) play an important role in the pathogenesis of inflammatory liver diseases leading to fibrosis. Anti-inflammatory drugs are only effective when administered at high doses that may cause side effects. Therefore, dexamethasone coupled to mannosylated albumin (Dexa(5)-Man(10)-HSA) was designed by us to selectively deliver this anti-inflammatory drug to the KC. The effectiveness of Dexa(5)-Man(10)-HSA was studied both in organ cultures and fibrosis induced by bile duct ligation (BDL) in rats. Dexa(5)-Man(10)-HSA accumulated in livers of both healthy and fibrotic rats (67% +/- 5% and 70% +/- 9% of the dose, respectively) and uptake was found almost exclusively in KC. Active dexamethasone was liberated from its carrier, because Dexa(5)-Man(10)-HSA could effectively inhibit nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) release in endotoxin-activated liver slices. In vivo, however, this was associated with increased collagen I and III depositions and enhanced tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression. This was accompanied by a decreased influx of reactive oxygen species (ROS) producing cells in the livers of BDL animals treated with Dexa(5)-Man(10)-HSA as compared with untreated BDL rats. Dexa(5)-Man(10)-HSA treatment also replenished the depleted glycogen stores in hepatocytes of BDL livers. In conclusion, our studies showed selective delivery of dexamethasone to KC with Dexa(5)-Man(10)-HSA. This conjugate reduced intrahepatic ROS in vivo and TNF-alpha production in vitro and prevented glycogen depletion in vivo, indicating effective pharmacologic targeting. Dexa(5)-Man(10)-HSA, however, also accelerated fibrogenesis, which was paralleled by TIMP-1 mRNA induction. Targeting of dexamethasone to KC provides evidence for a dual role of this cell type in fibrogenesis of BDL rats.

  16. Sinusoidal endothelial cell and hepatic stellate cell phenotype correlates with stage of fibrosis in chronic liver disease in dogs.

    Science.gov (United States)

    Vince, Andrew R; Hayes, M Anthony; Jefferson, Barbara J; Stalker, Margaret J

    2016-09-01

    We evaluated the extent of hepatic fibrosis in chronic liver disease of dogs using a modification of Ishak's staging criteria for human chronic liver disease, and examined the association of stage of fibrosis with immunophenotypic markers of transdifferentiation of hepatic sinusoidal endothelial cells and hepatic stellate cells. Formalin-fixed, paraffin-embedded, hematoxylin and eosin-stained liver biopsy specimens from 45 case dogs with chronic liver disease and 55 healthy control dogs were scored for the presence and extent of fibrosis. This stage score for fibrosis strongly correlated with upregulated von Willebrand factor (vWF) expression in lobular sinusoidal endothelial cells (Spearman correlation coefficient [SCC] = 0.57, p < 0.05). Immunoreactivity for vWF factor was identified in 68.9% of case biopsies, varying in distribution from periportal to diffuse, whereas vWF immunoreactivity was identified in only 14.5% of control specimens, and was restricted to the immediate periportal sinusoids. The majority of both case and control biopsies exhibited similar prominent lobular perisinusoidal expression of alpha-smooth muscle actin (α-SMA). A minority of specimens (17.8% of case biopsies, 1.8% of control biopsies) exhibited low perisinoidal α-SMA expression, and there was a weak negative correlation between α-SMA expression and stage of fibrosis (SCC = -0.29, p = 0.0037). These results document a method for staging the severity of fibrosis in canine liver biopsies, and show a strong association between fibrosis and increased expression of vWF in hepatic sinusoidal endothelial cells. © 2016 The Author(s).

  17. Investigations on in vitro anti-carcinogenic potential of L-carnosine in liver cancer cells.

    Science.gov (United States)

    Ding, Minghui; Jiao, Guihua; Shi, Haizhou; Chen, Yanrong

    2017-07-27

    This study was carried out to investigate the anti-carcinogenic effect of L-carnosine in human carcinoma cells (SNU-423). The SNU-423 cancer cells were cultured at a density of 2 × 104 cells/well in Dulbecco modified Eagle medium. After 24 h of adherence, the cells were treated with L-carnosine (0.2 and 1 mg/mL) for 48 h. Then, cell viability was assessed by sulforhodamine assay, while mitochondrial dysfunction was measured by fluorescence microscopy using chromatin-specific dye Hoechst 33258. Intracellular levels of ROS were assayed by fluorescence spectroscopy with 2',7'-dichlorofluorescein diacetate (DCFDA). L-Carnosine significantly inhibited the growth of the SNU-423 cells (p carcinogenic effects in human liver cancer cells.

  18. PDGFRB Promotes Liver Metastasis Formation of Mesenchymal-Like Colorectal Tumor Cells

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    Ernst J.A. Steller

    2013-02-01

    Full Text Available In epithelial tumors, the platelet-derived growth factor receptor B (PDGFRB is mainly expressed by stromal cells of mesenchymal origin. Tumor cells may also acquire PDGFRB expression following epithelial-to-mesenchymal transition (EMT, which occurs during metastasis formation. Little is known about PDGFRB signaling in colorectal tumor cells. We studied the relationship between PDGFRB expression, EMT, and metastasis in human colorectal cancer (CRC cohorts by analysis of gene expression profiles. PDGFRB expression in primary CRC was correlated with short disease-free and overall survival. PDGFRB was co-expressed with genes involved in platelet activation, transforming growth factor beta (TGFB signaling, and EMT in three CRC cohorts. PDGFRB was expressed in mesenchymal-like tumor cell lines in vitro and stimulated invasion and liver metastasis formation in mice. Platelets, a major source of PDGF, preferentially bound to tumor cells in a non-activated state. Platelet activation caused robust PDGFRB tyrosine phosphorylation on tumor cells in vitro and in liver sinusoids in vivo. Platelets also release TGFB, which is a potent inducer of EMT. Inhibition of TGFB signaling in tumor cells caused partial reversion of the mesenchymal phenotype and strongly reduced PDGFRB expression and PDGF-stimulated tumor cell invasion. These results suggest that PDGFRB may contribute to the aggressive phenotype of colorectal tumors with mesenchymal properties, most likely downstream of platelet activation and TGFB signaling.

