Nuestros investigadores

Laura Guembe Echarri

Publicaciones científicas más recientes (desde 2010)

Autores: Argemí Ballbé, José María; Kress, T. R.; Chang, Haisul C. Y.; et al.
ISSN 0016-5085  Vol. 152  Nº 5  2017  págs. 1203 - 1216.e15
BACKGROUND & AIMS: Liver regeneration after partial hepatectomy ( PH) increases the protein folding burden at the endoplasmic reticulum of remnant hepatocytes, resulting in induction of the unfolded protein response. We investigated the role of the core unfolded protein response transcription factor X-box binding protein 1 ( XBP1) in liver regeneration using genome-wide chromatin immunoprecipitation analysis. METHODS: We performed studies with C57Bl6-J ( control) and interleukin 6-knockout mice. Mice underwent PH or sham surgeries. In some mice, hepatic expression of XBP1 was knocked down by injection of adenoviral vectors encoding small hairpin RNAs against Xbp1 messenger RNA. Liver tissues were collected before surgery and at 6 and 48 hours after surgery and analyzed by chromatin immunoprecipitation followed by sequencing. We also performed functional analyses of HepG2 cells. RESULTS: Expression of XBP1 by hepatocytes increased immediately after PH ( priming phase of liver regeneration) in control mice, but this effect was delayed in interleukin 6-deficient mice. In mice with knockdown of XBP1, we observed of liver tissue persistent endoplasmic reticulum stress, defects in acute-phase response, and increased hepatocellular damage, compared with control mice. Chromatin immunoprecipitation analyses of liver tissue showed that at 6 hours after PH, liver XBP1 became bound to a large set of genes implicated in proteostasis, the acute-phase response, metabolism, and the DNA damage response ( DDR). At this time point, XBP1 bound the promoter of the signal transducer and activator of transcription 3 gene ( Stat3). Livers of XBP1-knockdown mice showed reduced expression of STAT3 and had lower levels of STAT3 phosphorylation at Ser727, a modification that promotes cell proliferation and the DDR. Regenerating livers from XBP1-knockdown mice expressed high levels of a marker of DNA double-strand breaks, phosphorylated histone 2A, member X ( H2AX), compared with control mice. The inhibition of XBP1 expression caused a reduced up-regulation of DDR messenger RNAs in regenerating hepatocytes. CONCLUSION: In livers of mice, we found that PH induces expression of XBP1, and that this activity requires interleukin 6. XBP1 expression regulates the unfolded protein response, acute-phase response, and DDR in hepatocytes. In regenerating livers, XBP1 deficiency leads to endoplasmic reticulum stress and DNA damage.
Autores: Murillo Sauca, Oihana; Moreno Luqui, Daniel; Gazquez López, Cristina; et al.
ISSN 1600-0641  Vol. 64  Nº 2  2016  págs. 419-26
Our data demonstrate that AAV8-AAT-ATP7B-mediated gene therapy provides long-term correction of copper metabolism in a clinically relevant animal model of WD providing support for future translational studies.
Autores: Larrea Leoz, María Esther (Autor de correspondencia); Echeverria Beistegui, Itziar; Riezu Boj, José Ignacio; et al.
ISSN 0168-8278  Vol. 60  Nº 3  2014  págs. 482 - 489
BACKGROUND & AIMS: Oncostatin M (OSM) is an inflammatory cytokine which interacts with a heterodimeric receptor formed by gp130 and either OSMRß or LIFR. Here we have analysed OSM and its receptors in livers with chronic hepatitis C (CHC) and studied the factors that regulate this system. METHODS: OSM, OSM receptors and OSM-target molecules were studied by immunohistochemistry and/or qPCR analysis in livers from CHC patients and controls. We determined the production of OSM by CD40L-stimulated antigen presenting cells (APC) and its biological effects on HuH7 cells containing HCV replicon (HuH7 Core-3'). RESULTS: OSM was upregulated in livers with CHC and its production was mapped to CD11c+ cells. OSM levels correlated directly with inflammatory activity and CD40L expression. In vitro studies showed that OSM is released by APC upon interaction with activated CD4+ T cells in a CD40L-dependent manner. Culture of HuH7 Core-3' cells with supernatant from CD40L-stimulated APC repressed HCV replication and induced IL-7 and IL-15R¿. These effects were dampened by antibodies blocking OSM or gp130 and by silencing OSMRß. In CHC livers OSMRß and LIFR were significantly downregulated and their values correlated with those of OSM-induced molecules. Experiments in HuH7 cells showed that impaired STAT3 signaling and exposure to TGFß1, two findings in CHC, are factors involved in repressing OSMRß and LIFR, respectively. CONCLUSIONS: OSM is a cytokine possessing vigorous antiviral and immunostimulatory properties which is released by APC upon interaction with CD40L present on activated CD4+ T cells. In livers with CHC, OSM is overexpressed but its biological activity appears to be hampered because of downregulation of its receptor subunits.
Autores: Larrea Leoz, María Esther (Autor de correspondencia); Riezu Boj, José Ignacio; Aldabe Arregui, Rafael; et al.
Revista: GUT
ISSN 0017-5749  Vol. 63  Nº 4  2014  págs. 665 - 673
Background IL-7 and IL-15 are produced by hepatocytes and are critical for the expansion and function of CD8 T cells. IL-15 needs to be presented by IL-15R¿ for efficient stimulation of CD8 T cells. Methods We analysed the hepatic levels of IL-7, IL-15, IL-15R¿ and interferon regulatory factors (IRF) in patients with chronic hepatitis C (CHC) (78% genotype 1) and the role of IRF1 and IRF2 on IL-7 and IL-15R¿ expression in Huh7 cells with or without hepatitis C virus (HCV) replicon. Results Hepatic expression of both IL-7 and IL-15R¿, but not of IL-15, was reduced in CHC. These patients exhibited decreased hepatic IRF2 messenger RNA levels and diminished IRF2 staining in hepatocyte nuclei. We found that IRF2 controls basal expression of both IL-7 and IL-15R¿ in Huh7 cells. IRF2, but not IRF1, is downregulated in cells with HCV genotype 1b replicon and this was accompanied by decreased expression of IL-7 and IL-15R¿, a defect reversed by overexpressing IRF2. Treating Huh7 cells with IFN¿ plus oncostatin M increased IL-7 and IL-15R¿ mRNA more intensely than either cytokine alone. This effect was mediated by strong upregulation of IRF1 triggered by the combined treatment. Induction of IRF1, IL-7 and IL-15R¿ by IFN¿ plus oncostatin M was dampened in replicon cells but the combination was more effective than either cytokine alone. Conclusions HCV genotype 1 infection downregulates IRF2 in hepatocytes attenuating hepatocellular expression of IL-7 and IL-15R¿. Our data reveal a new mechanism by which HCV abrogates specific T-cell responses and point to a novel therapeutic approach to stimulate anti-HCV immunity.
Autores: Balasiddaiah, A.; Moreno Luqui, Daniel; Guembe Echarri, Laura; et al.
ISSN 1138-7548  Vol. 69  Nº 4  2013  págs. 835 - 845
Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.
Autores: Moreno Luqui, Daniel; Balasiddaiah, A.; Lamas Longarela, Óscar; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 8  Nº 9  2013  págs. e74948
It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/-) gamma c(-/-) mice using an adenovirus encoding herpes virus thymidine kinase (AdTk) and two consecutive doses of ganciclovir (GCV). We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.
Autores: Pañeda Rodríguez, María Astrid; Collantes Martínez, María; Beattie, SG; et al.
Revista: Human Gene Therapy (Print)
ISSN 1043-0342  Vol. 22  Nº 8  2011  págs.  999-1009
Autores: Riezu Boj, José Ignacio; Larrea Leoz, María Esther; Aldabe Arregui, Rafael; et al.
ISSN 0168-8278  Vol. 54  Nº 3  2011  págs. 422 - 431
Autores: Sobrevals, Luciano Matías; Rodríguez Ortigosa, Carlos Manuel; Romero-Trevejo, JL; et al.
Revista: Hepatology
ISSN 0270-9139  Vol. 51  Nº 3  2010  págs. 912 - 921
We investigated whether gene transfer of insulin-like growth factor I (IGF-I) to the hepatic tissue was able to improve liver histology and function in established liver cirrhosis. Rats with liver cirrhosis induced by carbon tetrachloride (CCl4) given orally for 8 weeks were injected through the hepatic artery with saline or with Simian virus 40 vectors encoding IGF-I (SVIGF-I), or luciferase (SVLuc). Animals were sacrificed 8 weeks after vector injection. In cirrhotic rats we observed that, whereas IGF-I was synthesized by hepatocytes, IGF-I receptor was predominantly expressed by nonparenchymal cells, mainly in fibrous septa surrounding hepatic nodules. Rats treated with SVIGF-I showed increased hepatic levels of IGF-I, improved liver function tests, and reduced fibrosis in association with diminished ¿-smooth muscle actin expression, up-regulation of matrix metalloproteases (MMPs) and decreased expression of the tissue inhibitors of MMPs TIM-1 and TIM-2. SVIGF-I therapy induced down-regulation of the profibrogenic molecules transforming growth factor beta (TGFß), amphiregulin, platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), and vascular endothelium growth factor (VEGF) and induction of the antifibrogenic and cytoprotective hepatocyte growth factor (HGF). Furthermore, SVIGF-I-treated animals showed decreased expression of Wilms tumor-1 (WT-1; a nuclear factor involved in hepatocyte dedifferentiation) and up-regulation of hepatocyte nuclear factor 4 alpha (HNF4¿) (which stimulates hepatocellular differentiation). The therapeutic potential of SVIGF-I was also tested in rats with thioacetamide-induced liver cirrhosis. Also in this model, SVIGF-I improved liver function and reduced liver fibrosis in association with up-regulation of HGF and MMPs and down-regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1). Conclusion: IGF-I gene transfer to cirrhotic livers induces MMPs and hepatoprotective factors leading to reversion of fibrosis and improvement of liver function. IGF-I gene therapy may be a useful alternative therapy for patients with advanced cirrhosis without timely access to liver transplantation.
Autores: Casares Lagar, Noelia; Rudilla Salvador, Francesc; Arribillaga Arangoa, Laura; et al.
ISSN 0022-1767  Vol. 185  Nº 9  2010  págs. 5150 - 5159
Immunosuppressive activity of regulatory T cells (Treg) may contribute to the progression of cancer or infectious diseases by preventing the induction of specific immune responses. Using a phage-displayed random peptide library, we identified a 15-mer synthetic peptide, P60, able to bind to forkhead/winged helix transcription factor 3 (FOXP3), a factor required for development and function of Treg. P60 enters the cells, inhibits FOXP3 nuclear translocation, and reduces its ability to suppress the transcription factors NF-¿B and NFAT. In vitro, P60 inhibited murine and human-derived Treg and improved effector T cell stimulation. P60 administration to newborn mice induced a lymphoproliferative autoimmune syndrome resembling the reported pathology in scurfy mice lacking functional Foxp3. However, P60 did not cause toxic effects in adult mice and, when given to BALB/c mice immunized with the cytotoxic T cell epitope AH1 from CT26 tumor cells, it induced protection against tumor implantation. Similarly, P60 improved the antiviral efficacy of a recombinant adenovirus expressing NS3 protein from hepatitis C virus. Functional inhibition of Treg by the FOXP3-inhibitory peptide P60 constitutes a strategy to enhance antitumor and antiviral immunotherapies.