Browsing by MeSH term "Cell Death"
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Publication Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses(Nature Publishing Group, 2017-08-17) Cortegano, Isabel; Rodriguez-Garcia, Mercedes; Martín, Isabel; Prado-Zamora, Maria Carmen; Ruiz, Carolina; Hortigüela, Rafael; Alia, Mario; Vilar, Marçal; Mira, Helena; Cano, Eva; Dominguez-Rodriguez, Mercedes; Andres, Belen de; Gaspar, Maria Luisa; Instituto de Salud Carlos III; Ministerio de Economía, Industria y Competitividad (España)Aging has a strong impact on the activity of the immune system, enhancing susceptibility to pathogens and provoking a predominant pre-inflammatory status, whereas dampening responses to vaccines in humans and mice. Here, we demonstrate a loss of marginal zone B lymphocytes (MZ, CD19+CD45R+CD21++CD23lo) and a decrease of naive B cells (CD19+IgD+), whereas there is an enhancement of a CD19+CD45Rlo innate-like B cell population (B1REL) and the so-called aged B cell compartment (ABC, CD45R+CD21loCD23loCD5-CD11b-) in aged senescence-accelerated (SAMP8) mice but not in aged senescence-resistant (SAMR1) mice. These changes in aged SAMP8 mice were associated with lower IgG isotype levels, displaying low variable gene usage repertoires of the immunoglobulin heavy chain (VH) diversity, with a diminution on IgG1-memory B cells (CD11b-Gr1-CD138-IgM-IgD-CD19+CD38+IgG1+), an increase in T follicular helper (TFH, CD4+CXCR5+PD1+) cell numbers, and an altered MOMA-1 (metallophilic macrophages) band in primary follicles. LPS-mediated IgG1 responses were impaired in the B1REL and ABC cell compartments, both in vitro and in vivo. These data demonstrate the prominent changes to different B cell populations and in structural follicle organization that occur upon aging in SAMP8 mice. These novel results raise new questions regarding the importance of the cellular distribution in the B cell layers, and their effector functions needed to mount a coordinated and effective humoral response.Publication Autophagic-related cell death of Trypanosoma brucei induced by bacteriocin AS-48(Elsevier, 2018) Martínez-García, Marta; Bart, Jean Mathieu; Campos-Salinas, Jenny; Valdivia, Eva; Martínez-Bueno, Manuel; González-Rey, Elena; Navarro, Miguel; Maqueda, Mercedes; Cebrián, Rubén; Pérez-Victoria, José M; Ministerio de Economía y Competitividad (España); Regional Government of Andalusia (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Instituto de Salud Carlos IIIThe parasitic protozoan Trypanosoma brucei is the causative agent of human African trypanosomiasis (sleeping sickness) and nagana. Current drug therapies have limited efficacy, high toxicity and/or are continually hampered by the appearance of resistance. Antimicrobial peptides have recently attracted attention as potential parasiticidal compounds. Here, we explore circular bacteriocin AS-48's ability to kill clinically relevant bloodstream forms of T. brucei gambiense, T. brucei rhodesiense and T. brucei brucei. AS-48 exhibited excellent anti-trypanosomal activity in vitro (EC50 = 1-3 nM) against the three T. brucei subspecies, but it was innocuous to human cells at 104-fold higher concentrations. In contrast to its antibacterial action, AS-48 does not kill the parasite through plasma membrane permeabilization but by targeting intracellular compartments. This was evidenced by the fact that vital dye internalization-prohibiting concentrations of AS-48 could kill the parasite at 37 °C but not at 4 °C. Furthermore, AS-48 interacted with the surface of the parasite, at least in part via VSG, its uptake was temperature-dependent and clathrin-depleted cells were less permissive to the action of AS-48. The bacteriocin also caused the appearance of myelin-like structures and double-membrane autophagic vacuoles. These changes in the parasite's ultrastructure were confirmed by fluorescence microscopy as AS-48 induced the production of EGFP-ATG8.2-labeled autophagosomes. Collectively, these results indicate AS-48 kills the parasite through a mechanism involving clathrin-mediated endocytosis of VSG-bound AS-48 and the induction of autophagic-like cell death. As AS-48 has greater in vitro activity than the drugs currently used to treat T. brucei infection and does not present any signs of toxicity in mammalian cells, it could be an attractive lead compound for the treatment of sleeping sickness and nagana.Publication Cdk4 and Cdk6 cooperate in counteracting the INK4 family of inhibitors during murine leukemogenesis.