Hernandez-Alcoceba, R. (Rubén)

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    Oxaliplatin in combination with liver-specific expression of interleukin 12 reduces the immunosuppressive microenvironment of tumours and eradicates metastatic colorectal cancer in mice
    (2011) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Mancheño, U. (Uxua); Alzuguren, P. (Pilar); Hervas-Stubbs, S. (Sandra); Crettaz, J. (Julien); Prieto, J. (Jesús); Medina-Echeverz, J. (José); Hernandez-Alcoceba, R. (Rubén); Gonzalez-Aparicio, M. (Manuela); Mauleon, I. (Itsaso)
    BACKGROUND AND AIMS: New options are needed for the management and prevention of colorectal cancer liver metastases. Interleukin 12 (IL-12) is an immunostimulatory cytokine with proven antitumour effect in animal models. Despite evidence indicating its biological effect in humans, neither the recombinant protein nor gene therapy vectors expressing IL-12 have shown a relevant benefit in patients with cancer. OBJECTIVE: To develop a new approach to overcome the difficulties in obtaining a suitable expression pattern and the immunosuppressive milieu in the tumours which contribute to this poor performance. METHODS: A high-capacity ('gutless') adenoviral vector carrying a liver-specific, mifepristone (Mif)-inducible system for the expression of IL-12 (HC-Ad/RUmIL-12) was used in combination with chemotherapy. Tumours were established in the liver of C57BL/6 mice by inoculation of MC38 colon cancer cells. RESULTS: Intrahepatic injection of HC-Ad/RUmIL-12 and tailored induction regimens allowed the maintenance of safe and efficient levels of IL-12 in vivo. An individualised, stepwise increase in the dose of Mif (125-4000 μg/kg) was needed to compensate for the progressive but transient downregulation of the inducible system. Repeated cycles of Mif induction (every 24 h for 10 days) were needed for optimal tumour eradication. However, complete protection against tumour rechallenge was seen in < 25% of the animals. The administration of oxaliplatin (5 mg/kg intraperitoneally) 3 days before starting the induction regimen achieved efficient elimination of liver metastases with a single cycle of IL-12 induction, and improved protection against tumour rechallenge. This was associated with a shift in the tumour microenvironment towards a more pro-immunogenic phenotype, with an increase in the CD8+/T regulatory cell ratio and a reduction in myeloid-derived suppressor cells. These effects were not seen with 5-fluorouracil, irinotecan or gemcitabine.
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    Enhanced therapeutic effect using sequential administration of antigenically distinct oncolytic viruses expressing oncostatin M in a Syrian hamster orthotopic pancreatic cancer model
    (BioMed Central, 2015) González-Aseguinolaza, G. (Gloria); Bravo-Perez, C. (Carlos); Quetglas, J.I. (José Ignacio); Larrea, E. (Esther); Poutou, J. (Joanna); Nistal-Villan, E. (Estanislao); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Carte, B. (Beatriz); Gonzalez-Aparicio, M. (Manuela)
    The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed.
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    Intrahepatic injection of adenovirus reduces inflammation and increases gene transfer and therapeutic effect in mice
    (Wiley-Blackwell, 2006) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Rooijen, N. (Nico) van; Qian, C. (Cheng); Kochanek, S. (Stefan); Barajas, M. (Miguel); Shankar, V. (Vijay); Ochoa, L. (Laura); Crettaz, J. (Julien); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Mauleon, I. (Itsaso)
    Recombinant adenoviruses (Ad) are among the most extensively used vectors for liver gene transfer. One of the major limitations for the clinical application of these vectors is the inflammatory immune response associated with systemic administration of high dose of virus. We evaluated the effect of Ad administration route on the inflammatory immune response and liver transgene expression. We compared direct intrahepatic injection (IH) with the systemic administration via tail vein (IV). IH injection of Ad resulted in a lower inflammatory response and a higher transgene expression. When a relatively low dose of virus was used, IV administration resulted in no detectable protein expression but production of proinflammatory cytokines. In contrast, IH administration induced high levels of transgene expression and no inflammation, although we detected a transient hypertransaminemia, which fully resolved within days. Furthermore, IH injection resulted in a faster protein expression being more intense at the site of injection, whereas IV administration caused slower but diffuse liver expression. IH injection also reduced the spreading of the virus to other organs. Independently of the route, depletion of Kupffer cells significantly enhanced the transduction efficiency of Ad. This effect was stronger when using IV injection, indicating that IH injection partially overcomes Kupffer cell phagocytic activity. Moreover, the antitumor efficacy of high-capacity-Ad encoding murine interleukin-12 (IL-12) was significantly greater when the vector was administered by IH injection than when given IV. In conclusion, IH injection of adenovirus represents a safe and efficient administration route for clinical applications of gene therapy targeting the liver.