  19. Ex Vivo Transduction and Transplantation of Bone Marrow Cells for Liver Gene Delivery of α1-Antitrypsin

    OpenAIRE

    Li, Hong; Lu, Yuanqing; Witek, Rafal P.; Chang, Lung-Ji; Campbell-Thompson, Martha; Jorgensen, Marda,; Petersen, Bryon; Song, Sihong

    2010-01-01

    Adult stem cell–based gene therapy holds several unique advantages including avoidance of germline or other undesirable cell transductions. We have previously shown that liver progenitor (oval) cells can be used as a platform for liver gene delivery of human α1-antitrypsin (hAAT). However, this cell source cannot be used in humans for autologous transplantation. In the present study, we tested the feasibility of bone marrow (BM) cell–based liver gene delivery of hAAT. In vitro studies showed ...

  20. 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...

  1. 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.

  2. 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.

  3. Total intermittent Pringle maneuver during liver resection can induce intestinal epithelial cell damage and endotoxemia.

    Directory of Open Access Journals (Sweden)

    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.

  4. 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.

  5. 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.

  6. The interaction of bacterial magnetosomes and human liver cancer cells in vitro

    Science.gov (United States)

    Wang, Pingping; Chen, Chuanfang; Chen, Changyou; Li, Yue; Pan, Weidong; Song, Tao

    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.

  7. Cell Kinetics of Regenerating Liver After 70% Hepatectomy in Rats - 2-Color Flow Cytometric Analysis

    OpenAIRE

    Tamura, Jun; Tanaka, Junji; Fujita, Ken-Ichi; Yoshida, Masanori; Kasamatsu, Takayuki; Arii, Shigeki; Tobe, Takayoshi

    1992-01-01

    Two-color flow cytometric (FCM) analysis using anti-bromodeoxyuridine (BrdU) monoclonal antibody (MoAb) was used to investigate the cell kinetics of regenerating liver after 70% partial hepatectomy in rats. Three peaks were seen in DNA histograms of rat hepatocyte nuclei, corresponding to diploid(2c), tetraploid(4c), and octaploid(8c). These proportions changed in the course of regeneration which were clearly demonstrated by DNA histograms using flow cytometry. The proportion of d...

  8. effect of hepatitis-b virus co-infection on cd4 cell count and liver ...

    African Journals Online (AJOL)

    2014-06-01

    Jun 1, 2014 ... 1). Effect of Hepatitis B virus co-infection on CD4 cell count and liver function of HIV infected patients. The mean CD4 count of all the patients was 212. ±188/µl. The mean serum levels for ALT, AST, ALP, total protein and albumin for the study population were. 17±23IU/L, 25± 24IU/L, 56± 64IU/L, 75±9g/l and.

  9. Hepatic stellate cell-derived PDGFRα-enriched extracellular vesicles promote liver fibrosis in mice through SHP2.

    Science.gov (United States)

    Kostallari, Enis; Hirsova, Petra; Prasnicka, Alena; Verma, Vikas K; Yaqoob, Usman; Wongjarupong, Nicha; Roberts, Lewis R; Shah, Vijay H

    2018-01-23

    Liver fibrosis is characterized by the activation and migration of hepatic stellate cells (HSCs) followed by matrix deposition. Recently, several studies have shown the importance of extracellular vesicles (EVs) derived from liver cells, such as hepatocytes and endothelial cells, in liver pathobiology. While most of the studies describe how liver cells modulate HSC behavior, an important gap exists in the understanding of HSC-derived signals and more specifically HSC-derived EVs in liver fibrosis. Here, we investigated the molecules released through HSC-derived EVs, the mechanism of their release and the role of these EVs in fibrosis. Mass spectrometry analysis showed that platelet-derived growth factor (PDGF) receptor α (PDGFRα) was enriched in EVs derived from PDGF-BB-treated HSCs. Moreover, patients with liver fibrosis had increased PDGFRα levels in serum EVs compared to healthy individuals. Mechanistically, in vitro tyrosine720-to-phenylalanine mutation (Y720F) on PDGFRα sequence abolished enrichment of PDGFRα in EVs and redirected the receptor towards degradation. Congruently, the inhibition of Src homology 2 domain tyrosine phosphatase 2 (SHP2), the regulatory binding partner of phosphorylated Y720, also inhibited PDGFRα enrichment in EVs. EVs derived from PDGFRα-overexpressing cells promoted in vitro HSC migration and in vivo liver fibrosis. Finally, administration of SHP2 inhibitor, SHP099, to carbon tetrachloride-administered mice inhibited PDGFRα enrichment in serum EVs and reduced liver fibrosis. PDGFRα is enriched in EVs derived from PDGF-BB-treated HSCs in an SHP2-dependent manner and these PDGFRα-enriched EVs participate in development of liver fibrosis. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

  10. Silent information regulator 1 (SIRT1) ameliorates liver fibrosis via promoting activated stellate cell apoptosis and reversion.

    Science.gov (United States)

    Wu, Yuting; Liu, Xuejiao; Zhou, Qun; Huang, Cheng; Meng, Xiaoming; Xu, Fengyun; Li, Jun

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. 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...... specific growth rate of the strains was reduced by 54% and 33%, respectively, upon the introduction of the gene encoding aprotinin. Growing the strains in sequential shake flask cultivations for 250 generations led to an increased maximal specific growth rate and a decrease in the yield of aprotinin...

  12. 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

  13. Protective role of biliverdin against bile acid-induced oxidative stress in liver cells.

    Science.gov (United States)

    Gonzalez-Sanchez, Ester; Perez, Maria J; Nytofte, Nikolaj S; Briz, Oscar; Monte, Maria J; Lozano, Elisa; Serrano, Maria A; Marin, Jose J G

    2016-08-01

    The accumulation of bile acids affects mitochondria causing oxidative stress. Antioxidant defense is accepted to include biotransformation of biliverdin (BV) into bilirubin (BR) through BV reductase α (BVRα). The mutation (c.214C>A) in BLVRA results in a non-functional enzyme (mutBVRα). Consequently, homozygous carriers suffering from cholestasis develop green jaundice. Whether BVRα deficiency reduces BV-dependent protection against bile acids is a relevant question because a screening of the mut-BLVRA allele (a) in 311 individuals in Greenland revealed that this SNP was relatively frequent in the Inuit population studied (1% a/a and 4.5% A/a). In three human liver cell lines an inverse correlation between BVRα expression (HepG2>Alexander>HuH-7) and basal reactive oxygen species (ROS) levels was found, however the ability of BV to reduce oxidative stress and cell death induced by deoxycholic acid (DCA) or potassium dichromate (PDC) was similar in these cells. The transduction of BVRα or mutBVRα in human placenta JAr cells with negligible BVRα expression or the silencing of endogenous BVRα expression in liver cells had no effect on DCA-induced oxidative stress and cell death or BV-mediated cytoprotection. DCA stimulated both superoxide anion and hydrogen peroxide production, whereas BV only inhibited the latter. DCA and other dihydroxy-bile acids, but not PDC, induced up-regulation of both BVRα and heme oxygenase-1 (HO-1) in liver cells through a FXR independent and BV insensitive mechanism. In conclusion, BV exerts direct and BVRα-independent antioxidant and cytoprotective effects, whereas bile acid accumulation in cholestasis stimulates the expression of enzymes favoring the heme biotransformation into BV and BR. Copyright © 2016 Elsevier B.V. All rights reserved.

  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. Transplantation of Adipose-Derived Mesenchymal Stem Cells Efficiently Rescues Thioacetamide-Induced Acute Liver Failure in Mice.

    Science.gov (United States)

    Deng, L; Kong, X; Liu, G; Li, C; Chen, H; Hong, Z; Liu, J; Xia, J

    2016-01-01

    We aimed to investigate the efficacy of adipose-derived mesenchymal stem cell (ADMSC) transplantation in acute liver failure caused by thioacetamide in mice as well as its underlying mechanism by comparing transplantation routes. ADMSCs were isolated from inguinal fat pads of enhanced green fluorescent protein (EGFP) transgenic mice and analyzed regarding their surface markers and differentiation potential. Acute liver failure models were established by infusion of thioacetamide, and then we injected EGFP-ADMSCs or phosphate-buffered saline solution by intrasplenic or intravenous route. The restoration of biologic functions of the livers receiving transplantation was assessed by means of a variety of approaches, such as survival rates, live function parameters, histology, localization of EGFP-ADMSCs, and immunofluorescence analysis. ADMSCs were positive for CD90 and CD44 and negative for CD34 and had adipogenic and osteogenic differentiation potential. And they prevented the release of liver injury biomarkers. Transplantation via tail vein provided a significant survival benefit, but no significant differences were observed in the intrasplenic pathway and between the 2 pathways in our animal experiments. Furthermore, the transplanted cells were well integrated into injured livers and produced albumin and cytokeratin-8. Direct transplantation of ADMSCs is an effective treatment for acute liver failure rather than intrasplenic transplantation. The transplanted ADMSCs exhibit the potential to differentiate into hepatocyte-like cells in the injured livers. Thus, ADCMSCs would be a potential option for treatment of acute liver failure. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Distribution of hepatic stellate cells and their role in the development of parasitic fibrosis and liver cirrhosis in domestic animals

    Directory of Open Access Journals (Sweden)

    Kukolj Vladimir

    2015-01-01

    Full Text Available Increasing of the extracellular matrix in rats, as well as in humans, occurs as a consequence of hepatic stellate cells (HSCs activity. The objective of this work was to investigation the role of these cells in the development of fibrosis and liver cirrhosis which occurs as a consequence of infection of sheep and goats with large (Fasciola hepatica and small (Dicrocoelium dendriticum fluke. Liver samples taken from 12 cattle and 10 sheep infected under natural conditions with large and small fluke were fixed in formalin and embedded in paraffin. Paraffin clips were stained with hematoxylin- eosin and masson trichrome method, and immunohistochemical method for α-smooth muscle actin (α-SMA. All tested samples were divided into three groups according to histological criteria: livers of infected animals with the first degree of fibrosis, livers of infected animals with the second degree of fibrosis, and livers of infected animals with cirrhosis. Distribution of HSCs depended on the degree of liver fibrosis. Immunohistochemically reactive HSCs were predominantly placed in perisinusoidal space. In liver samples with cirrhosis, HSCs were placed on the periphery of pseudolobulus. Cells of a different shape and size were positive to α-SMA. HSCs play an important role in synthesis of components of extracellular matrix during the development of parasitic fibrosis and liver cirrhosis in domestic animals.

  17. 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.

  18. Biology and clinical implications of CD133{sup +} liver cancer stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Stephanie, E-mail: stefma@hku.hk [Department of Clinical Oncology, State Key Laboratory for Liver Research, LKS Faculty of Medicine, The University of Hong Kong (Hong Kong)

    2013-01-15

    Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver, accounting for 80%–90% of all liver cancers. The disease ranks as the fifth most common cancer worldwide and is the third leading cause of all cancer-associated deaths. Although advances in HCC detection and treatment have increased the likelihood of a cure at early stages of the disease, HCC remains largely incurable because of late presentation and tumor recurrence. Only 25% of HCC patients are deemed suitable for curative treatment, with the overall survival at just a few months for inoperable patients. Apart from surgical resection, loco-regional ablation and liver transplantation, current treatment protocols include conventional cytotoxic chemotherapy. But due to the highly resistant nature of the disease, the efficacy of the latter regimen is limited. The recent emergence of the cancer stem cell (CSC) concept lends insight into the explanation of why treatment with chemotherapy often may seem to be initially successful but results in not only a failure to eradicate the tumor but also possibly tumor relapse. Commonly used anti-cancer drugs in HCC work by targeting the rapidly proliferating and differentiated liver cancer cells that constitute the bulk of the tumor. However, a subset of CSCs exists within the tumor, which are more resistant and are able to survive and maintain residence after treatment, thus, growing and self-renewing to generate the development and spread of recurrent tumors in HCC. In the past few years, compelling evidence has emerged in support of the hierarchic CSC model for solid tumors, including HCC. And in particular, CD133 has drawn significant attention as a critical liver CSC marker. Understanding the characteristics and function of CD133{sup +} liver CSCs has also shed light on HCC management and treatment, including the implications for prognosis, prediction and treatment resistance. In this review, a detailed summary of the recent progress

  19. Implantation of healthy matrix-embedded endothelial cells rescues dysfunctional endothelium and ischaemic tissue in liver engraftment.

    Science.gov (United States)

    Melgar-Lesmes, Pedro; Balcells, Mercedes; Edelman, Elazer R

    2017-07-01

    Liver transplantation is limited by ischaemic injury which promotes endothelial cell and hepatocyte dysfunction and eventually organ failure. We sought to understand how endothelial state determines liver recovery after hepatectomy and engraftment. Matrix-embedded endothelial cells (MEECs) with retained healthy phenotype or control acellular matrices were implanted in direct contact with the remaining median lobe of donor mice undergoing partial hepatectomy (70%), or in the interface between the remaining median lobe and an autograft or isograft from the left lobe in hepatectomised recipient mice. Hepatic vascular architecture, DNA fragmentation and apoptosis in the median lobe and grafts, serum markers of liver damage and phenotype of macrophage and lymphocyte subsets in the liver after engraftment were analysed 7 days post-op. Healthy MEECs create a functional vascular splice in donor and recipient liver after 70% hepatectomy in mouse protecting these livers from ischaemic injury, hepatic congestion and inflammation. Macrophages recruited adjacent to the vascular nodes into the implants switched to an anti-inflammatory and regenerative profile M2. MEECs improved liver function and the rate of liver regeneration and prevented apoptosis in donor liver lobes, autologous grafts and syngeneic engraftment. Implants with healthy endothelial cells rescue liver donor and recipient endothelium and parenchyma from ischaemic injury after major hepatectomy and engraftment. This study highlights endothelial-hepatocyte crosstalk in hepatic repair and provides a promising new approach to improve regenerative medicine outcomes and liver transplantation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  20. The Role of Bone Marrow Mesenchymal Stem Cells in the Treatment of Acute Liver Failure

    Directory of Open Access Journals (Sweden)

    Shufang Yuan

    2013-01-01

    Full Text Available Objective. This study is to investigate the effects of bone marrow mesenchymal stem cell (BMSC transplantation on acute liver failure (ALF. Methods. BMSCs were separated from rat bone marrow, cultured, and identified by flow cytometry. Rat model with ALF was established by injecting D-galactosamine and lipopolysaccharide. Rats were randomly divided into the control group and BMSC transplantation group. The serum levels of alanine aminotransferase (ALT and aspartate aminotransferase (AST were measured at 24 h, 120 h, and 168 h after BMSC transplantation. Apoptosis was detected by TUNEL assay. The expression of VEGF and AFP proteins was detected by immunofluorescence. Caspase-1 and IL-18 proteins and mRNA were detected by immunohistochemistry and RT-PCR. Results. Compared with the control group, levels of ALT, AST, caspase-1 and IL-18 proteins, and mRNA in the transplantation group were significantly lower at 120 h and 168 h after BMSCs transplantation. Apoptosis was inhibited by BMSCs transplantation. The VEGF protein levels were increased with the improvement of liver function, and the AFP protein levels were increased with the deterioration of the liver function after BMSCs transplantation. Conclusions. BMSCs transplantation can improve liver function and inhibit hepatocyte apoptosis as well as promote hepatocyte proliferation in rat model with ALF.

  1. [Clinical application of mesenchymal stem cells in treatment of acute-on-chronic liver failure and related research advances].

    Science.gov (United States)

    Feng, L Y; Zhang, D Z

    2017-09-20

    Acute-on-chronic liver failure is a syndrome characterized by acute exacerbation of chronic hepatitis, organ failure, and high mortality. Clinical treatment of acute-on-chronic liver failure included comprehensive medical treatment, artificial liver support system, and liver transplantation, but such methods have their own shortcomings and patients tend to have a poor prognosis. Mesenchymal stem cells (MSCs), as a new type of cell therapy, have wide sources and are easy to extract and culture. Many studies have shown that MSC treatment not only helps to achieve a high survival rate, but also has good tolerability and safety; therefore, the clinical value of MSCs has become a hot research topic. This article reviews the clinical studies on acute-on-chronic liver failure, related mechanisms, and research advances, in order to provide a reference for future clinical trials and application.

  2. 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

    -activated protein kinase kinase (MEK) inhibitor UO126 prevented ADAM12 induction by TGF-beta, suggesting the involvement of PI3K and MEK activities. In vivo, the steady-state of both ADAM9 and ADAM12 mRNA levels was nearly undetectable in both normal livers and benign tumors and increased in hepatocellular...... 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......"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...

  3. Transplantation of human bone marrow mesenchymal stromal cells reduces liver fibrosis more effectively than Wharton?s jelly?mesenchymal stromal cells

    OpenAIRE

    Rengasamy, Mathiyazhagan; Singh, Gurbind; Fakharuzi, Noor Atiqah; Siddikuzzaman,; Balasubramanian, Sudha; Swamynathan, Priyanka; Thej, Charan; Sasidharan, Gopinath; Gupta, Pawan Kumar; Das, Anjan Kumar; Rahman, Ahmad Zuhairi Abd; Fakiruddin, Kamal Shaik; Nian, Lim Moon; Zakaria, Zubaidah; Majumdar, Anish S.

    2017-01-01

    Background Mesenchymal stromal cells (MSCs) from various tissues have shown moderate therapeutic efficacy in reversing liver fibrosis in preclinical models. Here, we compared the relative therapeutic potential of pooled, adult human bone marrow (BM)- and neonatal Wharton?s jelly (WJ)-derived MSCs to treat CCl4-induced liver fibrosis in rats. Methods Sprague-Dawley rats were injected with CCl4 for 8?weeks to induce irreversible liver fibrosis. Ex-vivo expanded, pooled human MSCs obtained from ...

  4. Innate Immune Responses in Viral Hepatitis: the role of Kupffer cells and liver-derived monocytes in shaping intrahepatic immunity in mice using the LCMV infection model

    NARCIS (Netherlands)

    D. Movita (Dowty)

    2014-01-01

    markdownabstract__Abstract__ This study was performed to elucidate the immunological role of the liver in viral hepatitis. The immune functions of the liver are shaped by the intrahepatic cells present during steady state condition, as well as the recruited immune cells during liver

  5. 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.

  6. Evaluation of a hybrid artificial liver module based on a spheroid culture system of embryonic stem cell-derived hepatic cells.

    Science.gov (United States)

    Mizumoto, Hiroshi; Hayashi, Shunsuke; Matsumoto, Kinya; Ikeda, Kaoru; Kusumi, Tomoaki; Inamori, Masakazu; Nakazawa, Kohji; Ijima, Hiroyuki; Funatsu, Kazumori; Kajiwara, Toshihisa

    2012-01-01

    Hybrid artificial liver (HAL) is an extracorporeal circulation system comprised of a bioreactor containing immobilized functional liver cells. It is expected to not only serve as a temporary liver function support system, but also to accelerate liver regeneration in recovery from hepatic failure. One of the most difficult problems in developing a hybrid artificial liver is obtaining an adequate cell source. In this study, we attempt to differentiate embryonic stem (ES) cells by hepatic lineage using a polyurethane foam (PUF)/spheroid culture in which the cultured cells spontaneously form spherical multicellular aggregates (spheroids) in the pores of the PUF. We also demonstrate the feasibility of the PUF-HAL system by comparing ES cells to primary hepatocytes in in vitro and ex vivo experiments. Mouse ES cells formed multicellular spheroids in the pores of PUF. ES cells expressed liver-specific functions (ammonia removal and albumin secretion) after treatment with the differentiation-promoting agent, sodium butyrate (SB). We designed a PUF-HAL module comprised of a cylindrical PUF block with many medium-flow capillaries for hepatic differentiation of ES cells. The PUF-HAL module cells expressed ammonia removal and albumin secretion functions after 2 weeks of SB culture. Because of high proliferative activity of ES cells and high cell density, the maximum expression level of albumin secretion function per unit volume of module was comparable to that seen in primary mouse hepatocyte culture. In the animal experiments with rats, the PUF-HAL differentiating ES cells appeared to partially contribute to recovery from liver failure. This outcome indicates that the PUF module containing differentiating ES cells may be a useful biocomponent of a hybrid artificial liver support system.

  7. Study of Silymarin and Vitamin E Protective Effects on Silver Nanoparticle Toxicity on Mice Liver Primary Cell Culture

    Directory of Open Access Journals (Sweden)

    Firouz Faedmaleki

    2016-03-01

    Full Text Available Nanotechnology is a most promising field for generating new applications in medicine, although, only few nano products are currently in use for medical purposes. A most prominent nanoproduct is nanosilver. Nano-silver has biological properties which are significant for consumer products, food technology, textiles, and medical applications (e.g. wound care products, implantable medical devices, in diagnosis, drug delivery, and imaging. For their antibacterial activity, silver nanoparticles (Ag NPs are largely used in various commercially available products. The use of nano-silver is becoming more and more widespread in medicine and related applications, and due to its increasing exposure, toxicological and environmental issues need to be raised. Cytotoxicity induced by silver nanoparticles (AgNPs and the role that oxidative stress plays in this process were demonstrated in human hepatoma cells AgNPs agglomerated in the cytoplasm and nuclei of treated cells, and they induced intracellular oxidative stress. AgNP reduced ATP content of the cell and caused damage to mitochondria and increased production of reactive oxygen species (ROS in a dose-dependent manner. Silymarin was known as a hepatoprotective agent that is used in the treatment of hepatic diseases including viral hepatitis, alcoholic liver diseases, Amanita mushroom poisoning, liver cirrhosis, toxic and drug-induced liver diseases. It promotes protein synthesis, helps in regenerating liver tissue, controls inflammation, enhances glucuronidation, and protects against glutathione depletion. Vitamin E is a well-known antioxidant and has hepatoprotective effect in liver diseases. In this study, we investigated the cytotoxic effects of Ag NPs on primary liver cells of mice. Cell viability (cytotoxicity was examined with MTT assay after primary liver cells of mice exposure to AgNPs at 1, 10, 50, 100, 150, 200, 400 ppm for 24h. AgNPs caused a concentration- dependent decrease of cell viability

  8. 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

  9. Low doses of paclitaxel enhance liver metastasis of breast cancer cells in the mouse model.

    Science.gov (United States)

    Li, Qi; Ma, Zhuang; Liu, Yinhua; Kan, Xiaoxi; Wang, Changjun; Su, Bingnan; Li, Yuchen; Zhang, Yingmei; Wang, Pingzhang; Luo, Yang; Na, Daxiang; Wang, Lanlan; Zhang, Guoying; Zhu, Xiaoxin; Wang, Lu

    2016-08-01

    Paclitaxel is the most commonly used chemotherapeutic agent in breast cancer treatment. In addition to its well-known cytotoxic effects, recent studies have shown that paclitaxel has tumor-supportive activities. Importantly, paclitaxel levels are not maintained at the effective concentration through one treatment cycle; rather, the concentration decreases during the cycle as a result of drug metabolism. Therefore, a comprehensive understanding of paclitaxel's effects requires insight into the dose-specific activities of paclitaxel and their influence on cancer cells and the host microenvironment. Here we report that a low dose of paclitaxel enhances metastasis of breast cancer cells to the liver in mouse models. We used microarray analysis to investigate gene expression patterns in invasive breast cancer cells treated with low or clinically relevant high doses of paclitaxel. We also investigated the effects of low doses of paclitaxel on cell migration, invasion and metastasis in vitro and in vivo. The results showed that low doses of paclitaxel promoted inflammation and initiated the epithelial-mesenchymal transition, which enhanced tumor cell migration and invasion in vitro. These effects could be reversed by inhibiting NF-κB. Furthermore, low doses of paclitaxel promoted liver metastasis in mouse xenografts, which correlated with changes in estrogen metabolism in the host liver. Collectively, these findings reveal the paradoxical and dose-dependent effects of paclitaxel on breast cancer cell activity, and suggest that increased consideration be given to potential adverse effects associated with low concentrations of paclitaxel during treatment. Gene expression microarray data are available in the GEO database under accession number GSE82048. © 2016 Federation of European Biochemical Societies.

  10. 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.

  11. Prolyl Oligopeptidase Inhibition Attenuates Steatosis in the L02 Human Liver Cell Line.

    Directory of Open Access Journals (Sweden)

    Da Zhou

    Full Text Available Prolyl oligopeptidase (POP is a serine endopeptidase that is widely distributed in vivo, particularly in the liver. Significant changes in functional mitochondrial proteins involved with mitochondrial oxidoreductases/transporters and nucleic acid binding proteins were observed after POP inhibition in the liver, which suggested a role of POP in regulating liver energy metabolism. Steatosis in nonalcoholic fatty liver disease (NAFLD is associated with disturbances in lipid and energy metabolism in hepatocytes. Here, we aimed to study the effect of POP on hepatocyte steatosis.The human liver cell line L02 was used to investigate the biological effects of POP. An in vitro cell model of steatosis was successfully induced with oleic acid and palmitic acid. L02 cells were also subjected to S17092 (a POP inhibitor at different concentrations for 24 or 48 h. Ac-SDKP levels and POP activity were measured to assess the rate of inhibition of POP by S17092. The POP gene and protein expression levels were detected using real-time PCR and Western blots, respectively. Oil red O staining was performed and the triglyceride levels in the L02 cells were also measured. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The expression of genes involved in lipid metabolism was detected using real-time PCR. The effects of POP inhibition on LC3B II were detected by Western blot.Compared with the control, the POP mRNA levels increased by approximately 30%, and the POP protein levels increased by almost 60% in the steatotic L02 cells. After S17092 (0.026~130 μM incubation for 24 or 48 h, cell proliferation was significantly decreased in the free fatty acid (FFA-treated cells at 26-130 μM; however, S17092 did not affect the proliferation of L02 cells after 24 h of incubation with S17092 at 0.026-65 μM without FFA treatment. S17092 treatment (13 and 26 μM also elicited no significant effect on apoptosis in normal L02 cells, but

  12. Toll-like receptor 8 agonist and bacteria trigger potent activation of innate immune cells in human liver.

    Directory of Open Access Journals (Sweden)

    Juandy Jo

    2014-06-01

    Full Text Available The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161(Bright mucosal-associated invariant T (MAIT and CD56(Bright NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.

  13. Toll-like receptor 8 agonist and bacteria trigger potent activation of innate immune cells in human liver.

    Science.gov (United States)

    Jo, Juandy; Tan, Anthony T; Ussher, James E; Sandalova, Elena; Tang, Xin-Zi; Tan-Garcia, Alfonso; To, Natalie; Hong, Michelle; Chia, Adeline; Gill, Upkar S; Kennedy, Patrick T; Tan, Kai Chah; Lee, Kang Hoe; De Libero, Gennaro; Gehring, Adam J; Willberg, Christian B; Klenerman, Paul; Bertoletti, Antonio

    2014-06-01

    The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161(Bright) mucosal-associated invariant T (MAIT) and CD56(Bright) NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.

  14. A new fluorescent probe for colorimetric and ratiometric detection of sulfur dioxide derivatives in liver cancer cells

    Science.gov (United States)

    Li, Dong-Peng; Wang, Zhao-Yang; Cui, Jie; Wang, Xin; Miao, Jun-Ying; Zhao, Bao-Xiang

    2017-03-01

    A new ratiometric fluorescent probe was constructed with hemicyanine and 7-nitrobenzofurazan for detection of sulfur dioxide derivatives (HSO3-/SO32-). The ratiometric response mode could be attributed to the efficient FRET (Förster resonance energy transfer) platform. The probe exbihited some desirable properties including fast response (within 2 minutes), good selectivity and high sensitivity. Moreover, the probe could detect endogenous HSO3- in liver cancer cells rather than normal liver cells, implying the diagnosal potential of the probe.

  15. 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.

  16. Effect of bone marrow mesenchymal stem cells transplantation on the serum and liver HMGB1 expression in rats with acute liver failure.

    Science.gov (United States)

    Zheng, Sheng; Yang, Juan; Tang, Yingmei; Yang, Jinhui; Shao, Qinghua; Guo, Ling; Liu, Qinghua

    2015-01-01

    This study aimed to investigate the effect of bone marrow mesenchymal stem cells (BMSCs) transplantation on the expression of high mobility group box 1 protein (HMGB1) in the serum and liver of rats with acute liver failure (ALF). Healthy male SD rats were randomly divided into control group, ALF group and BMSCs group. ALF was induced by intraperitoneal injection of 900 mg/kg D-GalN and 10 μg/kg LPS. In BMSCs group, rats received BMSCs (1.0×10(7)) transplantation via the tail vein at 2 h after ALF induction. Intraperitoneal injection of 900 mg/kg D-GalN and 10 μg/kg LPS was able to induce ALF in rats. In ALF group, serum ALT and AST increased gradually over time. At 72 h, the serum ALT and AST in BMSCs group were significantly different from those in ALF group. HMGB1 expression in the serum and liver remained at a low level at any time point in control group, but increased significantly in ALF group and BMSCs group. The serum and liver HMGB1 expression increased progressively in ALF group, but reduced gradually in BMSCs group. Significant difference in serum and liver HMGB1 expression was observed between ALF group and BMSCs group at 24 h and 72 h. In addition, there was marked difference in the survival rate among three groups at 24 h (χ (2) =21.098, Pliver function and liver pathology in ALF rats and decrease the serum and liver HMGB1.

  17. Combined strategies for the improvement of heterologous ...

    African Journals Online (AJOL)

    use

    2011-12-14

    Dec 14, 2011 ... The N-terminal histidine-tag brought convenience to purification. The molecular weight of His6-YlLip2 was about 38 ... Lip2 with an N-terminal of histidine-tag; PTM1, Pichia trace metals. pharmaceuticals and energy ... disadvantage to the purification process of YlLip2. In contrast, heterologous expression of ...

  18. Guided tissue regeneration with heterologous materials in primary subtalar arthrodesis: a case report.

    Science.gov (United States)

    Frangez, Igor; Kasnik, Tea; Cimerman, Matej; Smrke, Dragica Maja

    2016-05-03

    Calcaneal fractures are relatively rare and difficult to treat. Treatment options vary based on the type of fracture and the surgeon's experiences. In recent years, surgical procedures have increasingly been used due to the better long-term results. We present a case where guided tissue regeneration was performed in a calcaneal fracture that needed primary subtalar arthrodesis. We used the principles of guided tissue regeneration from oral surgery to perform primary subtalar arthrodesis and minimize the risk of non-union. We used a heterologous collagen membrane, which acts as a mechanical barrier and protects the bone graft from the invasion of unwanted cells that could lead to non-union. The collagenous membrane also has osteoconductive properties and is therefore able to increase the osteoblast proliferation rate. A 62-year-old Caucasian woman sustained multiple fractures of her lower limbs and spine after a fall from a ladder. Her left calcaneus had a comminuted multifragmental fracture (Sanders type IV) with severe destruction of the cartilage of her subtalar joint and depression of the Böhler's angle. Therefore, we performed primary arthrodesis of her subtalar joint with elevation of the Böhler's angle using a 7.3 mm titanium screw, a heterologous cortico-cancellous collagenated pre-hydrated bone mix, a heterologous cancellous collagenated bone wedge, and a heterologous collagen membrane (Tecnoss®, Italy). The graft was fully incorporated 12 weeks after the procedure and a year and a half later our patient walks without limping. We present a new use of guided tissue regeneration with heterologous materials that can be used to treat extensive bone defects after bone injuries. We believe that guided tissue regeneration using heterologous materials, including a heterologous collagen membrane that presents a mechanical barrier between soft tissues and bone as well as a stimulative component that enhances bone formation, could be more often used in bone surgery.

  19. 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

  20. 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

    2012-12-20

    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.

  1. Parallel Profiles of Inflammatory and Effector Memory T Cells in Visceral Fat and Liver of Obesity-Associated Cancer Patients.

    Science.gov (United States)

    Conroy, Melissa J; Galvin, Karen C; Doyle, Suzanne L; Kavanagh, Maria E; Mongan, Ann-Marie; Cannon, Aoife; Moore, Gillian Y; Reynolds, John V; Lysaght, Joanne

    2016-10-01

    In the midst of a worsening obesity epidemic, the incidence of obesity-associated morbidities, including cancer, diabetes, cardiac and liver disease is increasing. Insights into mechanisms underlying pathological obesity-associated inflammation are lacking. Both the omentum, the principal component of visceral fat, and liver of obese individuals are sites of excessive inflammation, but to date the T cell profiles of both compartments have not been assessed or compared in a patient cohort with obesity-associated disease. We have previously identified that omentum is enriched with inflammatory cytokines, chemokines and T cells. Here, we compared the inflammatory profile of T cells in the omentum and liver of patients with the obesity-associated malignancy oesophageal adenocarcinoma (OAC). Furthermore, we assessed the secreted cytokine profile in OAC patient serum, omentum and liver to assess systemic and local inflammation. We observed parallel T cell cytokine profiles and phenotypes in the omentum and liver of OAC patients, in particular CD69(+) and inflammatory effector memory T cells. This study reflects similar processes of inflammation and T cell activation in the omentum and liver, and may suggest common targets to modulate pathological inflammation at these sites.

  2. General Information about Liver (Hepatocellular) Cancer

    Science.gov (United States)

    ... Hepatitis C Hepatitis D Hepatitis E Hepatitis G Liver cancer is a disease in which malignant (cancer) cells ... Primary Liver Cancer Treatment Childhood Liver Cancer Treatment Liver cancer is not common in the United States. Liver ...

  3. Preservation of Cell Structure, Metabolism, and Biotransformation Activity of Liver-On-Chip Organ Models by Hypothermic Storage.

    Science.gov (United States)

    Gröger, Marko; Dinger, Julia; Kiehntopf, Michael; Peters, Frank T; Rauen, Ursula; Mosig, Alexander S

    2017-09-27

    The liver is a central organ in the metabolization of nutrition, endogenous and exogenous substances, and xenobiotic drugs. The emerging organ-on-chip technology has paved the way to model essential liver functions as well as certain aspects of liver disease in vitro in liver-on-chip models. However, a broader use of this technology in biomedical research is limited by a lack of protocols that enable the short-term preservation of preassembled liver-on-chip models for stocking or delivery to researchers outside the bioengineering community. For the first time, this study tested the ability of hypothermic storage of liver-on-chip models to preserve cell viability, tissue morphology, metabolism and biotransformation activity. In a systematic study with different preservation solutions, liver-on-chip function can be preserved for up to 2 d using a derivative of the tissue preservation solution TiProtec, containing high chloride ion concentrations and the iron chelators LK614 and deferoxamine, supplemented with polyethylene glycol (PEG). Hypothermic storage in this solution represents a promising method to preserve liver-on-chip function for at least 2 d and allows an easier access to liver-on-chip technology and its versatile and flexible use in biomedical research. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Transcriptional Regulation of Glucose Sensors in Pancreatic β-Cells and Liver: An Update

    Directory of Open Access Journals (Sweden)

    Jin-Sik Bae

    2010-05-01

    Full Text Available Pancreatic β-cells and the liver play a key role in glucose homeostasis. After a meal or in a state of hyperglycemia, glucose is transported into the β-cells or hepatocytes where it is metabolized. In the β-cells, glucose is metabolized to increase the ATP:ADP ratio, resulting in the secretion of insulin stored in the vesicle. In the hepatocytes, glucose is metabolized to CO2, fatty acids or stored as glycogen. In these cells, solute carrier family 2 (SLC2A2 and glucokinase play a key role in sensing and uptaking glucose. Dysfunction of these proteins results in the hyperglycemia which is one of the characteristics of type 2 diabetes mellitus (T2DM. Thus, studies on the molecular mechanisms of their transcriptional regulations are important in understanding pathogenesis and combating T2DM. In this paper, we will review a recent update on the progress of gene regulation of glucose sensors in the liver and β-cells.

  5. Transplantation of umbilical cord mesenchymal stem cells via different routes in rats with acute liver failure.

    Science.gov (United States)

    Zheng, Sheng; Yang, Juan; Yang, Jinhui; Tang, Yingmei; Shao, Qinghua; Guo, Ling; Liu, Qinghua

    2015-01-01

    This study aimed to compare the therapeutic efficacy of transplantation of human umbilical cord mesenchymal stem cells (hUCMSC) in different routes in acute hepatic failure (ALF) in rats. hUCMSCs were isolated and identified by detection of surface antigens via flow cytometry. In T group and H group, ALF rats received hUCMSC transplantation through the tail vein and intrahepatic injection, respectively. In hUCMSC group, healthy rats received hUCMSCs transplantation via the tail vein. In ALF group, rats received injection of normal saline through the tail vein. The TBil and ALT in ALF rats with and without transplantation were significantly higher than in healthy rats (Pcells, and liver pathology was improved in T group and H group as compared to ALF group. At 3 d after transplantation, CK18 expression was detectable in both H group and T group. At 1 w and 2 w, the mRNA expressions of CK8, CK18 and AFP in H group and T group were significantly different from those in ALF group (Pstem cells were comparable between H group and T group (P>0.05). hUCMSCs transplantation can improve the liver function and promote the liver repair following ALF. hUCMSCs transplantation via tail vein has similar therapeutic efficacy to that through intrahepatic injection.

  6. Therapy of primary and metastatic liver cancer by human iPS cell-derived myeloid cells producing interferon-β.

    Science.gov (United States)

    Sakisaka, Masataka; Haruta, Miwa; Komohara, Yoshihiro; Umemoto, Satoshi; Matsumura, Keiko; Ikeda, Tokunori; Takeya, Motohiro; Inomata, Yukihiro; Nishimura, Yasuharu; Senju, Satoru

    2017-02-01

    iPS-ML are myeloid lineage cells with a proliferative capacity derived from induced pluripotent stem (iPS) cells. This study aimed to examine therapeutic effect of iPS-ML producing interferon-β (iPS-ML/IFN-β) towards primary and metastatic liver cancer and investigate the mechanism of that effect. We established a xenograft model of liver metastasis by injecting the spleen of SCID mice with MKN-45 human gastric cancer cells and also a primary liver cancer model by injecting SK-HEP-1 human hepatocellular carcinoma cells into the liver. After cancer lesions were established, iPS-ML/IFN-β was administered by intraperitoneal injection, and therapeutic effect was evaluated. The i.p. injection of iPS-ML/IFN-β resulted in a significant retardation of cancer progression and prolonged mouse survival. The infiltration of i.p. administered iPS-ML into tumor lesions located below the liver capsule was observed, suggesting tumor-directed migration and penetration of the liver capsule by iPS-ML. The IFN-β concentration in the liver was maintained at levels sufficient to exert an anti-cancer effect for at least 3 days post-injection, accounting for the potent therapeutic effect obtained by injection two to three times per week. This study demonstrates the therapeutic potential of the iPS-ML/IFN-β in patients with liver cancer. © 2017 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

  7. Dexamethasone coupled to albumin is selectively taken up by rat nonparenchymal liver cells and attenuates LPS-induced activation of hepatic cells

    NARCIS (Netherlands)

    Melgert, BN; Olinga, P; Jack, VK; Molema, G; Meijer, Dirk; Poelstra, K

    Background/Aims: The human serum albumin (HSA) conjugate Dexa(10)-HSA was designed to specifically deliver the anti-inflammatory drug dexamethasone (Dexa) to nonparenchymal cells (NPC) in the rat liver. NPC play an important role in the pathogenesis of acute and chronic inflammatory liver diseases

  8. Multifaceted Therapeutic Benefits of Factors Derived From Dental Pulp Stem Cells for Mouse Liver Fibrosis.

    Science.gov (United States)

    Hirata, Marina; Ishigami, Masatoshi; Matsushita, Yoshihiro; Ito, Takanori; Hattori, Hisashi; Hibi, Hideharu; Goto, Hidemi; Ueda, Minoru; Yamamoto, Akihito

    2016-10-01

    : Chronic liver injury from various causes often results in liver fibrosis (LF). Although the liver possesses endogenous tissue-repairing activities, these can be overcome by sustained inflammation and excessive fibrotic scar formation. Advanced LF leads to irreversible cirrhosis and subsequent liver failure and/or hepatic cancer. Here, using the mouse carbon tetrachloride (CCl4)-induced LF model, we showed that a single intravenous administration of stem cells derived from human exfoliated deciduous teeth (SHEDs) or of SHED-derived serum-free conditioned medium (SHED-CM) resulted in fibrotic scar resolution. SHED-CM suppressed the gene expression of proinflammatory mediators, such as TNF-α, IL-1β, and iNOS, and eliminated activated hepatic stellate cells by inducing their apoptosis, but protected parenchymal hepatocytes from undergoing apoptosis. In addition, SHED-CM induced tissue-repairing macrophages that expressed high levels of the profibrinolytic factor, matrix metalloproteinase 13. Furthermore, SHED-CM suppressed the CCl4-induced apoptosis of primary cultured hepatocytes. SHED-CM contained a high level of hepatocyte growth factor (HGF). Notably, HGF-depleted SHED-CM (dHGF-CM) did not suppress the proinflammatory response or resolve fibrotic scarring. Furthermore, SHED-CM, but not dHGF-CM, inhibited CCl4-induced hepatocyte apoptosis. These results suggest that HGF plays a central role in the SHED-CM-mediated resolution of LF. Ta