(Elsevier, 2014-10-09) Rodríguez-Díez, Esther; Quereda, Victor; Bellutti, Florian; Prchal-Murphy, Michaela; Partida, David; Eguren, Manuel; Kollmann, Karoline; Gómez de Cedrón, Marta; Dubus, Pierre; Cañamero, Marta; Martinez Garcia, Maria Dolores; Sexl, Veronika; Malumbres Martinez, Marcos; FWF Austrian Science Fund; Gobierno de España; Fundación Ramón Areces; Comunidad de Madrid (España); Ministerio de Ciencia e Innovación (España); Unión EuropeaCdk4 and Cdk6 are related protein kinases that bind d-type cyclins and regulate cell-cycle progression. Cdk4/6 inhibitors are currently being used in advanced clinical trials and show great promise against many types of tumors. Cdk4 and Cdk6 are inhibited by INK4 proteins, which exert tumor-suppressing functions. To test the significance of this inhibitory mechanism, we generated knock-in mice that express a Cdk6 mutant (Cdk6 R31C) insensitive to INK4-mediated inhibition. Cdk6(R/R) mice display altered development of the hematopoietic system without enhanced tumor susceptibility, either in the presence or absence of p53. Unexpectedly, Cdk6 R31C impairs the potential of hematopoietic progenitors to repopulate upon adoptive transfer or after 5-fluorouracil-induced damage. The defects are overcome by eliminating sensitivity of cells to INK4 inhibitors by introducing the INK4-insensitive Cdk4 R24C allele, and INK4-resistant mice are more susceptible to hematopoietic and endocrine tumors. In BCR-ABL-transformed hematopoietic cells, Cdk6 R31C causes increased binding of p16(INK4a) to wild-type Cdk4, whereas cells harboring Cdk4 R24C and Cdk6 R31C are fully insensitive to INK4 inhibitors, resulting in accelerated disease onset. Our observations reveal that Cdk4 and Cdk6 cooperate in hematopoietic tumor development and suggest a role for Cdk6 in sequestering INK4 proteins away from Cdk4.Publication Clec2d Joins the Cell Death Sensor Ranks(Cell Press, 2020-01) del Fresno, Carlos; Sancho, David; Asociación Española Contra el Cáncer; Unión Europea. Comisión Europea. European Research Council (ERC); Unión Europea. Comisión Europea; Ministerio de Ciencia, Innovación y Universidades (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Instituto de Salud Carlos III; Fondation ACTERIA (Acting on European Research in Immunology and Allergology); Comunidad de Madrid (España); Atresmedia; Fundación La Marató TV3; Fundación ProCNICSensing tissue damage is an ancient function of immune cells that is central to the regulation of inflammation, tissue repair, and immunity. In this issue of Immunity, Lai et al. (2020) uncover a role for the C-type lectin receptor Clec2d as a sensor of cell death, which directly detects histones released during necrosis and thus contributes to inflammation and immunopathology.Publication Der p 1-based immunotoxin as potential tool for the treatment of dust mite respiratory allergy(Springer, 2020-07-23) Lázaro-Gorines, Rodrigo; López-Rodríguez, Juan Carlos; Benedé, Sara; González, Miguel; Mayorga, Cristobalina; Vogel, Lothar; Martínez-Del-Pozo, Álvaro; Lacadena, Javier; Villalba, Mayte; [Lázaro Gorines,R; López Rodríguez,JC; Benedé,S; Martínez del Pozo,A; Lacadena,J; Villalba,M] Biochemistry and Molecular Biology Department, Chemical Sciences Faculty, Complutense University of Madrid, Madrid, Spain. [González,M; Mayorga,C] Allergy Research Laboratory, IBIMA, Hospital Regional Universitario de Málaga, UMA, Málaga, Spain. [Mayorga,C] U.G.C. Allergy, IBIMA, Hospital Regional Universitario de Málaga, UMA, Málaga, Spain. [Vogel,L] Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany.Immunotoxins appear as promising therapeutic molecules, alternative to allergen-specific-immunotherapy. In this work, we achieved the development of a protein chimera able to promote specific cell death on effector cells involved in the allergic reaction. Der p 1 allergen was chosen as cell-targeting domain and the powerful ribotoxin α-sarcin as the toxic moiety. The resultant construction, named proDerp1αS, was produced and purified from the yeast Pichia pastoris. Der p 1-protease activity and α-sarcin ribonucleolytic action were effectively conserved in proDerp1αS. Immunotoxin impact was assayed by using effector cells sensitized with house dust mite-allergic sera. Cell degranulation and death, triggered by proDerp1αS, was exclusively observed on Der p 1 sera sensitized-humRBL-2H3 cells, but not when treated with non-allergic sera. Most notably, equivalent IgE-binding and degranulation were observed with both proDerp1αS construct and native Der p 1 when using purified basophils from sensitized patients. However, proDerp1αS did not cause any cytotoxic effect on these cells, apparently due to its lack of internalization after their surface IgE-binding, showing the complex in vivo panorama governing allergic reactions. In conclusion, herein we present proDerp1αS as a proof of concept for a potential and alternative new designs of therapeutic tools for allergies. Development of new, and more specific, second-generation of immunotoxins following proDerp1αS, is further discussed.Publication Dual functionality of the amyloid protein TasA in Bacillus physiology and fitness on the phylloplane(Springer, 2020) Cámara-Almirón, Jesús; Navarro, Yurena; Díaz-Martínez, Luis; Magno-Pérez-Bryan, María Concepción; Molina-Santiago, Carlos; Pearson, John R.; de Vicente, Antonio; Pérez-García, Alejandro; Romero, Diego; [Cámara-Almirón,J; Navarro,Y; Díaz-Martínez,L; Magno-Pérez-Bryan,MC; Molina-Santiago,C; de Vicente,A; Pérez-García,A; Romero,D] Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” – Departamento de Microbiología, Universidad de Málaga, Málaga, Spain. [Pearson,JR] Nano-imaging Unit, Andalusian Centre for Nanomedicine and Biotechnology, BIONAND, Málaga, Spain.Bacteria can form biofilms that consist of multicellular communities embedded in an extracellular matrix (ECM). In Bacillus subtilis, the main protein component of the ECM is the functional amyloid TasA. Here, we study further the roles played by TasA in B. subtilis physiology and biofilm formation on plant leaves and in vitro. We show that ΔtasA cells exhibit a range of cytological symptoms indicative of excessive cellular stress leading to increased cell death. TasA associates to the detergent-resistant fraction of the cell membrane, and the distribution of the flotillin-like protein FloT is altered in ΔtasA cells. We propose that, in addition to a structural function during ECM assembly and interactions with plants, TasA contributes to the stabilization of membrane dynamics as cells enter stationary phase.Publication Extra Virgin Oil Polyphenols Improve the Protective Effects of Hydroxytyrosol in an In Vitro Model of Hypoxia-Reoxygenation of Rat Brain(Multidisciplinary Digital Publishing Institute (MDPI), 2021-08-26) De La Cruz Cortés, José Pedro; Pérez de Algaba, Inmaculada; Martín-Aurioles, Esther; Arrebola, María Monsalud; Ortega-Hombrados, Laura; Rodríguez-Pérez, María Dolores; Fernández-Prior, María África; Bermúdez-Oria, Alejandra; Verdugo, Cristina; González-Correa, José Antonio; [De La Cruz Cortés,JP; Ortega-Hombrados,L; Rodríguez-Pérez,MD; Verdugo,C; González-Correa,JA] Departmento de Farmacología, Facultad de Medicina, Instituto de Investigación Biomédica (IBIMA), Universidad de Málaga, Málaga, Spain. [Pérez de Algaba,I] Clinical Laboratory Department, Hospital Público de Montilla, Córdoba, Spain. [Martín-Aurioles,E] UGC La Roca, Distrito Sanitario Málaga-Guadalhorce, Málaga, Spain. [Arrebola,MM] UGC Laboratorio Clínico, Hospital de la Axarquía, AGSEMA, Málaga, Spain. [Fernández-Prior,MÁ; Bermúdez-Oria,A] Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain.Hydroxytyrosol (HT) is the component primarily responsible for the neuroprotective effect of extra virgin olive oil (EVOO). However, it is less effective on its own than the demonstrated neuroprotective effect of EVOO, and for this reason, it can be postulated that there is an interaction between several of the polyphenols of EVOO. The objective of the study was to assess the possible interaction of four EVOO polyphenols (HT, tyrosol, dihydroxyphenylglycol, and oleocanthal) in an experimental model of hypoxia-reoxygenation in rat brain slices. The lactate dehydrogenase (LDH) efflux, lipid peroxidation, and peroxynitrite production were determined as measures of cell death, oxidative stress, and nitrosative stress, respectively. First, the polyphenols were incubated with the brain slices in the same proportions that exist in EVOO, comparing their effects with those of HT. In all cases, the cytoprotective and antioxidant effects of the combination were greater than those of HT alone. Second, we calculated the concentration-effect curves for HT in the absence or presence of each polyphenol. Tyrosol did not significantly modify any of the variables inhibited by HT. Dihydroxyphenylglycol only increased the cytoprotective effect of HT at 10 µM, while it increased its antioxidant effect at 50 and 100 µM and its inhibitory effect on peroxynitrite formation at all the concentrations tested. Oleocanthal increased the cytoprotective and antioxidant effects of HT but did not modify its inhibitory effect on nitrosative stress. The results of this study show that the EVOO polyphenols DHPG and OLC increase the cytoprotective effect of HT in an experimental model of hypoxia-reoxygenation in rat brain slices, mainly due to a possibly synergistic effect on HT's antioxidant action. These results could explain the greater neuroprotective effect of EVOO than of the polyphenols alone.Publication IIIG9 inhibition in adult ependymal cells changes adherens junctions structure and induces cellular detachment(Springer, 2021-09-17) Baeza, Victor; Cifuentes, Manuel; Martínez, Fernando; Ramírez, Eder; Nualart, Francisco; Ferrada, Luciano; Oviedo, María José; De Lima, Isabelle; Troncoso, Ninoschka; Saldivia, Natalia; Salazar, Katterine; [Baeza,V; Martínez,F; Ramírez,E; Nualart,F; Oviedo,MJ; De Lima,I; Troncoso,N; Saldivia,N; Salazar,K] Laboratory of Neurobiology and Stem Cells, NeuroCellT, Department of Cellular Biology, Faculty of Biological Sciences, University of Concepcion, Concepcion, Chile. [Nualart,F; Ferrada,L; Salazar,K] Faculty of Biological Sciences, Center for Advanced Microscopy CMA BIOBIO, University of Concepcion, Concepcion, Chile. [Cifuentes,M] Department of Cell Biology, Genetics and Physiology, University of Malaga, IBIMA, Malaga, Spain. [Cifuentes,M] Andalusian Center for Nanomedicine and Biotechnology, BIONAND, Malaga, Spain. [Cifuentes,M] Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, Malaga, Spain.Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. In cancer, the loss of ependymal cell polarity promotes the formation of different types of tumors, such as supratentorial anaplastic ependymomas, which are highly aggressive in children. IIIG9 (PPP1R32) is a protein restricted to adult ependymal cells located in cilia and in the apical cytoplasm and has unknown function. In this work, we studied the expression and localization of IIIG9 in the adherens junctions (cadherin/β-catenin-positive junctions) of adult brain ependymal cells using confocal and transmission electron microscopy. Through in vivo loss-of-function studies, ependymal denudation (single-dose injection experiments of inhibitory adenovirus) was observed, inducing the formation of ependymal cells with a "balloon-like" morphology. These cells had reduced cadherin expression (and/or delocalization) and cleavage of the cell death marker caspase-3, with "cilia rigidity" morphology (probably vibrational beating activity) and ventriculomegaly occurring prior to these events. Finally, after performing continuous infusions of adenovirus for 14 days, we observed total cell denudation and reactive parenchymal astrogliosis. Our data confirmed that IIIG9 is essential for the maintenance of adherens junctions of polarized ependymal cells. Eventually, altered levels of this protein in ependymal cell differentiation may increase ventricular pathologies, such as hydrocephalus or neoplastic transformation.Publication Inflammatory Cascade in Alzheimer's Disease Pathogenesis: A Review of Experimental Findings(Multidisciplinary Digital Publishing Institute (MDPI), 2021-09-28) de Oliveira, Jade; Kucharska, Ewa; Garcez, Michelle Lima; Rodrigues, Matheus Scarpatto; Quevedo, João; Moreno-Gonzalez, Ines; Budni, Josiane; [de Oliveira,J; Scarpatto Rodrigues,M] Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.[Kucharska,E] Faculty of Education, Institute of Educational Sciences, Jesuit University Ignatianum in Krakow, Krakow, Poland. [Lima Garcez,M] Department of Biochemistry, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil. [Quevedo,J] Translational Psychiatry Program, Faillace Department of Psychiatry and Behavioral Sciences, McGovern Medical School, The University of Texas Health Science Center at Houston. [Quevedo,J] Center of Excellence on Mood Disorders, Faillace Department of Psychiatry and Behavioral Sciences, McGovern Medical School, The University of Texas Health Science Center at Houston (UTHealth), Houston, USA. [Quevedo,J] Neuroscience Graduate Program, Graduate School of Biomedical Sciences, MD Anderson Cancer Center, UTHealth, The University of Texas Houston, Houston, TX, USA. [Quevedo,J] Graduate Program in Health Sciences, Translational Psychiatry Laboratory, University of Southern Santa Catarina (UNESC), Criciuma, Brazil. [Moreno-Gonzalez,I] Department of Cell Biology, Faculty of Sciences, University of Malaga, IBIMA, Malaga, Spain. [Moreno-Gonzalez,I] Networking Research Center on Neurodegenerative Diseases (CIBERNED), Malaga, Spain. [Moreno-Gonzalez,I] Department of Neurology, McGovern Medical School, The University of Texas Health Science Center at Houston (UTHealth), Houston, USA. [Budni,J] Programa de Pós-Graduação em Ciências da Saúde, Laboratório de Neurologia Experimental, Universidade do Extremo Sul Catarinense, Criciuma, Brazil.Alzheimer's disease (AD) is the leading cause of dementia worldwide. Most AD patients develop the disease in late life, named late onset AD (LOAD). Currently, the most recognized explanation for AD pathology is the amyloid cascade hypothesis. It is assumed that amyloid beta (Aβ) aggregation and deposition are critical pathogenic processes in AD, leading to the formation of amyloid plaques, as well as neurofibrillary tangles, neuronal cell death, synaptic degeneration, and dementia. In LOAD, the causes of Aβ accumulation and neuronal loss are not completely clear. Importantly, the blood-brain barrier (BBB) disruption seems to present an essential role in the induction of neuroinflammation and consequent AD development. In addition, we propose that the systemic inflammation triggered by conditions like metabolic diseases or infections are causative factors of BBB disruption, coexistent inflammatory cascade and, ultimately, the neurodegeneration observed in AD. In this regard, the use of anti-inflammatory molecles could be an interesting strategy to treat, delay or even halt AD onset and progression. Herein, we review the inflammatory cascade and underlying mechanisms involved in AD pathogenesis and revise the anti-inflammatory effects of compounds as emerging therapeutic drugs against AD.Publication JUNB/AP-1 controls IFN-γ during inflammatory liver disease.(American Society for Clinical Investigation (ASCI), 2013-12) Thomsen, Martin K; Bakiri, Latifa; Hasenfuss, Sebastian C; Hamacher, Rainer; Martinez Garcia, Maria Dolores; Wagner, Erwin F; Fundación BBVA; Gobierno de España; Unión Europea. Comisión Europea. European Research Council (ERC); Instituto de Salud Carlos III; European Molecular Biology Organization; German Research Foundation (DFG)Understanding the molecular pathogenesis of inflammatory liver disease is essential to design efficient therapeutic approaches. In hepatocytes, the dimeric transcription factor c-JUN/AP-1 is a major mediator of cell survival during hepatitis, although functions for other JUN proteins in liver disease are less defined. Here, we found that JUNB was specifically expressed in human and murine immune cells during acute liver injury. We analyzed the molecular function of JUNB in experimental models of hepatitis, including administration of concanavalin A (ConA) or α-galactosyl-ceramide, which induce liver inflammation and injury. Mice specifically lacking JUNB in hepatocytes displayed a mild increase in ConA-induced liver damage. However, targeted deletion of Junb in immune cells and hepatocytes protected against hepatitis in experimental models that involved NK/NKT cells. The absence of JUNB in immune cells decreased IFN-γ expression and secretion from NK and NKT cells, leading to reduced STAT1 pathway activation. Systemic IFN-γ treatment or adenovirus-based IRF1 delivery to Junb-deficient mice restored hepatotoxicity, and we demonstrate that Ifng is a direct transcriptional target of JUNB. These findings demonstrate that JUNB/AP-1 promotes cell death during acute hepatitis by regulating IFN-γ production in NK and NKT cells and thus functionally antagonizes the hepatoprotective function of c-JUN/AP-1 in hepatocytes.Publication Microglia activated by microbial neuraminidase contributes to ependymal cell death(BioMed Central (BMC), 2021-03-23) Fernández-Arjona, María Del Mar; León-Rodríguez, Ana; López-Ávalos, María Dolores; Grondona, Jesús M.; [Fernández-Arjona,MDM; León-Rodríguez,A; López-Ávalos,MD; Grondona,JM] Laboratorio de Fisiología Animal, Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Universidad de Málaga, Instituto de Investigación Biomédica de Málaga-IBIMA, Málaga, Spain.The administration of microbial neuraminidase into the brain ventricular cavities of rodents represents a model of acute aseptic neuroinflammation. Ependymal cell death and hydrocephalus are unique features of this model. Here we demonstrate that activated microglia participates in ependymal cell death. Co-cultures of pure microglia with ependymal cells (both obtained from rats) were performed, and neuraminidase or lipopolysaccharide were used to activate microglia. Ependymal cell viability was unaltered in the absence of microglia or inflammatory stimulus (neuraminidase or lipopolysaccharide). The constitutive expression by ependymal cells of receptors for cytokines released by activated microglia, such as IL-1β, was demonstrated by qPCR. Besides, neuraminidase induced the overexpression of both receptors in ventricular wall explants. Finally, ependymal viability was evaluated in the presence of functional blocking antibodies against IL-1β and TNFα. In the co-culture setting, an IL-1β blocking antibody prevented ependymal cell death, while TNFα antibody did not. These results suggest that activated microglia are involved in the ependymal damage that occurs after the administration of neuraminidase in the ventricular cavities, and points to IL-1β as possible mediator of such effect. The relevance of these results lies in the fact that brain infections caused by neuraminidase-bearing pathogens are frequently associated to ependymal death and hydrocephalus.Publication Oridonin enhances antitumor effects of doxorubicin in human osteosarcoma cells(Springer, 2021-08-24) Kazantseva, Liliya; Becerra, José; Santos-Ruiz, Leonor; [Kazantseva,L; Becerra,J; Santos-Ruiz,L] Andalusian Centre for Nanomedicine and Biotechnology‑BIONAND, Universidad de Málaga, Campanillas Málaga, Spain. [Kazantseva,L; Becerra,J; Santos-Ruiz,L] Centro de Investigación Biomédica en Red, Biotecnología, Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain. [Kazantseva,L; Becerra,J; Santos-Ruiz,L] Instituto de Investigación Biomédica de Málaga-IBIMA, Málaga, Spain. [Becerra,J; Santos-Ruiz,L] Departamento de Biología Celular, Genética y Fisiología Facultad de Ciencias, Universidad de Málaga, Málaga, Spain.Background: Doxorubicin is the chemotherapeutic drug of choice in osteosarcoma treatment, but its cumulative administration causes dilated cardiomyopathy. Combination therapy represents a potential strategy to reduce the therapeutic dosage of the chemotherapeutic agent and minimize its side effects. The aim of this study was to evaluate the potential of oridonin, a natural product from the medicinal herb Rabdosia rubescens, to act in combination with doxorubicin for osteosarcoma treatment. To date, there are no reports of the simultaneous administration of both drugs in osteosarcoma therapy. Methods: The combined administration of different doses of oridonin and doxorubicin, as compared with the drugs alone, were tested in an in vitro model of osteosarcoma. The synergistic effect of the drugs on cell death was assessed by alamarBlue™ and by CompuSyn software. Early and late apoptosis markers (JC-1 fluorescence and Annexin V immunofluorescence), as well as the production of reactive oxygen species, were evaluated by flow cytometry. Western blot was used to assess the expression of anti-apoptotic proteins. Results: Oridonin and doxorubicin presented a synergistic cytotoxic effect in osteosarcoma cells. In the presence of sub-cytotoxic concentrations of the natural product, there was an increased accumulation of intracellular doxorubicin, increased levels of reactive oxygen species (ROS), alteration of mitochondria membrane potential and a higher rate of apoptosis. Conclusion: The combined use of oridonin and doxorubicin could help to reduce the clinical dosage of doxorubicin and its dangerous side effects.Publication Oxidative Stress in Drug-Induced Liver Injury (DILI): From Mechanisms to Biomarkers for Use in Clinical Practice(Multidisciplinary Digital Publishing Institute (MDPI), 2021-03-05) Villanueva-Paz, Marina; Morán, Laura; López-Alcántara, Nuria; Freixo, Cristiana; Andrade, Raúl J.; Lucena, M Isabel; Cubero, Francisco Javier; [Villanueva-Paz,M; Andrade,RJ; Lucena,MI] Unidad de Gestión Clínica de Gastroenterología, Servicio de Farmacología Clínica, Instituto de Investigación Biomédica de Málaga-IBIMA, Hospital Universitario Virgen de la Victoria, Universidad de Málaga, CIBERehd, Málaga, Spain. [Morán,L; López-Alcántara,N; Cubero,FJ] Department of Immunology, Ophthalmology and ENT, Complutense University School of Medicine, Madrid, Spain. [Morán,L] Health Research Institute Gregorio Marañón (IiSGM), Madrid, Spain. [Freixo,C] CINTESIS, Center for Health Technology and Services Research, do Porto University School of Medicine, Porto, Portugal. [Cubero,FJ] 12 de Octubre Health Research Institute (imas12), Madrid, Spain.Idiosyncratic drug-induced liver injury (DILI) is a type of hepatic injury caused by an uncommon drug adverse reaction that can develop to conditions spanning from asymptomatic liver laboratory abnormalities to acute liver failure (ALF) and death. The cellular and molecular mechanisms involved in DILI are poorly understood. Hepatocyte damage can be caused by the metabolic activation of chemically active intermediate metabolites that covalently bind to macromolecules (e.g., proteins, DNA), forming protein adducts-neoantigens-that lead to the generation of oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress, which can eventually lead to cell death. In parallel, damage-associated molecular patterns (DAMPs) stimulate the immune response, whereby inflammasomes play a pivotal role, and neoantigen presentation on specific human leukocyte antigen (HLA) molecules trigger the adaptive immune response. A wide array of antioxidant mechanisms exists to counterbalance the effect of oxidants, including glutathione (GSH), superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), which are pivotal in detoxification. These get compromised during DILI, triggering an imbalance between oxidants and antioxidants defense systems, generating oxidative stress. As a result of exacerbated oxidative stress, several danger signals, including mitochondrial damage, cell death, and inflammatory markers, and microRNAs (miRNAs) related to extracellular vesicles (EVs) have already been reported as mechanistic biomarkers. Here, the status quo and the future directions in DILI are thoroughly discussed, with a special focus on the role of oxidative stress and the development of new biomarkers.Publication PGC-1α deficiency causes spontaneous kidney inflammation and increases the severity of nephrotoxic AKI.(Wiley, 2019-09) Fontecha-Barriuso, Miguel; Martín-Sánchez, Diego; Martinez-Moreno, Julio M; Carrasco, Susana; Ruiz-Andrés, Olga; Monsalve, Maria; Sanchez-Ramos, Cristina; Gómez, Manuel J; Ruiz-Ortega, Marta; Sánchez-Niño, Maria D; Cannata-Ortiz, Pablo; Cabello, Ramiro; Gonzalez-Enguita, Carmen; Ortiz, Alberto; Sanz, Ana BPGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α, PPARGC1A) regulates the expression of genes involved in energy homeostasis and mitochondrial biogenesis. Here we identify inactivation of the transcriptional regulator PGC-1α as a landmark for experimental nephrotoxic acute kidney injury (AKI) and describe the in vivo consequences of PGC-1α deficiency over inflammation and cell death in kidney injury. Kidney transcriptomic analyses of WT mice with folic acid-induced AKI revealed 1398 up- and 1627 downregulated genes. Upstream transcriptional regulator analyses pointed to PGC-1α as the transcription factor potentially driving the observed expression changes with the highest reduction in activity. Reduced PGC-1α expression was shared by human kidney injury. Ppargc1a-/- mice had spontaneous subclinical kidney injury characterized by tubulointerstitial inflammation and increased Ngal expression. Upon AKI, Ppargc1a-/- mice had lower survival and more severe loss of renal function, tubular injury, and reduction in expression of mitochondrial PGC-1α-dependent genes in the kidney, and an earlier decrease in mitochondrial mass than WT mice. Additionally, surviving Ppargc1a-/- mice showed higher rates of tubular cell death, compensatory proliferation, expression of proinflammatory cytokines, NF-κB activation, and interstitial inflammatory cell infiltration. Specifically, Ppargc1a-/- mice displayed increased M1 and decreased M2 responses and expression of the anti-inflammatory cytokine IL-10. In cultured renal tubular cells, PGC-1α targeting promoted spontaneous cell death and proinflammatory responses. In conclusion, PGC-1α inactivation is a key driver of the gene expression response in nephrotoxic AKI and PGC-1α deficiency promotes a spontaneous inflammatory kidney response that is magnified during AKI. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Publication Pharmacological blockade of the fatty acid amide hydrolase (FAAH) alters neural proliferation, apoptosis and gliosis in the rat hippocampus, hypothalamus and striatum in a negative energy context.(Frontiers Media, 2015-03-27) Rivera, Patricia; Bindila, Laura; Pastor, Antoni; Pérez-Martín, Margarita; Pavón, Francisco-Javier; Serrano, Antonia; de la Torre, Rafael; Lutz, Beat; Rodríguez de Fonseca, Fernando; Suárez, Juan; [Rivera,P; Pavón,FJ; Serrano,A; Rodríguez de Fonseca,F; Suárez,J] UGC Salud Mental, Instituto de Investigación Biomédica (IBIMA), Universidad de Málaga-Hospital Universitario Regional de Málaga, Málaga, Spain. [Rivera,P; Pavón,FJ; Serrano,A; de la Torre,R; Rodríguez de Fonseca,F; Suárez,J] CIBER OBN, Instituto de Salud Carlos III, Madrid, Spain. [Bindila,L, Lutz,B] Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg-University of Mainz, Mainz, Germany. [Pastor,A; de la Torre,R] Institut Hospital del Mar d'Investigacions Mediques, Barcelona, Spain. [Pastor,A] Facultat de Medicina, Universitat Autonoma de Barcelona, Barcelona, Spain. [Pérez-Martín,M] Departamento de Biología Celular, Genética y Fisiología, Instituto de Investigación Biomédica (IBIMA), Universidad de Málaga, Málaga, Spain. [de la Torre, R] Facultat de Ciencies de la Salut i de la Vida, Universitat Pompeu Fabra (CEXS-UPF), Barcelona, Spain.Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3(+) or BrdU(+) cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3(+)), astroglia (GFAP(+)), and microglia (Iba1(+) cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3(+) and BrdU(+) subgranular cells as well as GFAP(+), Iba1(+) and cleaved caspase-3(+) cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3(+), GFAP(+) and 3-weeks-old BrdU(+) cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context.Publication POLD3 Is Haploinsufficient for DNA Replication in Mice(Cell Press, 2016-09-01) Murga, Matilde; Lecona, Emilio; Kamileri, Irene; Díaz, Marcos; Lugli, Natalia; Sotiriou, Sotirios K; Anton, Marta E; Mendez, Juan; Halazonetis, Thanos D; Fernandez-Capetillo, Oscar; Botín Foundation; Banco Santander; Ministerio de Economía y Competitividad (España); Fundación La Marató TV3; Howard Hughes Medical Institute; Unión Europea. Comisión Europea. European Research Council (ERC)The Pold3 gene encodes a subunit of the Polδ DNA polymerase complex. Pold3 orthologs are not essential in Saccharomyces cerevisiae or chicken DT40 cells, but the Schizosaccharomyces pombe ortholog is essential. POLD3 also has a specialized role in the repair of broken replication forks, suggesting that POLD3 activity could be particularly relevant for cancer cells enduring high levels of DNA replication stress. We report here that POLD3 is essential for mouse development and is also required for viability in adult animals. Strikingly, even Pold3(+/-) mice were born at sub-Mendelian ratios, and, of those born, some presented hydrocephaly and had a reduced lifespan. In cells, POLD3 deficiency led to replication stress and cell death, which were aggravated by the expression of activated oncogenes. Finally, we show that Pold3 deletion destabilizes all members of the Polδ complex, explaining its major role in DNA replication and the severe impact of its deficiency.Publication Retama monosperma n-hexane extract induces cell cycle arrest and extrinsic pathway-dependent apoptosis in Jurkat cells(BioMed Central (BMC), 2014-01-24) Belayachi, Lamiae; Aceves-Luquero, Clara; Merghoub, Nawel; Bakri, Youssef; Fernandez de Mattos, Silvia; Amzazi, Saaid; Villalonga, PriamBackground: Retama monosperma L. (Boiss.) or Genista monosperma L. (Lam.), locally named as R'tam, is an annual and spontaneous plant belonging to the Fabaceae family. In Morocco, Retama genus is located in desert regions and across the Middle Atlas and it has been widely used in traditional medicine in many countries. In this study, we show that Retama monosperma hexane extract presents significant anti-leukemic effects against human Jurkat cells. Methods: Human Jurkat cells, together with other cell lines were screened with different concentrations of Retama monosperma hexane extract at different time intervals. Growth inhibition was determined using luminescent-based viability assays. Cell cycle arrest and apoptosis were measured by flow cytometry analysis. Combined caspase 3 and 7 activities were measured using luminometric caspase assays and immunoblots were performed to analyze expression of relevant pro-and anti-apoptotic proteins. GC-MS were used to determine the chemical constituents of the active extract. Results: Retama monosperma hexane extract (Rm-HE) showed significant cytotoxicity against Jurkat cells, whereas it proved to be essentially ineffective against both normal mouse fibroblasts (NIH3T3) and normal lymphocytes (TK-6). Cytometric analysis indicated that Rm-HE promoted cell cycle arrest and apoptosis induction accompanied by DNA damage induction indicated by an increase in p-H2A. X levels. Rm-HE induced apoptosis was partially JNK-dependent and characterized by an increase in Fas-L levels together with activation of caspases 8, 3, 7 and 9, whereas neither the pro-apoptotic nor anti-apoptotic mitochondrial membrane proteins analyzed were significantly altered. Chemical identification analysis indicated that a-linolenic acid, campesterol, stigmasterol and sitosterol were the major bioactive components within the extract. Conclusions: Our data suggest that bioactive compounds present in Rm-HE show significant anti leukemic activity inducing cell cycle arrest and cell death that operates, at least partially, through the extrinsic apoptosis pathway.Publication SIRT1 Controls Acetaminophen Hepatotoxicity by Modulating Inflammation and Oxidative Stress.