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    Evaluation of monocytes as carriers for armed oncolytic adenoviruses in murine and Syrian hamster models of cancer
    (Mary Ann Liebert, 2012) Garcia-Aragoncillo, E. (Eva); Quetglas, J.I. (José Ignacio); Ortiz-de-Solorzano, C. (Carlos); Hervas-Stubbs, S. (Sandra); Prieto, J. (Jesús); Bortolanza, S. (Sergia); Hernandez-Alcoceba, R. (Rubén); Benavides, C. (Carolina); Buñuales, M. (María); Raquel
    Replication-competent (oncolytic) adenoviruses (OAV) can be adapted as vectors for the delivery of therapeutic genes, with the aim of extending the antitumor effect beyond direct cytolysis. Transgene expression using these vectors is usually intense but short-lived, and repeated administrations are hampered by the rapid appearance of neutralizing antibodies (NAbs). We have studied the performance of monocytes as cell carriers to improve transgene expression in cancer models established in athymic mice and immunocompetent Syrian hamsters. Human and hamster monocytic cell lines (MonoMac6 and HM-1, respectively) were loaded with replication-competent adenovirus-expressing luciferase. Intravenous administration of these cells caused a modest increase in transgene expression in tumor xenografts, but this effect was virtually lost in hamsters. In contrast, intratumoral administration of HM-1 cells allowed repeated cycles of expression and achieved partial protection from NAbs in preimmunized hamsters bearing pancreatic tumors. To explore the therapeutic potential of this approach, HM-1 cells were loaded with a hypoxia-inducible OAV expressing the immunostimulatory cytokine interleukin-12 (IL-12). Three cycles of treatment achieved a significant antitumor effect in the hamster model, and transgene expression was detected following each administration, in contrast with the rapid neutralization of the free virus. We propose monocytes as carriers for multiple intratumoral administrations of armed OAVs.
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    Alterations of the hippocampal neurogenic niche in a mouse model of dravet syndrome
    (Frontiers, 2020) Encinas, J.M. (Juan Manuel); Ricobaraza, A. (Ana); Martín-Suárez, S. (Soraya); Abiega, O. (Oihane); Hernandez-Alcoceba, R. (Rubén)
    Hippocampal neurogenesis, the process by which neural stem cells (NSCs) continuously generate new neurons in the dentate gyrus (DG) of most mammals including humans, is chiefly regulated by neuronal activity. Thus, severe alterations have been found in samples from epilepsy patients and in the hippocampal neurogenic niche in mouse models of epilepsy. Reactive-like and gliogenic NSCs plus aberrant newborn neurons with altered migration, morphology, and functional properties are induced by seizures in experimental models of temporal lobe epilepsy. Hippocampal neurogenesis participates in memory and learning and in the control of anxiety and stress. It has been therefore hypothesized that part of the cognitive symptoms associated with epilepsy could be promoted by impaired hippocampal neurogenesis. We here analyze for the first time the alterations of the neurogenic niche in a novel mouse model of Dravet syndrome (DS), a genetic encephalopathy with severe epilepsy in infancy and multiple neurological comorbidities. Scn1aWT/A1783V mice, hereafter referred to as DS, carrying a heterozygous and clinically relevant SCN1A mutation (A1783V) recapitulate the disease at the genetic and phenotypic levels. We demonstrate that in the neurogenic niche of young adult DS mice there are fewer NSCs, they have impaired cell division and bear reactive-like morphology. In addition, there is significant aberrant neurogenesis. Newborn immature neurons migrate abnormally, and several morphological features are drastically changed. Thus, this study shows for the first time important modifications in hippocampal neurogenesis in DS and opens venues for further research on this topic.
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    Treatment of pancreatic cancer with an oncolytic adenovirus expressing Interleukin-12 in syrian hamsters
    (Nature Publishing Group, 2009) González-Aseguinolaza, G. (Gloria); Ortiz-de-Solorzano, C. (Carlos); Otano, I. (Itziar); Prieto, J. (Jesús); Bortolanza, S. (Sergia); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Perez-Martin, D. (Daniel)
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    Identification of replication-competent HSV-1 Cgal+ strain signaling targets in human hepatoma cells by functional organelle proteomics
    (American Society for Biochemistry and Molecular Biology, 2009) Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Epstein, A.L. (Alberto L.); Potel, C. (Corinne); Fernandez-Irigoyen, J. (Joaquín); Carro-Roldan, E. (Elvira); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Santamaria, E. (Enrique)
    In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal(+)) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal(+) infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca(2+)-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal(+) infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK(1/2), ERK(1/2)) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal(+) in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.
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    An oncolytic adenovirus controlled by a modified telomerase promoter is attenuated in telomerase-negative cells, but shows reduced activity in cancer cells
    (Springer, 2005) Gomar, C. (Celia); Qian, C. (Cheng); Kramer, M.G. (María Gabriela); Farinati, F. (F.); Prieto, J. (Jesús); Bortolanza, S. (Sergia); Hernandez-Alcoceba, R. (Rubén)
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    Evaluation of bioluminescent imaging for noninvasive monitoring of colorectal cancer progression in the liver and its response to immunogene therapy
    (BioMed Central, 2009) González-Aseguinolaza, G. (Gloria); Zabala, M. (Maider); Ortiz-de-Solorzano, C. (Carlos); Alzuguren, P. (Pilar); Crettaz, J. (Julien); Kramer, M.G. (María Gabriela); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Benavides, C. (Carolina); Gonzalez-Aparicio, M. (Manuela)
    BACKGROUND: Bioluminescent imaging (BLI) is based on the detection of light emitted by living cells expressing a luciferase gene. Stable transfection of luciferase in cancer cells and their inoculation into permissive animals allows the noninvasive monitorization of tumor progression inside internal organs. We have applied this technology for the development of a murine model of colorectal cancer involving the liver, with the aim of improving the pre-clinical evaluation of new anticancer therapies. RESULTS: A murine colon cancer cell line stably transfected with the luciferase gene (MC38Luc1) retains tumorigenicity in immunocompetent C57BL/6 animals. Intrahepatic inoculation of MC38Luc1 causes progressive liver infiltration that can be monitored by BLI. Compared with ultrasonography (US), BLI is more sensitive, but accurate estimation of tumor mass is impaired in advanced stages. We applied BLI to evaluate the efficacy of an immunogene therapy approach based on the liver-specific expression of the proinflammatory cytokine interleukin-12 (IL-12). Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival. CONCLUSION: We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver. Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.
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    Gene therapy of liver cancer
    (WJG Press, 2006) Sangro, B. (Bruno); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén)