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  • 1.
    Alberti, Esteban
    et al.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Los, Marek Jan
    Interfaculty Institute for Biochemistry, University of Tübingen, Germany; BioApplications Enterprises, Winnipeg, MB, Canada.
    Garcia, Rocio
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Fraga, JL
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Serrano, T.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Hernandez, E.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Klonisch, Thomas
    Department of Human Anatomy and Cell Sciences, and Manitoba Institute of Child Health, Winnipeg, Canada.
    Macías, R.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Martinez, L.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Castillo, L.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    de la Cuétara, K.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.
    Prolonged Survival and expression of neural markers by bone marrow-derived stem cells transplanted into brain lesions2009In: Medical Science Monitor, ISSN 1234-1010, E-ISSN 1643-3750, Vol. 15, no 2, p. BR47-BR54Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Bone marrow-derived stem cell transplantation is a potentially viable therapeutic option for the treatment of neurodegenerative disease. MATERIAL/METHODS: We have isolated bone marrow stem cells by standard method. We then evaluated the survival of rats' bone marrow mononuclear cells implanted in rats' brain. The cells were extracted from rats' femurs, and marked for monitoring purposes by adenoviral transduction with Green Fluorescent Protein (GFP). Labeled cells were implanted within the area of rats' striatum lesions that were induced a month earlier employing quinolinic acid-based method. The implants were phenotyped by monitoring CD34; CD38; CD45 and CD90 expression. Bone marrow stromal cells were extracted from rats' femurs and cultivated until monolayer bone marrow stromal cells were obtained. The ability of bone marrow stromal cells to express NGF and GDNF was evaluated by RT-PCR. RESULTS: Implanted cells survived for at least one month after transplantation and dispersed from the area of injection towards corpus callosum and brain cortex. Interestingly, passaged rat bone marrow stromal cells expressed NGF and GDNF mRNA. CONCLUSIONS: The bone marrow cells could be successfully transplanted to the brain either for the purpose of trans-differentiation, or for the expression of desired growth factors.

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  • 2.
    Alizadeh, Javad
    et al.
    University of Manitoba, Canada.
    Zeki, Amir A.
    Centre Comparat Resp Biol and Med, CA USA.
    Mirzaei, Nima
    University of Manitoba, Canada.
    Tewary, Sandipan
    University of Manitoba, Canada.
    Rezaei Moghadam, Adel
    University of Manitoba, Canada; University of Manitoba, Canada.
    Glogowska, Aleksandra
    University of Manitoba, Canada.
    Nagakannan, Pandian
    University of Manitoba, Canada.
    Eftekharpour, Eftekhar
    University of Manitoba, Canada.
    Wiechec, Emilia
    Linköping University, Department of Clinical and Experimental Medicine, Division of Speech language pathology, Audiology and Otorhinolaryngology. Linköping University, Faculty of Medicine and Health Sciences.
    Gordon, Joseph W.
    University of Manitoba, Canada; University of Manitoba, Canada; University of Manitoba, Canada.
    Xu, Fred. Y.
    University of Manitoba, Canada; University of Manitoba, Canada.
    Field, Jared T.
    University of Manitoba, Canada.
    Yoneda, Ken Y.
    Centre Comparat Resp Biol and Med, CA USA.
    Kenyon, Nicholas J.
    Centre Comparat Resp Biol and Med, CA USA.
    Hashemi, Mohammad
    Zehedan University of Medical Science, Iran.
    Hatch, Grant M.
    University of Manitoba, Canada; University of Manitoba, Canada.
    Hombach-Klonisch, Sabine
    University of Manitoba, Canada.
    Klonisch, Thomas
    University of Manitoba, Canada.
    Ghavami, Saeid
    University of Manitoba, Canada; University of Manitoba, Canada; Shiraz University of Medical Science, Iran.
    Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells2017In: Scientific Reports, E-ISSN 2045-2322, Vol. 7, article id 44841Article in journal (Refereed)
    Abstract [en]

    The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP-and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB231( breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines.

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  • 3.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Vicente-Carrillo, Alejandro
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Exogenous Individual Lecithin-Phospholipids (Phosphatidylcholine and Phosphatidylglycerol) Cannot Prevent the Oxidative Stress Imposed by Cryopreservation of Boar Sperm.2017In: Journal of veterinary medicine and surgery, ISSN 2574-2868, Vol. 1, no 1Article in journal (Refereed)
    Abstract [en]

    Objective: Despite the use of high proportions of the chemically undefined lipoprotein/phospholipid-rich egg-yolk in extenders, boar sperm are highly sensitive to cooling, which induces ROS generation and disrupts the plasma membrane.

    Here, we studied whether replacement of hen egg-yolk by commercially defined lecithin phospholipids, derived from egg (LPGE: phosphatidyl glycerol, LPCE: phosphatidyl choline) or soybean (LPCS: phosphatidyl choline), could individually ameliorate such oxidative effects during cryopreservation of ejaculated (sperm rich fraction, SRF) or of cauda-epididymal sperm, retrieved post-mortem from the same males.

    Methods: A conventional extender (lactose buffer, with 20% egg-yolk, 0.5% OEP and 3% glycerol) was used as control. Cryodamage was assessed as loss of sperm motility, membrane and acrosome intactness, early membrane destabilization changes, mitochondrial potential, superoxide and ROS production, to finally determine lipid peroxidation (LPO) using specific probes.

    Results and conclusion: In general, the exogenous phospholipids assayed were unable of maintaining neither sperm motility nor viability post-thaw compared to controls, owing to increased ROS production and lipid peroxidation. In our study, mitochondrial superoxide production resulted in very high levels for all groups, whereas both ROS production and lipid peroxidation were reduced in the control group, containing emulsified hen egg yolk. Further studies using various dosage and combination of LPCS should be followed for their eventual protective effect.

    Keywords: Cryodamage; Sperm; Boar; Mitochondrial activation; Mitochondrial superoxide; ROS production; Lipid peroxidation

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  • 4. Order onlineBuy this publication >>
    Amirhosseini, Mehdi
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Aseptic Loosening of Orthopedic Implants: Osteoclastogenesis Regulation and Potential Therapeutics2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Aseptic loosening is the main cause of failure of orthopedic prostheses. With no pharmaceuticals to prevent or mitigate periprosthetic bone degradation, a surgery to replace the loose implant with a new one is the only choice to restore patients’ function. Most studies on mechanisms for aseptic loosening investigate wear debris particle-induced osteolysis. However, pathological loading conditions around unstable implants can also trigger osteoclast differentiation and bone loss.

    In the first study, global gene expression changes induced by mechanical instability of implants, and by titanium particles were compared in a validated rat model for aseptic loosening. Microarray analysis showed that similar signaling pathways and gene expression patterns are involved in particle- and instability-induced periprosthetic osteolysis with an early onset innate immune response as a hallmark of osteolysis induced by mechanical instability.

    Further, effects of potential therapeutics on restriction of excessive osteoclast differentiation were evaluated. Wnt signaling pathway is known to regulate bone remodeling. In the second study, effects of inactivation of glycogen synthase kinase 3 beta (GSK-3β), a negative regulator of canonical Wnt signaling, on instability-induced periprosthetic osteolysis were examined using our rat model for aseptic loosening. Inhibition of GSK-3β led to a decrease in osteoclast numbers in the periprosthetic bone tissue exposed to mechanical instability while osteoblast perimeter showed an increase. This was accompanied by higher bone volume fraction (BV/TV) in animals treated with the GSK-3β inhibitor.

    In the third study, potential beneficial effects of two selective inhibitors of cyclindependent kinase 8/19 (CDK8/19) on bone tissue were evaluated. CDK8/19 is a Mediator complex-associated transcriptional regulator involved in several signaling pathways. CDK8/19 inhibitors, mainly under investigation as treatments for tumors, are reported to enhance osteoblast differentiation and bone formation. We show in this study, for the first time, that inhibition of CDK8/19 led to marked suppression of osteoclast differentiation from bone marrow macrophages in vitro through disruption of the RANK signaling. In mouse primary osteoblasts downregulation of osteopontin mRNA, a negative regulator of mineralization, together with increased alkaline phosphatase activity and calcium deposition indicated that osteoblast mineralization was promoted by CDK8/19 inhibition. Moreover, local administration of a CDK8/19 inhibitor promoted cancellous bone regeneration in a rat model for bone healing.

    These studies contribute to better understanding of mechanisms behind mechanical instability-induced periprosthetic osteolysis and propose potential therapeutics to restrict bone loss with effects on both osteoclasts and osteoblasts.

    List of papers
    1. Mechanical instability and titanium particles induce similar transcriptomic changes in a rat model for periprosthetic osteolysis and aseptic loosening
    Open this publication in new window or tab >>Mechanical instability and titanium particles induce similar transcriptomic changes in a rat model for periprosthetic osteolysis and aseptic loosening
    2017 (English)In: Bone Reports, ISSN 2352-1872, Vol. 7, p. 17-25Article in journal (Refereed) Published
    Abstract [en]

    Wear debris particles released from prosthetic bearing surfaces and mechanical instability of implants are two main causes of periprosthetic osteolysis. While particle-induced loosening has been studied extensively, mechanisms through which mechanical factors lead to implant loosening have been less investigated. This study compares the transcriptional profiles associated with osteolysis in a rat model for aseptic loosening, induced by either mechanical instability or titanium particles. Rats were exposed to mechanical instability or titanium particles. After 15 min, 3, 48 or 120 h from start of the stimulation, gene expression changes in periprosthetic bone tissue was determined by microarray analysis. Microarray data were analyzed by PANTHER Gene List Analysis tool and Ingenuity Pathway Analysis (IPA). Both types of osteolytic stimulation led to gene regulation in comparison to unstimulated controls after 3, 48 or 120 h. However, when mechanical instability was compared to titanium particles, no gene showed a statistically significant difference (fold change = ± 1.5 and adjusted p-value = 0.05) at any time point. There was a remarkable similarity in numbers and functional classification of regulated genes. Pathway analysis showed several inflammatory pathways activated by both stimuli, including Acute Phase Response signaling, IL-6 signaling and Oncostatin M signaling. Quantitative PCR confirmed the changes in expression of key genes involved in osteolysis observed by global transcriptomics. Inflammatory mediators including interleukin (IL)-6, IL-1ß, chemokine (C-C motif) ligand (CCL)2, prostaglandin-endoperoxide synthase (Ptgs)2 and leukemia inhibitory factor (LIF) showed strong upregulation, as assessed by both microarray and qPCR. By investigating genome-wide expression changes we show that, despite the different nature of mechanical implant instability and titanium particles, osteolysis seems to be induced through similar biological and signaling pathways in this rat model for aseptic loosening. Pathways associated to the innate inflammatory response appear to be a major driver for osteolysis. Our findings implicate early restriction of inflammation to be critical to prevent or mitigate osteolysis and aseptic loosening of orthopedic implants.