(Mary Ann Liebert, 2018-05-01) Rada, Patricia; Pardo, Virginia; Mobasher, Maysa A; García-Martínez, Irma; Ruiz, Laura; González-Rodríguez, Águeda; Sanchez-Ramos, Cristina; Muntané, Jordi; Alemany, Susana; James, Laura P; Simpson, Kenneth J; Monsalve, María; Valdecantos, Maria Pilar; Valverde, Ángela MAIMS Sirtuin 1 (SIRT1) is a key player in liver physiology and a therapeutic target against hepatic inflammation. We evaluated the role of SIRT1 in the proinflammatory context and oxidative stress during acetaminophen (APAP)-mediated hepatotoxicity. RESULTS SIRT1 protein levels decreased in human and mouse livers following APAP overdose. SIRT1-Tg mice maintained higher levels of SIRT1 on APAP injection than wild-type mice and were protected against hepatotoxicity by modulation of antioxidant systems and restrained inflammatory responses, with decreased oxidative stress, proinflammatory cytokine messenger RNA levels, nuclear factor kappa B (NFκB) signaling, and cell death. Mouse hepatocytes stimulated with conditioned medium of APAP-treated macrophages (APAP-CM) showed decreased SIRT1 levels; an effect mimicked by interleukin (IL)1β, an activator of NFκB. This negative modulation was abolished by neutralizing IL1β in APAP-CM or silencing p65-NFκB in hepatocytes. APAP-CM of macrophages from SIRT1-Tg mice failed to downregulate SIRT1 protein levels in hepatocytes. In vivo administration of the NFκB inhibitor BAY 11-7082 preserved SIRT1 levels and protected from APAP-mediated hepatotoxicity. INNOVATION Our work evidenced the unique role of SIRT1 in APAP hepatoprotection by targeting oxidative stress and inflammation. CONCLUSION SIRT1 protein levels are downregulated by IL1β/NFκB signaling in APAP hepatotoxicity, resulting in inflammation and oxidative stress. Thus, maintenance of SIRT1 during APAP overdose by inhibiting NFκB might be clinically relevant. Rebound Track: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16:293-296, 2012) with the following serving as open reviewers: Rafael de Cabo, Joaquim Ros, Kalervo Hiltunen, and Neil Kaplowitz. Antioxid. Redox Signal. 28, 1187-1208.Publication β3 adrenergic receptor selective stimulation during ischemia/reperfusion improves cardiac function in translational models through inhibition of mPTP opening in cardiomyocytes.(Springer, 2014-07) García-Prieto, Jaime; García-Ruiz, Jose Manuel; Sanz-Rosa, David; Pun, Andrés; García-Alvarez, Ana; Davidson, Sean M; Fernández-Friera, Leticia; Nuno-Ayala, Mario; Fernández-Jiménez, Rodrigo; Bernal, Juan A; Izquierdo-Garcia, José Luis; Jimenez-Borreguero, Jesús; Pizarro, Gonzalo; Ruiz-Cabello, Jesús; Macaya, Carlos; Fuster, Valentín; Yellon, Derek M; Ibáñez, BorjaSelective stimulation of β3 adrenergic-receptor (β3AR) has been shown to reduce infarct size in a mouse model of myocardial ischemia/reperfusion. However, its functional long-term effect and the cardioprotective mechanisms at the level of cardiomyocytes have not been elucidated, and the impact of β3AR stimulation has not been evaluated in a more translational large animal model. This study aimed at evaluating pre-perfusion administration of BRL37344 both in small and large animal models of myocardial ischemia/reperfusion. Pre-reperfusion administration of the β3AR agonist BRL37344 (5 μg/kg) reduced infarct size at 2-and 24-h reperfusion in wild-type mice. Long-term (12-weeks) left ventricular (LV) function assessed by echocardiography and cardiac magnetic resonance (CMR) was significantly improved in β3AR agonist-treated mice. Incubation with β3AR agonist (BRL37344, 7 μmol/L) significantly reduced cell death in isolated adult mouse cardiomyocytes during hypoxia/reoxygenation and decreased susceptibility to deleterious opening of the mitochondrial permeability transition pore (mPTP), via a mechanism dependent on the Akt-NO signaling pathway. Pre-reperfusion BRL37344 administration had no effect on infarct size in cyclophilin-D KO mice, further implicating mPTP in the mechanism of protection. Large-white pigs underwent percutaneous coronary ischemia/reperfusion and 3-T CMR at 7 and 45 days post-infarction. Pre-perfusion administration of BRL37344 (5 μg/kg) decreased infarct size and improved long-term LV contractile function. A single-dose administration of β3AR agonist before reperfusion decreased infarct size and resulted in a consistent and long-term improvement in cardiac function, both in small and large animal models of myocardial ischemia/reperfusion. This protection appears to be executed through inhibition of mPTP opening in cardiomyocytes.