    Place, publisher, year, edition, pages
    Elsevier, 2017
    Keywords
    Aseptic loosening; Implant; Instability; Microarray; Wear debris
    National Category
    Cell and Molecular Biology Orthopaedics
    Identifiers
    urn:nbn:se:liu:diva-146297 (URN)10.1016/j.bonr.2017.07.003 (DOI)28795083 (PubMedID)
    Available from: 2018-04-07 Created: 2018-04-07 Last updated: 2019-03-08
    2. GSK-3 beta inhibition suppresses instability-induced osteolysis by a dual action on osteoblast and osteoclast differentiation
    Open this publication in new window or tab >>GSK-3 beta inhibition suppresses instability-induced osteolysis by a dual action on osteoblast and osteoclast differentiation
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    2018 (English)In: Journal of Cellular Physiology, ISSN 0021-9541, E-ISSN 1097-4652, Vol. 233, no 3, p. 2398-2408Article in journal (Refereed) Published
    Abstract [en]

    Currently, there are no medications available to treat aseptic loosening of orthopedic implants. Using osteoprotegerin fusion protein (OPG-Fc), we previously blocked instability-induced osteoclast differentiation and peri-prosthetic osteolysis. Wnt/beta-catenin signaling, which regulates OPG secretion from osteoblasts, also modulates the bone tissue response to mechanical loading. We hypothesized that activating Wnt/beta-catenin signaling by inhibiting glycogen synthase kinase-3 beta (GSK-3 beta) would reduce instability-induced bone loss through regulation of both osteoblast and osteoclast differentiation. We examined effects of GSK-3 beta inhibition on regulation of RANKL and OPG in a rat model of mechanical instability-induced peri-implant osteolysis. The rats were treated daily with a GSK-3 beta inhibitor, AR28 (20 mg/kg bw), for up to 5 days. Bone tissue and blood serum were assessed by qRT-PCR, immunohistochemistry, and ELISA on days 3 and 5, and by micro-CT on day 5. After 3 days of treatment with AR28, mRNA levels of beta-catenin, Runx2, Osterix, Col1 alpha 1, and ALP were increased leading to higher osteoblast numbers compared to vehicle-treated animals. BMP-2 and Wnt16 mRNA levels were downregulated by mechanical instability and this was rescued by GSK-3 beta inhibition. Osteoclast numbers were decreased significantly after 3 days of GSK-3 beta inhibition, which correlated with enhanced OPG mRNA expression. This was accompanied by decreased serum levels of TRAP5b on days 3 and 5. Treatment with AR28 upregulated osteoblast differentiation, while osteoclastogenesis was blunted, leading to increased bone mass by day 5. These data suggest that GSK-3 beta inactivation suppresses osteolysis through regulating both osteoblast and osteoclast differentiation in a rat model of instability-induced osteolysis.

    Place, publisher, year, edition, pages
    WILEY, 2018
    Keywords
    bone implant; GSK-3 beta; mechanical instability; osteolysis; Wnt signaling
    National Category
    Pharmacology and Toxicology
    Identifiers
    urn:nbn:se:liu:diva-148660 (URN)10.1002/jcp.26111 (DOI)000433519300056 ()28731198 (PubMedID)
    Note

    Funding Agencies|VINNOVA [2012-04409]; National Institutes of Health [AR056802]; Vetenskapsradet [K2014-7X-22506-01-3]; Swedish Research Council; Swedish Governmental Agency for Innovation Systems

    Available from: 2018-06-18 Created: 2018-06-18 Last updated: 2019-04-08
    3. Cyclin-dependent kinase 8/19 inhibition suppresses osteoclastogenesis by downregulating RANK and promotes osteoblast mineralization and cancellous bone healing.
    Open this publication in new window or tab >>Cyclin-dependent kinase 8/19 inhibition suppresses osteoclastogenesis by downregulating RANK and promotes osteoblast mineralization and cancellous bone healing.
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    2019 (English)In: Journal of Cellular Physiology, ISSN 0021-9541, E-ISSN 1097-4652, Vol. 234, no 9, p. 16503-16516Article in journal (Refereed) Published
    Abstract [en]

    Cyclin-dependent kinase 8 (CDK8) is a mediator complex-associated transcriptional regulator that acts depending on context and cell type. While primarily under investigation as potential cancer therapeutics, some inhibitors of CDK8-and its paralog CDK19-have been reported to affect the osteoblast lineage and bone formation. This study investigated the effects of two selective CDK8/19 inhibitors on osteoclastogenesis and osteoblasts in vitro, and further evaluated how local treatment with a CDK8/19 inhibitor affects cancellous bone healing in rats. CDK8/19 inhibitors did not alter the proliferation of neither mouse bone marrow-derived macrophages (BMMs) nor primary mouse osteoblasts. Receptor activator of nuclear factor κΒ (NF-κB) ligand (RANKL)-induced osteoclastogenesis from mouse BMMs was suppressed markedly by inhibition of CDK8/19, concomitant with reduced tartrate-resistant acid phosphatase (TRAP) activity and C-terminal telopeptide of type I collagen levels. This was accompanied by downregulation of PU.1, RANK, NF-κB, nuclear factor of activated T-cells 1 (NFATc1), dendritic cell-specific transmembrane protein (DC-STAMP), TRAP, and cathepsin K in RANKL-stimulated BMMs. Downregulating RANK and its downstream signaling in osteoclast precursors enforce CDK8/19 inhibitors as anticatabolic agents to impede excessive osteoclastogenesis. In mouse primary osteoblasts, CDK8/19 inhibition did not affect differentiation but enhanced osteoblast mineralization by promoting alkaline phosphatase activity and downregulating osteopontin, a negative regulator of mineralization. In rat tibiae, a CDK8/19 inhibitor administered locally promoted cancellous bone regeneration. Our data indicate that inhibitors of CDK8/19 have the potential to develop into therapeutics to restrict osteolysis and enhance bone regeneration.

    Keywords
    CDK8, RANK, osteoblasts, osteoclasts
    National Category
    Cell and Molecular Biology Medicinal Chemistry
    Identifiers
    urn:nbn:se:liu:diva-154927 (URN)10.1002/jcp.28321 (DOI)000470174200186 ()30793301 (PubMedID)
    Note

    Funding agencies: Vetenskapsradet [521-2013-2593, 2016-06097, K2015-99x-10363-23-4, 2016-01822]; Swedish Research Council

    Available from: 2019-03-05 Created: 2019-03-05 Last updated: 2019-07-03
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    Aseptic Loosening of Orthopedic Implants: Osteoclastogenesis Regulation and Potential Therapeutics
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  • 5.
    Assenhöj, Maria
    et al.
    Linköping University, Department of Health, Medicine and Caring Sciences, Division of Prevention, Rehabilitation and Community Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Medicine Center, Occupational and Environmental Medicine Center.
    Eriksson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, Faculty of Science & Engineering.
    Donnes, Pierre
    SciCross AB, Sweden.
    Ljunggren, Stefan
    Linköping University, Department of Health, Medicine and Caring Sciences, Division of Prevention, Rehabilitation and Community Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Medicine Center, Occupational and Environmental Medicine Center.
    Marcusson-Stahl, Maritha
    Res Inst Sweden RISE, Sweden.
    Du Rietz, Anna
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, Faculty of Science & Engineering.
    Uvdal, Kajsa
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, Faculty of Science & Engineering.
    Karlsson, Helen
    Linköping University, Department of Health, Medicine and Caring Sciences, Division of Prevention, Rehabilitation and Community Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Medicine Center, Occupational and Environmental Medicine Center.
    Cederbrant, Karin
    Res Inst Sweden RISE, Sweden.
    Protein interaction, monocyte toxicity and immunogenic properties of cerium oxide crystals with 5% or 14% gadolinium, cobalt oxide and iron oxide nanoparticles - an interdisciplinary approach2021In: Nanotoxicology, ISSN 1743-5390, E-ISSN 1743-5404, Vol. 15, no 8, p. 1035-1058Article in journal (Refereed)
    Abstract [en]

    Metal oxide nanoparticles are widely used in both consumer products and medical applications, but the knowledge regarding exposure-related health effects is limited. However, it is challenging to investigate nanoparticle interaction processes with biological systems. The overall aim of this project was to improve the possibility to predict exposure-related health effects of metal oxide nanoparticles through interdisciplinary collaboration by combining workflows from the pharmaceutical industry, nanomaterial sciences, and occupational medicine. Specific aims were to investigate nanoparticle-protein interactions and possible adverse immune reactions. Four different metal oxide nanoparticles; CeOx nanocrystals with 5% or 14% Gd, Co3O4, and Fe2O3, were characterized by dynamic light scattering and high-resolution transmission electron microscopy. Nanoparticle-binding proteins were identified and screened for HLA-binding peptides in silico. Monocyte interaction with nanoparticle-protein complexes was assessed in vitro. Herein, for the first time, immunogenic properties of nanoparticle-binding proteins have been characterized. The present study indicates that especially Co3O4-protein complexes can induce both danger signals, verified by the production of inflammatory cytokines and simultaneously bind autologous proteins, which can be presented as immunogenic epitopes by MHC class II. The clinical relevance of these findings should be further evaluated to investigate the role of metal oxide nanoparticles in the development of autoimmune disease. The general workflow identified experimental difficulties, such as nanoparticle aggregate formation and a lack of protein-free buffers suitable for particle characterization, protein analyses, as well as for cell studies. This confirms the importance of future interdisciplinary collaborations.

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  • 6. Order onlineBuy this publication >>
    Backteman, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Inflammation Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Immunology and Transfusion Medicine.
    T Cells and NK Cells in Coronary Artery Disease: Longitudinal and methodological studies in humans2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Coronary artery disease (CAD) is the leading cause of death worldwide and most often due to atherosclerosis. Atherosclerosis is a chronic inflammatory process that involves the arteries, inclouding those that supply blood to the heart muscle. Although inflammation is an important contributing factor to atherosclerosis, the mechanisms are not fully understood. One mechanism contributing to atherogenesis may involve some infectious microorganisms such as cytomegalovirus (CMV). In atherosclerosis, the arterial wall becomes infiltrated with lipids followed by different types of leukocytes and inflammatory mediators (atherogenesis). Leukocytes recirculate continuously between the blood and lymphoid organs, such as lymph nodes, where the adaptive immune response is started and regulated.

    The general aim of this thesis was to increase the understanding of associations between lymphocyte populations and different conditions of CAD (unstable and stable). To assess changes over time, a longitudinal follow up design was mostly used. Therefore, also perspectives of longitudinal variation were included in the thesis.

    Paper I showed that flow cytometric evaluation of lymphocyte populations is a robust technique that can be used in longitudinal studies, both in clinical and research settings. It was also shown that the time of sampling over the year did not have a major impact on the findings.

    In paper II, thoracic lymph nodes were investigated to assess whether CAD-associated changes were more prominent in comparison with blood. As expected, there were several major differences in lymphocyte composition between lymph nodes and blood. However, the analysis of thoracic lymph nodes did not reveal any further changes that were not detected in blood. Thus, blood is still the most reliable compartment for studies of lymphocyte populations in CAD since it is not possible to examine the local findings in the artery wall.

    Natural killer (NK) cells are innate lymphocytes with both regulatory and effector functions. In paper II and III we confirmed previous findings that CAD patients have lower proportions of NK cells in blood. However, the NK subtype and cytokine profile (paper III, measured by subtype markers and intra-cellular cytokine staining) did not differ between patients and controls. During a 12-month follow-up, the proportions of NK cells increased, although not in all patients. Failure to reconstitute NK cell levels was associated with several components of the metabolic syndrome and with a persistent low-grade inflammation as measured by plasma IL-6 levels. The findings support the notion of a protective role for NK cells in inflammation.

    CD4+ but not CD8+ T cells were significantly increased in patients with both unstable and stable conditions compared with healthy individuals (paper IV). Subpopulations of CD4+ T cells (CD4+CD28null) have previously been associated with CAD. However, we show that CD28null and CD28null57+ cells within the CD4+ and CD8+ T cell populations were similar in CAD patients and healthy controls. Instead, CMV seropositivity was the major determinant of expanded CD28null and CD57+ T cell fractions in both patients and healthy individuals. During the 1 year follow up the proportion of CD4+CD28null and CD8+CD28null cells increased in patients, which may reflect an accelerated immunological ageing occurring after the cardiac event.

    List of papers
    1. Biological and methodological variation of lymphocyte subsets in blood of human adults
    Open this publication in new window or tab >>Biological and methodological variation of lymphocyte subsets in blood of human adults
    2007 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 322, no 1-2, p. 20-27Article in journal (Refereed) Published
    Abstract [en]

    Although lymphocyte populations are often monitored over time, information about the biological variation over time is limited. Three-colour-flow cytometry was used to investigate the biological and methodological variation of lymphocyte populations in blood. Fifteen healthy individuals (11 females and 4 males) were longitudinally monitored for 2-8 years. Blood samples were drawn monthly when possible. In total, 493 observations were included. Absolute counts and proportions were determined for T-cells (CD3+), T-helper cells (CD3+ CD4+), cytolytic T-cells (CD3+ CD8+), B-cells (CD3- CD19+) and NK-cells (CD3- CD16+/56+). As to variation over the year, ANOVA testing showed only a minor monthly variation for absolute counts of the CD8+ population (p < 0.05) for October compared with June and July, whereas no significant differences were found for the other populations or in the proportions of lymphocyte subsets. Although lower than the longitudinal variation, the methodological variation, expressed as coefficient of variation (CV %), was in a similar range as the variation over time, indicating that the normal biological variation should not be overestimated, while the methodological inter-assay should be taken into consideration in longitudinal studies or monitoring of patients. © 2007 Elsevier B.V. All rights reserved.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-41169 (URN)10.1016/j.jim.2007.01.021 (DOI)55291 (Local ID)55291 (Archive number)55291 (OAI)
    Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2020-01-16
    2. Lymphocyte Subpopulations in Lymph Nodes and Peripheral Blood: A Comparison between Patients with Stable Angina and Acute Coronary Syndrome
    Open this publication in new window or tab >>Lymphocyte Subpopulations in Lymph Nodes and Peripheral Blood: A Comparison between Patients with Stable Angina and Acute Coronary Syndrome
    2012 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 7, no 3Article in journal (Refereed) Published
    Abstract [en]

    Objective: Atherosclerosis is characterized by a chronic inflammatory response involving activated T cells and impairment of natural killer (NK) cells. An increased T cell activity has been associated with plaque instability and risk of acute cardiac events. Lymphocyte analyses in blood are widely used to evaluate the immune status. However, peripheral blood contains only a minor proportion of lymphocytes. In this study, we hypothesized that thoracic lymph nodes from patients with stable angina (SA) and acute coronary syndrome (ACS) might add information to peripheral blood analyses. less thanbrgreater than less thanbrgreater thanMethods: Peripheral blood and lymph nodes were collected during coronary by-pass surgery in 13 patients with SA and 13 patients with ACS. Lymphocyte subpopulations were assessed by flow cytometry using antibodies against CD3, CD4, CD8, CD19, CD16/56, CD25, Foxp3, CD69, HLA-DR, IL-18 receptor (R) and CCR4. less thanbrgreater than less thanbrgreater thanResults: Lymph nodes revealed a lymphocyte subpopulation profile substantially differing from that in blood including a higher proportion of B cells, lower proportions of CD8(+) T cells and NK cells and a 2-fold higher CD4/CD8 ratio. CD4(+)CD69(+) cells as well as Foxp3(+) regulatory T cells were markedly enriched in lymph nodes (p andlt; 0.001) while T helper 1-like (CD4(+)IL-18R(+)) cells were more frequent in blood (p andlt; 0.001). The only significant differences between ACS and SA patients involved NK cells that were reduced in the ACS group. However, despite being reduced, the NK cell fraction in ACS patients contained a significantly higher proportion of IL-18R(+) cells compared with SA patients (p andlt; 0.05). less thanbrgreater than less thanbrgreater thanConclusion: There were several differences in lymphocyte subpopulations between blood and lymph nodes. However, the lymphocyte perturbations in peripheral blood of ACS patients compared with SA patients were not mirrored in lymph nodes. The findings indicate that lymph node analyses in multivessel coronary artery disease may not reveal any major changes in the immune response that are not detectable in blood.

    Place, publisher, year, edition, pages
    Public Library of Science, 2012
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-77542 (URN)10.1371/journal.pone.0032691 (DOI)000303005000033 ()
    Note
    Funding Agencies|Swedish Heart-Lung Foundation|20090489|Swedish Research Council|2008-2282|Available from: 2012-05-25 Created: 2012-05-22 Last updated: 2021-06-14
    3. Natural killer (NK) cell deficit in coronary artery disease: no aberrations in phenotype but sustained reduction of NK cells is associated with low-grade inflammation
    Open this publication in new window or tab >>Natural killer (NK) cell deficit in coronary artery disease: no aberrations in phenotype but sustained reduction of NK cells is associated with low-grade inflammation
    2014 (English)In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 175, no 1, p. 104-112Article in journal (Refereed) Published
    Abstract [en]

    Although reduced natural killer (NK) cell levels have been reported consistently in patients with coronary artery disease (CAD), the clinical significance and persistence of this immune perturbation is not clarified. In this study we characterized the NK cell deficit further by determining (i) differentiation surface markers and cytokine profile of NK cell subsets and (ii) ability to reconstitute NK cell levels over time. Flow cytometry was used to analyse NK cell subsets and the intracellular cytokine profile in 31 patients with non-ST elevation myocardial infarction (non-STEMI), 34 patients with stable angina (SA) and 37 healthy controls. In blood collected prior to coronary angiography, the proportions of NK cells were reduced significantly in non-STEMI and SA patients compared with controls, whereas NK cell subset analyses or cytokine profile measurements did not reveal any differences across groups. During a 12-month follow-up, the proportions of NK cells increased, although not in all patients. Failure to reconstitute NK cell levels was associated with several components of metabolic syndrome. Moreover, interleukin (IL)-6 levels remained high in patients with sustained NK cell deficit, whereas a decline in IL-6 (P < 0·001) was seen in patients with a pronounced increase in NK cells. In conclusion, we found no evidence that reduction of NK cells in CAD patients was associated with aberrations in NK cell phenotype at any clinical stage of the disease. Conversely, failure to reconstitute NK cell levels was associated with a persistent low-grade inflammation, suggesting a protective role of NK cells in CAD.

    Place, publisher, year, edition, pages
    Wiley-Blackwell, 2014
    Keywords
    coronary artery disease; cytokines; inflammation; leukocytes; natural killer cell
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-103363 (URN)10.1111/cei.12210 (DOI)000329165400012 ()24298947 (PubMedID)
    Available from: 2014-01-17 Created: 2014-01-17 Last updated: 2020-01-16Bibliographically approved
    4. Cytomegalovirus seropositivity is a major determinant of CD28null T cell expansion in patients with coronary artery disease
    Open this publication in new window or tab >>Cytomegalovirus seropositivity is a major determinant of CD28null T cell expansion in patients with coronary artery disease
    2014 (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Objective: Accumulation of CD4+28null cells, with a proinflammatory and senescent phenotype, has been associated with unstable conditions of coronary artery disease (CAD). Human cytomegalovirus (HCMV) is known to exert profound effects on T cells, including loss of CD28. Here, we longitudinally assessed the proportions of CD28null and CD28nullCD57+ cells in CD4+ and CD8+ T cell populations of patients with CAD and related the findings to HCMV seropositivity.

    Methods: HCMV antibody levels and expression of CD28 and CD57 on CD4+ and CD8+ T cells were analysed in 31 patients with acute coronary syndrome (ACS), 34 patients with stable angina (SA) and 37 healthy controls. Samples were taken prior to 34 coronary angiography and after 3 and 12 months. In a subsample, HCMV-specific IFN-γ and  TNF production was assessed ex vivo.

    Results: Increased proportions of CD4+CD28null, but not CD8+CD28null cells, were significantly associated with presence of CAD. Significant increases in CD28null 37 and CD28nullCD57+ cells occurred within CD4+ and CD8+ T cell compartments in both ACS and SA patients during 12-month follow-up. HCMV was the major determinant of CD28null and CD28nullCD57+ T cell levels in both patients and controls (p <0.001). There were no obvious signs of CMV reactivation in patients.

    Conclusion: HCMV was a major determinant of the presence of CD28null and CD28nullCD57+ T cells in patients with CAD, independent of clinical stage. Findings also indicate that HCMV might have a large impact on the T cell aging process that occurred in patients after a cardiac event.

    Keywords
    Coronary artery disease, acute coronary syndrome, CD28null T cells, CD57+ 49 T cells, Human cytomegalovirus
    National Category
    Clinical Medicine Immunology
    Identifiers
    urn:nbn:se:liu:diva-111049 (URN)
    Available from: 2014-10-06 Created: 2014-10-06 Last updated: 2020-01-16Bibliographically approved
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    T Cells and NK Cells in Coronary Artery Disease: Longitudinal and methodological studies in humans
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  • 7. Order onlineBuy this publication >>
    Bahrampour, Shahrzad
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Genetic mechanisms regulating proliferation and cell specification in the Drosophila embryonic CNS2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The central nervous system (CNS) consists of an enormous number of cells, and large cellular variance, integrated into an elaborate network. The CNS is the most complex animal organ, and therefore its establishment must be controlled by many different genetic programs. Considering the high level of complexity in the human CNS, addressing issues related to human neurodevelopment represents a major challenge. Since comparative studies have revealed that neurodevelopmental programs are well conserved through evolution, on both the genetic and functional levels, studies on invertebrate neurodevelopmental programs are often translatable to vertebrates. Indeed, the basis of our current knowledge about vertebrate CNS development has been greatly aided by studies on invertebrates, and in particular on the Drosophila melanogaster (fruit fly) model system.

    This thesis attempted to identify novel genes regulating neural cell specification and proliferation in the CNS, using the Drosophila model system. Moreover, I aimed to address how those genes govern neural progenitor cells (neuroblasts; NBs) to obtain/maintain their stemness identity and proliferation capacity, and how they drive NBs through temporal windows and series of programmed asymmetric division, which gradually reduces their stemness identity in favor of neural differentiation, resulting in appropriate lineage progression. In the first project, we conducted a forward genetic screen in Drosophila embryos, aimed at isolating genes involved in regulation of neural proliferation and specification, at the single cell resolution. By taking advantage of the restricted expression of the neuropeptide FMRFa in the last-born cell of the NB lineage 5-6T, the Ap4 neuron, we could monitor the entire lineage progression. This screen succeeded in identifying 43 novel genes controlling different aspects of CNS development. One of the genes isolated, Ctr9, displayed extra Ap4/FMRFa neurons. Ctr9 encodes a component of the RNA polymerase II complex Paf1, which is involved in a number of transcriptional processes. The Paf1C, including Ctr9, is highly conserved from yeast to human, and in the past couple of years, its importance for transcription has become increasingly appreciated. However, studies in the Drosophila system have been limited. In the screen, we isolated the first mutant of Drosophila Ctr9 and conducted the first detailed phenotypic study on its function in the Drosophila embryonic CNS. Loss of function of Ctr9 leads to extra NB numbers, higher proliferation ratio and lower expression of neuropeptides. Gene expression analysis identified several other genes regulated by Ctr9, which may explain the Ctr9 mutant phenotypes. In summary, we identified Ctr9 as an essential gene for proper CNS development in Drosophila, and this provides a platform for future study on the Drosophila Paf1C. Another interesting gene isolated in the screen was worniou (wor), a member of the Snail family of transcription factors. In contrast to Ctr9, whichdisplayed additional Ap4/FMRFa neurons, wor mutants displayed a loss of these neurons. Previous studies in our group have identified many genes acting to stop NB lineage progression, but how NBs are pushed to proliferate and generate their lineages was not well known. Since wor may constitute a “driver” of proliferation, we decided to study it further. Also, we identified five other transcription factors acting together with Wor as pro-proliferative in both NBs and their daughter cells. These “drivers” are gradually replaced by the previously identified late-acting “stoppers.” Early and late factors regulate each other and the cell cycle, and thereby orchestrate proper neural lineage progression.

    List of papers
    1. Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
    Open this publication in new window or tab >>Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
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    2015 (English)In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 200, no 4, p. 1229-1244Article in journal (Refereed) Published
    Abstract [en]

    The expression of neuropeptides is often extremely restricted in the nervous system, making them powerful markers for addressing cell specification . In the developing Drosophila ventral nerve cord, only six cells, the Ap4 neurons, of some 10,000 neurons, express the neuropeptide FMRFamide (FMRFa). Each Ap4/FMRFa neuron is the last-born cell generated by an identifiable and well-studied progenitor cell, neuroblast 5-6 (NB5-6T). The restricted expression of FMRFa and the wealth of information regarding its gene regulation and Ap4 neuron specification makes FMRFa a valuable readout for addressing many aspects of neural development, i.e., spatial and temporal patterning cues, cell cycle control, cell specification, axon transport, and retrograde signaling. To this end, we have conducted a forward genetic screen utilizing an Ap4-specific FMRFa-eGFP transgenic reporter as our readout. A total of 9781 EMS-mutated chromosomes were screened for perturbations in FMRFa-eGFP expression, and 611 mutants were identified. Seventy-nine of the strongest mutants were mapped down to the affected gene by deficiency mapping or whole-genome sequencing. We isolated novel alleles for previously known FMRFa regulators, confirming the validity of the screen. In addition, we identified novel essential genes, including several with previously undefined functions in neural development. Our identification of genes affecting most major steps required for successful terminal differentiation of Ap4 neurons provides a comprehensive view of the genetic flow controlling the generation of highly unique neuronal cell types in the developing nervous system.

    Place, publisher, year, edition, pages
    Genetics Society of America, 2015
    Keywords
    Drosophila; CNS development; neural cell fate specification; forward genetic screening; FMRFamide
    National Category
    Clinical Medicine
    Identifiers
    urn:nbn:se:liu:diva-121318 (URN)10.1534/genetics.115.178483 (DOI)000359917000020 ()26092715 (PubMedID)
    Available from: 2015-09-16 Created: 2015-09-14 Last updated: 2019-03-13Bibliographically approved
    2. Ctr9, a Key Component of the Paf1 Complex, Affects Proliferation and Terminal Differentiation in the Developing Drosophila Nervous System
    Open this publication in new window or tab >>Ctr9, a Key Component of the Paf1 Complex, Affects Proliferation and Terminal Differentiation in the Developing Drosophila Nervous System
    2016 (English)In: G3: Genes, Genomes, Genetics, E-ISSN 2160-1836, Vol. 6, no 10, p. 3229-3239Article in journal (Refereed) Published
    Abstract [en]

    The Paf1 protein complex (Paf1C) is increasingly recognized as a highly conserved and broadly utilized regulator of a variety of transcriptional processes. These include the promotion of H3K4 and H3K36 trimethylation, H2BK123 ubiquitination, RNA Pol II transcriptional termination, and also RNA-mediated gene silencing. Paf1C contains five canonical protein components, including Paf1 and Ctr9, which are critical for overall complex integrity, as well as Rtf1, Leo1, and Cdc73/Parafibromin(Hrpt2)/Hyrax. In spite of a growing appreciation for the importance of Paf1C from yeast and mammalian studies, there has only been limited work in Drosophila. Here, we provide the first detailed phenotypic study of Ctr9 function in Drosophila. We found that Ctr9 mutants die at late embryogenesis or early larval life, but can be partly rescued by nervous system reexpression of Ctr9. We observed a number of phenotypes in Ctr9 mutants, including increased neuroblast numbers, increased nervous system proliferation, as well as downregulation of many neuropeptide genes. Analysis of cell cycle and regulatory gene expression revealed upregulation of the E2f1 cell cycle factor, as well as changes in Antennapedia and Grainy head expression. We also found reduction of H3K4me3 modification in the embryonic nervous system. Genome-wide transcriptome analysis points to additional downstream genes that may underlie these Ctr9 phenotypes, revealing gene expression changes in Notch pathway target genes, cell cycle genes, and neuropeptide genes. In addition, we find significant effects on the gene expression of metabolic genes. These findings reveal that Ctr9 is an essential gene that is necessary at multiple stages of nervous system development, and provides a starting point for future studies of the Paf1C in Drosophila.

    Place, publisher, year, edition, pages
    Genetics Society of America, 2016
    Keywords
    neuroblast, lineage tree, cell cycle, epigenetics, terminal differentiation, FlyBook
    National Category
    Genetics
    Identifiers
    urn:nbn:se:liu:diva-132856 (URN)10.1534/g3.116.034231 (DOI)000386581200018 ()27520958 (PubMedID)
    Note

    Funding Agencies|Swedish Research Council [621-2013-5258]; Knut and Alice Wallenberg Foundation [KAW2011.0165]; Swedish Cancer Foundation [120531]; Swedish Royal Academy of Sciences

    Available from: 2016-12-06 Created: 2016-11-30 Last updated: 2024-01-17
    3. Neural Lineage Progression Controlled by a Temporal Proliferation Program.
    Open this publication in new window or tab >>Neural Lineage Progression Controlled by a Temporal Proliferation Program.
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    2017 (English)In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 43, no 3, p. 332-348Article in journal (Refereed) Published
    Abstract [en]

    Great progress has been made in identifying transcriptional programs that establish stem cell identity. In contrast, we have limited insight into how these programs are down-graded in a timely manner to halt proliferation and allow for cellular differentiation. Drosophila embryonic neuroblasts undergo such a temporal progression, initially dividing to bud off daughters that divide once (type I), then switching to generating non-dividing daughters (type 0), and finally exiting the cell cycle. We identify six early transcription factors that drive neuroblast and type I daughter proliferation. Early factors are gradually replaced by three late factors, acting to trigger the type I→0 daughter proliferation switch and eventually to stop neuroblasts. Early and late factors regulate each other and four key cell-cycle genes, providing a logical genetic pathway for these transitions. The identification of this extensive driver-stopper temporal program controlling neuroblast lineage progression may have implications for studies in many other systems.less thanbr /greater than (Copyright © 2017 Elsevier Inc. All rights reserved.)

    Place, publisher, year, edition, pages
    Cell Press, 2017
    National Category
    Developmental Biology
    Identifiers
    urn:nbn:se:liu:diva-143117 (URN)10.1016/j.devcel.2017.10.004 (DOI)000414584300011 ()29112852 (PubMedID)
    Note

    Funding agencies: Swedish Research Council [621-2013-5258]; Knut and Alice Wallenberg Foundation [KAW2011.0165, KAW2012.0101]; Swedish Cancer Foundation [140780, 150633]

    Available from: 2017-11-20 Created: 2017-11-20 Last updated: 2017-11-20Bibliographically approved
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  • 8.
    Barczyk, K.
    et al.
    Department of Immunology, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland; Institute of Experimental Dermatology, University of Münster, Münster, Germany.
    Kreuter, M.
    Department of Medicine/Hematology and Oncology, University of Münster, Münster, Germany.
    Pryjma, J.
    Department of Immunology, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland.
    Booy, Evan P.
    Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, Univ. Manitoba, Winnipeg, Canada.
    Maddika, Subbareddy
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Department of Biochemistry and Medical Genetics,University of Manitoba, Winnipeg, Canada .
    Ghavami, Saeid
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Berdel, W. E.
    Department of Medicine/Hematology and Oncology, University of Münster, Münster, Germany.
    Roth, J.
    Institute of Experimental Dermatology, University of Münster, Münster, Germany.
    Los, Marek Jan
    Institute of Experimental Dermatology, University of Münster, Münster, Germany Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Serum cytochrome c indicates in vivo apoptosis and can serve as a prognostic marker during cancer therapy2005In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 116, no 2, p. 167-173Article in journal (Refereed)
    Abstract [en]

    Despite significant progress in cancer therapy, the outcome of the treatment is often unfavorable. Better treatment monitoring would not only allow an individual more effective, patient-adjusted therapy, but also it would eliminate some of the side effects. Using a cytochrome c ELISA that was modified to increase sensitivity, we demonstrate that serum cytochrome c is a sensitive apoptotic marker in vivo reflecting therapy-induced cell death burden. Furthermore, increased serum cytochrome c level is a negative prognostic marker. Cancer patients whose serum cytochrome c level was normal 3 years ago have a twice as high probability to be still alive, as judged from sera samples collected for years, analyzed recently and matched with survival data. Moreover, we show that serum cytochrome c and serum LDH-activity reflect different stages and different forms of cell death. Cellular cytochrome c release is specific for apoptosis, whereas increased LDH activity is an indicator of (secondary) necrosis. Whereas serum LDH activity reflects the "global" degree of cell death over a period of time, the sensitive cytochrome c-based method allows confirmation of the individual cancer therapy-induced and spontaneous cell death events. The combination of cytochrome c with tissue-specific markers may provide the foundation for precise monitoring of apoptosis in vivo, by "lab-on-the-chip" technology. (c) 2005 Wiley-Liss, Inc.

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  • 9.
    Baumgartner, Johanna
    et al.
    Linköping University, Faculty of Science & Engineering. Linköping University, Department of Physics, Chemistry and Biology, Sensor and Actuator Systems.
    Jönsson, Jan-Ingvar
    Linköping University, Faculty of Medicine and Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Division of Hematopoiesis and Developmental Biology.
    Jager, Edwin W. H.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor and Actuator Systems. Linköping University, Faculty of Science & Engineering.
    Switchable presentation of cytokines on electroactive polypyrrole surfaces for hematopoietic stem and progenitor cells2018In: Journal of Materials Chemistry B, ISSN 2050-750X, Vol. 6, no 28, p. 4665-4675Article in journal (Refereed)
    Abstract [en]

    Hematopoietic stem cells are used in transplantations for patients with hematologic malignancies. Scarce sources require efficient strategies of expansion, including polymeric biomaterials mimicking architectures of bone marrow tissue. Tissue microenvironment and mode of cytokine presentation strongly influence cell fate. Although several cytokines with different functions as soluble or membrane-bound mediators have already been identified, their precise roles have not yet been clarified. A need exists for in vitro systems that mimic the in vivo situation to enable such studies. One way is to establish surfaces mimicking physiological presentation using protein-immobilization onto polymer films. However these films merely provide a static presentation of the immobilized proteins. It would be advantageous to also dynamically change protein presentation and functionality to better reflect the in vivo conditions. The electroactive polymer polypyrrole shows excellent biocompatibility and electrochemically alters its surface properties, becoming an interesting choice for such setups. Here, we present an in vitro system for switchable presentation of membrane-bound cytokines. We use interleukin IL-3, known to affect hematopoiesis, and show that when immobilized on polypyrrole films, IL-3 is bioavailable for the bone marrow-derived FDC-P1 progenitor cell line. Moreover, IL-3 presentation can be successfully altered by changing the redox state of the film, in turn influencing FDC-P1 cell viability. This novel in vitro system provides a valuable tool for stimuli-responsive switchable protein presentation allowing the dissection of relevant mediators in stem and progenitor cell behavior.

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    Switchable presentation of cytokines on electroactive polypyrrole surfaces for hematopoietic stem and progenitor cells
  • 10.
    Belogurov, G A
    et al.
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Fabrichniy, I P
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Pohjanjoki, P
    Department of Biochemistry, University of Turku, Turku, Finland.
    Kasho, V N
    Center for Ulcer Research and Education, Department of Medicine, University of California, Los Angeles, California, USA.
    Lehtihuhta, E
    Department of Biochemistry, University of Turku, Turku, Finland.
    Turkina, Maria V
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Cooperman, B S
    Department of Chemistry, University of Pennsylvania, Pennsylvania, USA.
    Goldman, A
    Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
    Baykov, A A
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Lahti, R
    Department of Biochemistry, University of Turku, Turku, Finland.
    Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase2000In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, no 45, p. 13931-13938Article in journal (Refereed)
    Abstract [en]

    Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.

  • 11.
    Belogurov, Georgiy A
    et al.
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland; A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow, Russia.
    Malinen, Anssi M
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Turkina, Maria V
    A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow, Russia.
    Jalonen, Ulla
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Rytkönen, Kalle
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Baykov, Alexander A
    A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow, Russia.
    Lahti, Reijo
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 6, p. 2088-2096Article in journal (Refereed)
    Abstract [en]

    Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.

  • 12.
    Belogurov, Georgiy A
    et al.
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland; A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Turkina, Maria V
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Penttinen, Anni
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Huopalahti, Saila
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Baykov, Alexander A
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Lahti, Reijo
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 25Article in journal (Refereed)
    Abstract [en]

    H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.

  • 13.
    Bettiga, Arianna
    et al.
    IRCCS Osped San Raffaele, Italy.
    Aureli, Massimo
    University of Milan, Italy.
    Colciago, Giorgia
    IRCCS Osped San Raffaele, Italy.
    Murdica, Valentina
    University of Milan, Italy.
    Moschini, Marco
    IRCCS Osped San Raffaele, Italy.
    Luciano, Roberta
    IRCCS Osped San Raffaele, Italy.
    Canals, Daniel
    SUNY Stony Brook, NY 11794 USA.
    Hannun, Yusuf
    SUNY Stony Brook, NY 11794 USA.
    Hedlund, Petter
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pharmacology. IRCCS Osped San Raffaele, Italy.
    Lavorgna, Giovanni
    IRCCS Osped San Raffaele, Italy.
    Colombo, Renzo
    IRCCS Osped San Raffaele, Italy.
    Bassi, Rosaria
    University of Milan, Italy.
    Samarani, Maura
    University of Milan, Italy.
    Montorsi, Francesco
    IRCCS Osped San Raffaele, Italy; University of Vita Salute San Raffaele, Italy.
    Salonia, Andrea
    University of Vita Salute San Raffaele, Italy.
    Benigni, Fabio
    IRCCS Osped San Raffaele, Italy.
    Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism2017In: Scientific Reports, E-ISSN 2045-2322, Vol. 7, article id 42157Article in journal (Refereed)
    Abstract [en]

    The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p amp;lt; 0.01) Gb3 ganglioside (-50 +/- 3%) and sphingosine 1-phosphate (S1P, -40 +/- 4%), which ended up to reduction in cell motility (-46 +/- 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility.

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  • 14. Order onlineBuy this publication >>
    Bivik Stadler, Caroline
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Genetic pathways controlling CNS development: The role of Notch signaling in regulating daughter cell proliferation in Drosophila2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The human central nervous system (CNS) displays the greatest cellular diversity of any organ system, consisting of billions of neurons, of numerous cell sub-types, interconnected in a vast network. Given this enormous complexity, decoding the genetic programs controlling the multistep process of CNS development remains a major challenge. While great progress has been made with respect to understanding sub-type specification, considerably less is known regarding how the generation of the precise number of each sub-type is controlled.

    The aim of this thesis was to gain deeper knowledge into the regulatory programs controlling cell specification and proliferation. To address these questions I have studied the Drosophila embryonic CNS as a model system, to thereby be able to investigate the genetic mechanisms at high resolution. Despite the different size and morphology between the Drosophila and the mammalian CNS, the lineages of their progenitors share similarity. Importantly for this thesis, both species progenitors show elaborate variations in their proliferation modes, either giving rise to daughters that can directly differentiate into neurons or glia (type 0), divide once (type I), or multiple times (type II).

    The studies launched off with a comprehensive chemical forward genetic screen, for the very last born cell in the well-studied lineage of progenitor NB5-6T: the Ap4 neuron, which expresses the neuropeptide FMRFa. NB5-6T is a powerful model to use, because it undergoes a programmed type I>0 daughter cell proliferation switch. An FMRF-eGFP transgenic reporter was utilized as readout for successful terminal differentiation of Ap4/FMRFa and thereby proper lineage progression of the ∼20 cells generated. The strongest mutants were mapped to genes with both known and novel essential functions e.g., spatial and temporal patterning, cell cycle control, cell specification and chromatin modification. Subsequently, we focused on some of the genes that showed a loss of function phenotype with an excess of lineage cells. We found that Notch is critical for the type I>0 daughter cell proliferation switch in the NB5-6T lineage and globally as well. When addressing the broader relevance of these findings, and to further decipher the Notch pathway, we discovered that selective groups of E(spl) genes is controlling the switch in a close interplay with four key cell cycle factors: Cyclin E, String, E2F and Dacapo, in most if not all embryonic progenitors. The Notch mediation of the switch is likely to be by direct transcriptional regulation. Furthermore, another gene identified in the screen, sequoia, was investigated. The analysis revealed that sequoia is also controlling the daughter cell switch in the CNS, and this partly through context dependent interactions with the Notch pathway.

    Taken together, the findings presented in this thesis demonstrate that daughter cell proliferation switches in Drosophila neural lineages are genetically programmed, and that Notch contributes to the triggering of these events. Given that early embryonic processes is frequently shown to be evolutionary conserved, you can speculate that changeable daughter proliferation programs could be applied to mammals, and contribute to a broader understanding of proliferation processes in humans as well.

     

    List of papers
    1. Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
    Open this publication in new window or tab >>Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
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    2015 (English)In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 200, no 4, p. 1229-1244Article in journal (Refereed) Published
    Abstract [en]

    The expression of neuropeptides is often extremely restricted in the nervous system, making them powerful markers for addressing cell specification . In the developing Drosophila ventral nerve cord, only six cells, the Ap4 neurons, of some 10,000 neurons, express the neuropeptide FMRFamide (FMRFa). Each Ap4/FMRFa neuron is the last-born cell generated by an identifiable and well-studied progenitor cell, neuroblast 5-6 (NB5-6T). The restricted expression of FMRFa and the wealth of information regarding its gene regulation and Ap4 neuron specification makes FMRFa a valuable readout for addressing many aspects of neural development, i.e., spatial and temporal patterning cues, cell cycle control, cell specification, axon transport, and retrograde signaling. To this end, we have conducted a forward genetic screen utilizing an Ap4-specific FMRFa-eGFP transgenic reporter as our readout. A total of 9781 EMS-mutated chromosomes were screened for perturbations in FMRFa-eGFP expression, and 611 mutants were identified. Seventy-nine of the strongest mutants were mapped down to the affected gene by deficiency mapping or whole-genome sequencing. We isolated novel alleles for previously known FMRFa regulators, confirming the validity of the screen. In addition, we identified novel essential genes, including several with previously undefined functions in neural development. Our identification of genes affecting most major steps required for successful terminal differentiation of Ap4 neurons provides a comprehensive view of the genetic flow controlling the generation of highly unique neuronal cell types in the developing nervous system.

    Place, publisher, year, edition, pages
    Genetics Society of America, 2015
    Keywords
    Drosophila; CNS development; neural cell fate specification; forward genetic screening; FMRFamide
    National Category
    Clinical Medicine
    Identifiers
    urn:nbn:se:liu:diva-121318 (URN)10.1534/genetics.115.178483 (DOI)000359917000020 ()26092715 (PubMedID)
    Available from: 2015-09-16 Created: 2015-09-14 Last updated: 2019-03-13Bibliographically approved
    2. Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression
    Open this publication in new window or tab >>Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression
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    2012 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 139, no 4, p. 678-689Article in journal (Refereed) Published
    Abstract [en]

    During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. Here, we identify two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers.

    Place, publisher, year, edition, pages
    Company of Biologists, 2012
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-74790 (URN)10.1242/dev.074500 (DOI)000300259800005 ()
    Note

    funding agencies|Swedish Research Council||Knut and Alice Wallenberg foundation||Swedish Cancer Foundation||

    Available from: 2012-02-08 Created: 2012-02-08 Last updated: 2019-03-13
    3. Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling
    Open this publication in new window or tab >>Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling
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    2016 (English)In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 4, article id e1005984Article in journal (Refereed) Published
    Abstract [en]

    The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21(CIP1)/p27(KIP1)/p57(Kip2)). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems.

    Place, publisher, year, edition, pages
    PUBLIC LIBRARY SCIENCE, 2016
    National Category
    Clinical Medicine
    Identifiers
    urn:nbn:se:liu:diva-128759 (URN)10.1371/journal.pgen.1005984 (DOI)000375231900032 ()27070787 (PubMedID)
    Note

    Funding Agencies|Knut and Alice Wallenberg Foundation [KAW2012.0101]; Swedish Research Council [621-2010-5214]; Swedish Cancer Foundation [120531]

    Available from: 2016-05-31 Created: 2016-05-30 Last updated: 2022-09-13
    4. sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system
    Open this publication in new window or tab >>sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system
    2016 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 143, no 20, p. 3774-3784Article in journal (Refereed) Published
    Abstract [en]

    Neural progenitors typically divide asymmetrically to renew themselves, while producing daughters with more limited potential. In the Drosophila embryonic ventral nerve cord, neuroblasts initially produce daughters that divide once to generate two neurons/glia (type I proliferation mode). Subsequently, many neuroblasts switch to generating daughters that differentiate directly (type 0). This programmed type I&gt;0 switch is controlled by Notch signaling, triggered at a distinct point of lineage progression in each neuroblast. However, how Notch signaling onset is gated was unclear. We recently identified Sequoia (Seq), a C2H2 zinc-finger transcription factor with homology to Drosophila Tramtrack (Ttk) and the positive regulatory domain (PRDM) family, as important for lineage progression. Here, we find that seq mutants fail to execute the type I&gt;0 daughter proliferation switch and also display increased neuroblast proliferation. Genetic interaction studies reveal that seq interacts with the Notch pathway, and seq furthermore affects expression of a Notch pathway reporter. These findings suggest that seq may act as a context-dependent regulator of Notch signaling, and underscore the growing connection between Seq, Ttk, the PRDM family and Notch signaling.

    Place, publisher, year, edition, pages
    The Company of Biologists Ltd, 2016
    Keywords
    Lineage tree, Cell cycle, Asymmetric division, Combinatorial control, Notch
    National Category
    Cell and Molecular Biology Biochemistry and Molecular Biology Cell Biology Medical Biotechnology
    Identifiers
    urn:nbn:se:liu:diva-132739 (URN)10.1242/dev.139998 (DOI)000393452500013 ()27578794 (PubMedID)
    Note

    Funding agencies: Swedish Research Council (Vetenskapsradet); Knut and Alice Wallenberg Foundation (Knut och Alice Wallenbergs Stiftelse); Swedish Cancer Foundation (Cancerfonden)

    Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2019-03-13Bibliographically approved
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    Genetic pathways controlling CNS development: The role of Notch signaling in regulating daughter cell proliferation in Drosophila
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  • 15.
    Bivik Stadler, Caroline
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Hematopoiesis and Developmental Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Arefin, Md Badrul
    Linköping University, Department of Clinical and Experimental Medicine, Division of Hematopoiesis and Developmental Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Ekman, Helen
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology, Infection and Inflammation. Linköping University, Faculty of Medicine and Health Sciences.
    Thor, Stefan
    Linköping University, Department of Clinical and Experimental Medicine, Division of Hematopoiesis and Developmental Biology. Linköping University, Faculty of Medicine and Health Sciences. Univ Queensland, Australia.
    PIP degron-stabilized Dacapo/p21(Cip)(1) and mutations in ago act in an anti- versus pro-proliferative manner, yet both trigger an increase in Cyclin E levels2019In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 146, no 13, article id UNSP dev175927Article in journal (Refereed)
    Abstract [en]

    During cell cycle progression, the activity of the CycE-Cdk2 complex gates S-phase entry. CycE-Cdk2 is inhibited by CDK inhibitors (CKIs) of the Cip/Kip family, which include the human p21(Cip)(1) and Drosophila Dacapo (Dap) proteins. Both the CycE and Cip/Kip family proteins are under elaborate control via protein degradation, mediated by the Cullin-RING ligase (CRL) family of ubiquitin ligase complexes. The CRL complex SCFFoxw7/Ago targets phosphorylated CycE, whereas p21(Cip)(1) and Dap are targeted by the CRLCdf2 complex, binding to the PIP degron. The role of CRL-mediated degradation of CycE and Cip/Kip proteins during CNS development is not well understood. Here, we analyse the role of ago (Fbxw7)-mediated CycE degradation, and of Dap and p21(Cip)(1) degradation during Drosophila CNS development. We find that ago mutants display over-proliferation, accompanied by elevated CycE expression levels. By contrast, expression of PIP degron mutant Dap and p21(Cip)(1) transgenes inhibit proliferation. However, surprisingly, this is also accompanied by elevated CycE levels. Hence, ago mutation and PIP degron Cip/Kip transgenic expression trigger opposite effects on proliferation, but similar effects on CycE levels.

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  • 16.
    Björk Wilhelms, Daniel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Fever: Role of brain endothelial prostaglandins2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Fever and loss of appetite are two of the most fundamental manifestations of disease. These disease symptoms, which lead to deviations from normal body temperature and food intake patterns, are seen in a vast array of infectious and inflammatory conditions. It is known that peripheral signals from the immune system are essential triggers for these responses, which are orchestrated by neuronal circuits in the brain. Due to the blood‐brain barrier, peripheral inflammatory signals require a specific mode of transmission into the brain. Such mechanisms have been proposed, but interventional studies of these mechanisms have never rendered conclusive results. In this thesis, we present the first functional evidence of cyclooxygenase 2 (COX‐2) and microsomal prostaglandin E synthase type 1 (mPGES‐1) mediated prostaglandin E2 synthesis in the blood‐brain barrier endothelial cells as a signaling mechanism in the initiation of inflammatory fever. We also show that one of the world’s most widely used antipyretics, paracetamol, acts by inhibition of COX‐2. Combined with the finding that COX‐2 and mPGES‐1 in brain endothelial cells play a key role in inflammatory fever, this finding suggests that paracetamol inhibits fever by specifically blocking prostaglandin E2 synthesis in blood‐brain barrier endothelium. In another symptom of inflammation, anorexia, the cellular origin of peripheral signals triggering acute anorexia are largely unknown. We show that the expression of myeloid differentiation primary response gene 88 (Myd88) in myeloid cells is important for the initiation of acute inflammatory anorexia and the maintenance of cancer anorexia‐cachexia.

    Taken together, these findings provide a significant advancement of our understanding of the mechanisms triggering acute inflammatory fever and anorexia and also explain the antipyretic effect of paracetamol.

    List of papers
    1. Inflammation- and tumor-induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells
    Open this publication in new window or tab >>Inflammation- and tumor-induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells
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    2013 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 27, no 5, p. 1973-1980Article in journal (Refereed) Published
    Abstract [en]

    Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll-like and IL-1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell-specific deletions. We found that MyD88-knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)-induced anorexia, displayed anorexia when transplanted with wild-type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS-induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild-type mice. Furthermore, in a model for cancer-induced anorexia-cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88-dependent signaling within the brain is not required for eliciting inflammation-induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical component for acute inflammatory-driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease.

    Place, publisher, year, edition, pages
    Federation of American Society of Experimental Biology (FASEB), 2013
    Keywords
    lipopolysaccharide; methylcholanthrene-induced sarcoma; food intake; chimeric mice; Cre-LoxP; inducible cell-specific deletion
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-96147 (URN)10.1096/fj.12-225433 (DOI)000318226100017 ()
    Available from: 2013-08-14 Created: 2013-08-14 Last updated: 2024-01-10
    2. Acetaminophen reduces lipopolysaccharide-induced fever by inhibiting cyclooxygenase-2
    Open this publication in new window or tab >>Acetaminophen reduces lipopolysaccharide-induced fever by inhibiting cyclooxygenase-2
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    2013 (English)In: Neuropharmacology, ISSN 0028-3908, E-ISSN 1873-7064, Vol. 71, p. 124-129Article in journal (Refereed) Published
    Abstract [en]

    Acetaminophen is one of the world's most commonly used drugs to treat fever and pain, yet its mechanism of action has remained unclear. Here we tested the hypothesis that acetaminophen blocks fever through inhibition of cyclooxygenase-2 (Cox-2), by monitoring lipopolysaccharide induced fever in mice with genetic manipulations of enzymes in the prostaglandin cascade. We exploited the fact that lowered levels of a specific enzyme make the system more sensitive to any further inhibition of the same enzyme. Mice were immune challenged by an intraperitoneal injection of bacterial wall lipopolysaccharide and their body temperature recorded by telemetry. We found that mice heterozygous for Cox-2, but not for microsomal prostaglandin E synthase-1 (mPGES-1), displayed attenuated fever, indicating a rate limiting role of Cox-2. We then titrated a dose of acetaminophen that did not inhibit the lipopolysaccharide-induced fever in wild-type mice. However, when the same dose of acetaminophen was given to Cox-2 heterozygous mice, the febrile response to lipopolysaccharide was strongly attenuated, resulting in an almost normalized temperature curve, whereas no difference was seen between wild-type and heterozygous mPGES-1 mice. Furthermore, the fever to intracerebrally injected prostaglandin E2 was unaffected by acetaminophen treatment. These findings reveal that acetaminophen, similar to aspirin and other non-steroidal anti-inflammatory drugs, is antipyretic by inhibiting cyclooxygenase-2, and not by inhibiting mPGES-1 or signaling cascades downstream of prostaglandin E2.

    Place, publisher, year, edition, pages
    Elsevier, 2013
    Keywords
    Fever; Cyclooxygenase-2; Cyclooxygenase-1; Microsomal prostaglandin E synthase-1; Gene dosage; Hypothalamus
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-96170 (URN)10.1016/j.neuropharm.2013.03.012 (DOI)000320424200012 ()
    Available from: 2013-08-14 Created: 2013-08-14 Last updated: 2024-01-10
    3. Deletion of Prostaglandin E-2 Synthesizing Enzymes in Brain Endothelial Cells Attenuates Inflammatory Fever
    Open this publication in new window or tab >>Deletion of Prostaglandin E-2 Synthesizing Enzymes in Brain Endothelial Cells Attenuates Inflammatory Fever
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    2014 (English)In: Journal of Neuroscience, ISSN 0270-6474, E-ISSN 1529-2401, Vol. 34, no 35, p. 11684-11690Article in journal (Refereed) Published
    Abstract [en]

    Fever is a hallmark of inflammatory and infectious diseases. The febrile response is triggered by prostaglandin E-2 synthesis mediated by induced expression of the enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1). The cellular source for pyrogenic PGE(2) remains a subject of debate; several hypotheses have been forwarded, including immune cells in the periphery and in the brain, as well as the brain endothelium. Here we generated mice with selective deletion of COX-2 and mPGES1 in brain endothelial cells. These mice displayed strongly attenuated febrile responses to peripheral immune challenge. In contrast, inflammation-induced hypoactivity was unaffected, demonstrating the physiological selectivity of the response to the targeted gene deletions. These findings demonstrate that PGE(2) synthesis in brain endothelial cells is critical for inflammation-induced fever.

    Place, publisher, year, edition, pages
    Society for Neuroscience, 2014
    Keywords
    COX-2; endothelium; fever; mPGES-1; PGE(2); prostaglandin
    National Category
    Cell and Molecular Biology Neurosciences
    Identifiers
    urn:nbn:se:liu:diva-111281 (URN)10.1523/JNEUROSCI.1838-14.2014 (DOI)000341314900017 ()25164664 (PubMedID)
    Note

    Funding Agencies|Swedish Medical Research Council; Swedish Cancer Foundation; European Research Council; Knut and Alice Wallenberg Foundation; Swedish Brain foundation; County Council of stergotland; Wenner-Gren Fellowship

    Available from: 2014-10-14 Created: 2014-10-14 Last updated: 2024-01-10
    4. Cyclooxygenase isoform exchange blocks inflammatory symptoms
    Open this publication in new window or tab >>Cyclooxygenase isoform exchange blocks inflammatory symptoms
    2014 (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Cyclooxygenase‐2 (COX‐2) is the main source of inducible prostaglandin E2 production and mediates inflammatory symptoms including fever, loss of appetite and hyperalgesia. In contrast, COX‐1 is dispensable for most inflammatory symptoms. Global deletion of COX‐2 leads to a blockade of inflammation‐induced fever and appetite loss but also to high rates of fetal mortality. The latter is unfortunate since mice without COX‐2 are powerful tools in the study of inflammation and cardiovascular medicine. The differential functionality of the COX isoforms could be due to differences in regulatory regions of the genes, leading to different expression patterns, or to differences in the coding sequence, leading to distinct functional properties of the proteins. To study this in the context of inflammatory symptoms, we used mice in which the coding sequence of COX‐2 was replaced by the corresponding sequence of COX‐1. In these mice, COX‐1 mRNA was induced by inflammation but COX‐1 protein expression did not fully mimic inflammation‐induced COX‐2 expression. Just like mice globally lacking COX‐2, these mice showed a complete lack of fever and inflammation‐induced anorexia. However, as previously reported, they displayed close to normal survival rates. This shows that the COX activity generated from the hybrid gene was strong enough to allow survival but not strong enough to mediate inflammatory symptoms, making the line an interesting alternative to COX‐2 knockouts for the study of inflammation. Our results also show that the functional differences between COX‐1 and COX‐2 in the context of inflammatory symptoms is not only dependent on the features of the promoter regions. Instead they indicate that there are fundamental differences between the isoforms at translational or posttranslational levels, which make hybrid genes less functional.

    National Category
    Cell Biology Rheumatology and Autoimmunity
    Identifiers
    urn:nbn:se:liu:diva-111725 (URN)
    Available from: 2014-10-29 Created: 2014-10-29 Last updated: 2024-01-10Bibliographically approved
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  • 17.
    Björk Wilhelms, Daniel
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Mirrasekhian, Elahe
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Engblom, David
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Cyclooxygenase isoform exchange blocks inflammatory symptoms2014Manuscript (preprint) (Other academic)
    Abstract [en]

    Cyclooxygenase‐2 (COX‐2) is the main source of inducible prostaglandin E2 production and mediates inflammatory symptoms including fever, loss of appetite and hyperalgesia. In contrast, COX‐1 is dispensable for most inflammatory symptoms. Global deletion of COX‐2 leads to a blockade of inflammation‐induced fever and appetite loss but also to high rates of fetal mortality. The latter is unfortunate since mice without COX‐2 are powerful tools in the study of inflammation and cardiovascular medicine. The differential functionality of the COX isoforms could be due to differences in regulatory regions of the genes, leading to different expression patterns, or to differences in the coding sequence, leading to distinct functional properties of the proteins. To study this in the context of inflammatory symptoms, we used mice in which the coding sequence of COX‐2 was replaced by the corresponding sequence of COX‐1. In these mice, COX‐1 mRNA was induced by inflammation but COX‐1 protein expression did not fully mimic inflammation‐induced COX‐2 expression. Just like mice globally lacking COX‐2, these mice showed a complete lack of fever and inflammation‐induced anorexia. However, as previously reported, they displayed close to normal survival rates. This shows that the COX activity generated from the hybrid gene was strong enough to allow survival but not strong enough to mediate inflammatory symptoms, making the line an interesting alternative to COX‐2 knockouts for the study of inflammation. Our results also show that the functional differences between COX‐1 and COX‐2 in the context of inflammatory symptoms is not only dependent on the features of the promoter regions. Instead they indicate that there are fundamental differences between the isoforms at translational or posttranslational levels, which make hybrid genes less functional.

  • 18.
    Bolshakova, A.V.
    et al.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Petukhova, O.A.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Pinaev, G.P.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Magnusson, Karl-Erik
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    The comparative analysis of subcellular fractionation methods for revealing of a-actinin 1 and a-actinin 4 in A431 cells2009In: Tsitologiya, ISSN 0041-3771, Vol. 51, no 2, p. 122-129Article in journal (Refereed)
    Abstract [en]

    a-Actinin 1 and a-actinin 4 belong to a family of actin-binding proteins with shared structural function and regulation of several processes in a cell. Based on previous data on different distribution of these proteins in the nucleus and cytoplasm, we have explored in detail the distribution of a-actinin 1 and a-actinin 4 in subcellular fractions in A431 cells spread on fibronectin. Several methods of subcellular fractionation were used. Complex approach allowed resuming that revealing of a-actinin isoforms in fractions depended on the composition of lysis buffer and preliminary low-temperature freezing of the cells. We have drawn a conclusion that a-actinin 4 can be found in all cytoplasmic and nuclear subfractions, while a-actinin 1 is characterized by cytoplasmic and membrane localization with specificity of its distribution tightly to the nuclear membrane.

  • 19.
    Bosagna, Carlos Guerrero
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Developmental and Epigenetic Origins of Male Reproductive Pathologies2015In: The Epigenome and Developmental Origins of Health and Disease / [ed] Cheryl Rosenfeld, Elsevier, 2015, 1, p. 171-189Chapter in book (Refereed)
    Abstract [en]

    Transgenerational epigenetic inheritance has gained increased attention due to the possibility that exposure to environmental toxicants or other stressors can induce long-lasting changes in lineages of organisms. The mechanism involves exposure of pregnant females and induction of germline epigenetic alterations in their developing embryos. This early developmental exposure generates phenotypic alterations in the adults. The germline epigenomic changes produced are then transmitted to future generations and associate with disease phenotypes in the unexposed individuals of subsequent generations. Exposures to environmental toxicants such as fungicides, pesticides, or plastic compounds have been shown in rodents to produce abnormal reproductive or metabolic phenotypes that are transgenerationally transmitted. These include transgenerational increases in the incidence of obesity, polycystic ovary syndrome (PCOS)-like symptoms, pregnancy defects, or germ cell apoptosis. Importantly, the increased incidence of these transgenerationally transmitted diseases in response to environmental exposures in animal models is sometimes drastic. The current evidence on transgenerational epigenetic inheritance observed in animal models allows predicting that environmental exposures of today's inhabitants of the world may affect the incidence of noninfectious diseases in future generations, which would be correlated with long-lasting alterations in the epigenome. The present chapter summarizes the evidence to date for transgenerational epigenetic inheritance, in both humans and animal models.

  • 20.
    Brannmark, Cecilia
    et al.
    Univ Gothenburg, Sweden.
    Kay, Emma I
    Univ Gothenburg, Sweden; AstraZeneca, Sweden.
    Örtegren Kugelberg, Unn
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Neurobiology. Linköping University, Faculty of Medicine and Health Sciences.
    Chanclon, Belen
    Univ Gothenburg, Sweden.
    Shrestha, Man Mohan
    Univ Gothenburg, Sweden.
    Asterholm, Ingrid Wernstedt
    Univ Gothenburg, Sweden.
    Strålfors, Peter
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Neurobiology. Linköping University, Faculty of Medicine and Health Sciences.
    Olofsson, Charlotta S.
    Univ Gothenburg, Sweden.
    Adiponectin is secreted via caveolin 1-dependent mechanisms in white adipocytes2020In: Journal of Endocrinology, ISSN 0022-0795, E-ISSN 1479-6805, Vol. 247, no 1, p. 25-38Article in journal (Refereed)
    Abstract [en]

    Here we have investigated the role of the protein caveolin 1 (Cav1) and caveolae in the secretion of the white adipocyte hormone adiponectin. Using mouse primary subcutaneous adipocytes genetically depleted of Cav1, we show that the adiponectin secretion, stimulated either adrenergically or by insulin, is abrogated while basal (unstimulated) release of adiponectin is elevated. Adiponectin secretion is similarly affected in wildtype mouse and human adipocytes where the caveolae structure was chemically disrupted. The altered ex vivo secretion in adipocytes isolated from Cav1 null mice is accompanied by lowered serum levels of the high-molecular weight (HMW) form of adiponectin, whereas the total concentration of adiponectin is unaltered. Interestingly, levels of HMW adiponectin are maintained in adipose tissue from Cav1-depleted mice, signifying that a secretory defect is present. The gene expression of key regulatory proteins known to be involved in cAMP/adrenergically triggered adiponectin exocytosis (the beta-3-adrenergic receptor and exchange protein directly activated by cAMP) remains intact in Cav1 null adipocytes. Microscopy and fractionation studies indicate that adiponectin vesicles do not co-localise with Cav1 but that some vesicles are associated with a specific fraction of caveolae. Our studies propose that Cav1 has an important role in secretion of HMW adiponectin, even though adiponectin-containing vesicles are not obviously associated with this protein. We suggest that Cav1, and/or the caveolae domain, is essential for the organisation of signalling pathways involved in the regulation of HMW adiponectin exocytosis, a function that is disrupted in Cav1/caveolae-depleted adipocytes.

  • 21. Order onlineBuy this publication >>
    Brodin Patcha, Veronika
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Pro- and anti-inflammatory regulation of β2 integrin signalling in human neutrophils2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The body is under constant attack from pathogens trying to slip by our immune defence. If the barrier is breached, invading pathogens enter the tissues and cause inflammation. During this process neutrophils, constituting the first line of defence, leave the bloodstream and seek out and kill the invading pathogens. The mechanisms leading to activation of receptors on neutrophils must be closely orchestrated. Pro- and anti-inflammatory substances can influence the outcome of the inflammation process by affecting the involved players. If not well balanced, inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, can be the outcome.

    The aim of this thesis was to elucidate the effect of pro- (fMLP, Leukotriene B4, and Interleukin-8) and anti- (lipoxins, aspirin and statins) inflammatory substances on the β2 integrins, mediating adhesion of neutrophils both under “normal” conditions and during coronary artery disease. More specifically, the effect of these substances on the β2 integrins were studied in regard to: i) the activity (i.e. affinity and avidity) of β2 integrins, ii) the signalling capacity of β2 integrins (i.e. detected as release of arachidonic acid, and the production of reactive oxygen species, and iii) the signal transduction mediated by the β2 integrins (i.e. phosphorylation of Pyk2).

    The pro-inflammatory substances belong to the family of chemoattractants that induces transmigration and chemotaxis. A hierarchy exists between the different family members; the end-target chemoattractants (e.g. fMLP) being more potent than intermediary chemoattractants (e.g. IL-8 and LTB4). It was found that intermediary chemoattractants regulate β2 integrins by mainly affecting the avidity of β2 integrins. End-target chemoattractants on the other hand, affected the β2 integrins by increasing the avidity and the affinity, as well as their signalling capacity.

    The anti-inflammatory substances used in this study were the exogenous aspirin and statins, and the endogenous lipoxins. In the presence of aspirin, stable analogues of lipoxin (i.e. epi-lipoxins) are formed in a trans-cellular process. Lipoxin inhibited the signalling capacity of β2 integrins mediated by intermediary chemoattractants, as well as the signal transduction induced by end-target chemoattractants. Moreover, the signalling capacity of β2 integrins in neutrophils from patients suffering from coronary artery disease (CAD) was impaired. Arachidonic acid, the precursor for both pro- and anti-inflammatory eicosanoid, induced an increase in the β2 integrin activity (both affinity and avidity), but had no effect on the signal transduction.

    In conclusion, different “roles” were observed for end-target and intermediary chemoattractants in the regulation of β2 integrins. The inhibitory effects of the anti-inflammatory lipoxins support earlier studies suggesting that these agents function as “stop signals” in inflammation. This is also confirmed by our findings in CAD patients, who have elevated levels of epi-lipoxins due to aspirin treatment. Moreover, Pyk2 was identified as a possible target for the inhibitory effect of anti-inflammatory drugs.

    List of papers
    1. Differential inside-out activation of β2 integrins by leukotriene B4 and fMLP in human neutrophils
    Open this publication in new window or tab >>Differential inside-out activation of β2 integrins by leukotriene B4 and fMLP in human neutrophils
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    2004 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 300, no 2, p. 308-319Article in journal (Refereed) Published
    Abstract [en]

    We have investigated how LTB4, an endogenous chemoattractant encountered early in the inflammatory process, and fMLP, a bacteria-derived chemotactic peptide emanating from the site of infection, mediate inside-out regulation of the β2-integrin. The role of the two chemoattractants on β2-integrin avidity was investigated by measuring their effect on β2-integrin clustering and surface mobility, whereas their effect on β2-integrin affinity was measured by the expression of a high affinity epitope, a ligand-binding domain on β2-integrins, and by integrin binding to s-ICAM. We find that the two chemoattractants modulate the β2-integrin differently. LTB4 induces an increase in integrin clustering and surface mobility, but only a modest increase in integrin affinity. fMLP evokes a large increase in β2-integrin affinity as well as in clustering and mobility. Lipoxin, which acts as a stop signal for the functions mediated by pro-inflammatory agents, was used as a tool for further examining the inside-out mechanisms. While LTB4-induced integrin clustering and mobility were inhibited by lipoxin, only a minor inhibition of fMLP-induced β2-integrin avidity and no inhibition of integrin affinity were detected. The different modes of the inside-out regulation of β2-integrins suggest that distinct mechanisms are involved in the β2-integrin modulation induced by various chemoattractants.

    Keywords
    β2-Integrins, Cell adhesion, Chemotactic factors, Eicosanoids, Inflammation, Leukotriene B4, Lipoxins, Human Neutrophils, Signal transduction
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-14629 (URN)10.1016/j.yexcr.2004.07.015 (DOI)
    Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2017-12-13Bibliographically approved
    2. LIpoxin A4 inhibits the fMet-Leu-Phe-induced, but not the β2 integrin-induced activation of the non-receptor tyrosine kinase Pyk2 in Human Leukemia 60 cells
    Open this publication in new window or tab >>LIpoxin A4 inhibits the fMet-Leu-Phe-induced, but not the β2 integrin-induced activation of the non-receptor tyrosine kinase Pyk2 in Human Leukemia 60 cells
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:liu:diva-14630 (URN)
    Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2010-01-13
    3. Inside-out regulated β2-integrin-induced release of arachidonic acid in Human Leukemia 60 cells
    Open this publication in new window or tab >>Inside-out regulated β2-integrin-induced release of arachidonic acid in Human Leukemia 60 cells
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:liu:diva-14631 (URN)
    Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2010-01-13
    4. Inactivation of Cdc42 is nessecary for depolymerization of phagosomal F-actin and subsequent phagosomal maturation
    Open this publication in new window or tab >>Inactivation of Cdc42 is nessecary for depolymerization of phagosomal F-actin and subsequent phagosomal maturation
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    2007 (English)In: Journal of Immunology, ISSN 0022-1767, Vol. 178, no 11, p. 7357-7365Article in journal (Refereed) Published
    Abstract [en]

    Phagocytosis is a complex process involving the activation of various signaling pathways, such as the Rho GTPases, and the subsequent reorganization of the actin cytoskeleton. In neutrophils, Rac and Cdc42 are activated during phagocytosis but less is known about the involvement of these GTPases during the different stages of the phagocytic process. The aim of this study was to elucidate the role of Cdc42 in phagocytosis and the subsequent phagosomal maturation. Using a TAT-based protein transduction technique, we introduced dominant negative and constitutively active forms of Cdc42 into neutrophil-like HL60 (human leukemia) cells that were allowed to phagocytose IgG-opsonized yeast particles. Staining of cellular F-actin in cells transduced with constitutively active Cdc42 revealed that the activation of Cdc42 induced sustained accumulation of periphagosomal actin. Moreover, the fusion of azurophilic granules with the phagosomal membrane was prevented by the accumulated F-actin. In contrast, introducing dominant negative Cdc42 impaired the translocation per se of azurophilic granules to the periphagosomal area. These results show that efficient phagosomal maturation and the subsequent eradication of ingested microbes in human neutrophils is dependent on a strictly regulated Cdc42. To induce granule translocation, Cdc42 must be in its active state but has to be inactivated to allow depolymerization of the F-actin cage around the phagosome, a process essential for phagolysosome formation.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-14632 (URN)
    Available from: 2007-09-13 Created: 2007-09-13
    5. Neutrophil activation status in stable coronary artery disease.
    Open this publication in new window or tab >>Neutrophil activation status in stable coronary artery disease.
    Show others...
    2007 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 2, no 10, p. e1056-Article in journal (Refereed) Published
    Abstract [en]

    Background: During the last years, neutrophils have emerged as important players in atherogenesis. They are highly activated in peripheral blood of patients with unstable angina. Moreover, a primed state of circulating neutrophils has been proposed in patients with stable angina. Our aim was to investigate the neutrophil activation status in patients with stable coronary artery disease (CAD) at conventional drug treatment.

    Methodology and principal findings: Thirty patients with stable CAD and 30 healthy controls were included using a paired design. The neutrophil expression of CD18 and high-affinity state of CD11b was analysed by flow cytometry before and after stimulation with chemoattractants. Also, the production of reactive oxygen species (ROS) was determined by chemiluminescence. During basal conditions, the neutrophil expression of CD18 or high-affinity state of CD11b did not differ between patients and controls. Chemoattractants (Interleukin-8 and Leukotriene B(4)) did not increase either the expression or the amount of high-affinity CD11b/CD18-integrins in CAD patients compared to controls, and had no effect on the production of ROS. On the other hand, the ROS production in response to C3bi-opsonised yeast particles and the neutrophils' inherent capacity to produce ROS were both significantly decreased in patients.

    Conclusion/Significance: We could not find any evidence that neutrophils in patients with stable CAD were primed, i.e. more prone to activation, compared to cells from healthy controls. According to our data, the circulating neutrophils in CAD patients rather showed an impaired activation status. It remains to be elucidated whether the neutrophil dysfunction in CAD is mainly a marker of chronic disease, an atherogenic factor or a consequence of the drug treatment.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-17246 (URN)10.1371/journal.pone.0001056 (DOI)17957240 (PubMedID)
    Available from: 2009-03-12 Created: 2009-03-12 Last updated: 2010-01-14
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  • 22.
    Buechel, David
    et al.
    Univ Basel, Switzerland.
    Sugiyama, Nami
    Univ Basel, Switzerland.
    Rubinstein, Natalia
    Univ Buenos Aires, Argentina.
    Saxena, Meera
    Univ Basel, Switzerland.
    Kalathur, Ravi K. R.
    Univ Basel, Switzerland; Royal Childrens Hosp, Australia.
    Luond, Fabiana
    Univ Basel, Switzerland.
    Vafaizadeh, Vida
    Univ Basel, Switzerland.
    Valenta, Tomas
    Univ Zurich, Switzerland.
    Hausmann, George
    Univ Zurich, Switzerland.
    Cantù, Claudio
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Molecular Medicine and Virology. Linköping University, Faculty of Medicine and Health Sciences.
    Basler, Konrad
    Univ Zurich, Switzerland.
    Christofori, Gerhard
    Univ Basel, Switzerland.
    Parsing beta-catenins cell adhesion and Wnt signaling functions in malignant mammary tumor progression2021In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 118, no 34, article id e2020227118Article in journal (Refereed)
    Abstract [en]

    During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, beta-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bd9 and Pygo-pus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of beta-catenins transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of beta-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of beta-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of beta-catenins transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by beta-catenins transcriptional activities upon stimulation with Wnt3a or during TGF-beta-induced EMT. Our results uncouple the signaling from the adhesion function of beta-catenin and underline the importance of Wnt/beta-catenin-dependent transcription in malignant tumor progression of breast cancer.

  • 23.
    Cantù, Claudio
    et al.
    Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy.
    Grande, Vito
    Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy.
    Alborelli, Ilaria
    Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy.
    Cassinelli, Letizia
    Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy.
    Cantù, Ileana
    Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy.
    Colzani, Maria Teresa
    Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy.
    Ierardi, Rossella
    San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, 20132 Milan, Italy.
    Ronzoni, Luisa
    Dipartimento di Medicina Interna, Università di Milano, Fondazione Policlinico Mangiagalli, Regina Elena, IRCCS, Milano, Italy.
    Cappellini, Maria Domenica
    Dipartimento di Medicina Interna, Università di Milano, Fondazione Policlinico Mangiagalli, Regina Elena, IRCCS, Milano, Italy.
    Ferrari, Giuliana
    San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, 20132 Milan // Vita-Salute San Raffaele University, Milan, Italy.
    Ottolenghi, Sergio
    Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy.
    Ronchi, Antonella
    Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy.
    A highly conserved SOX6 double binding site mediates SOX6 gene downregulation in erythroid cells2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 2, p. 486-501Article in journal (Refereed)
    Abstract [en]

    The Sox6 transcription factor plays critical roles in various cell types, including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells, we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar, evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo, and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation, mediated by the double Sox6 binding site within its own promoter, may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation.

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  • 24.
    Chaabane, Wiem
    et al.
    Linköping University, Faculty of Medicine and Health Sciences. Linköping University, Department of Medical and Health Sciences, Division of Drug Research.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Interconnections between apoptotic and autophagic pathways during thiopurine-induced toxicity in cancer cells: the role of reactive oxygen species2016In: Oncotarget, E-ISSN 1949-2553, Vol. 7, no 46, p. 75616-75634Article in journal (Refereed)
    Abstract [en]

    Thiopurines (azathioprine, 6-mercaptopurine and 6-thioguanine) are a class of genotoxic drugs extensively used in the treatment of various illnesses including leukemia. Their underlying molecular mechanism of action involves the activation of apoptosis and autophagy but remains widely unclear. Here we present evidence that autophagy induction by thiopurines is a survival mechanism that antagonizes apoptosis and is involved in degrading damaged mitochondria through mitophagy. On the other hand, apoptosis is the main cell death mechanism by thiopurines as its inhibition prohibited cell death. Thus a tight interplay between apoptosis and autophagy controls cell fate in response to thiopurine treatment. Moreover, thiopurines disrupt mitochondrial function and induce a loss of the mitochondrial transmembrane potential. The involvement of the mitochondrial pathway in thiopurine-induced apoptosis was further confirmed by increased formation of reactive oxygen species (ROS). Inhibiting oxidative stress protected the cells from thiopurine-induced cell death and ROS scavenging prohibited autophagy induction by thiopurines. Our data indicate that the anticarcinogenic effects of thiopurines are mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy.

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  • 25.
    Chen, Zhenzhong
    et al.
    School of Mechanical Engineering, Sungkyunkwan University (SKKU), Suwon, South Korea.
    Han, Seokgyu
    School of Mechanical Engineering, Sungkyunkwan University (SKKU), Suwon, South Korea.
    Sanny, Arleen
    Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR), Singapore.
    Chan, Dorothy Leung-Kwan
    Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR), Singapore.
    van Noort, Danny
    Linköping University, Department of Physics, Chemistry and Biology, Biophysics and bioengineering. Linköping University, Faculty of Science & Engineering. Centro de Investigación en Bioingeniería, Universidad de Ingenieria y Tecnologia - UTEC, Lima, Peru.
    Lim, Wanyoung
    Department of Biomedical Engineering, Sungkyunkwan University (SKKU), Suwon, South Korea.
    Tan, Andy Hee-Meng
    Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR), Singapore.
    Park, Sungsu
    School of Mechanical Engineering, Sungkyunkwan University (SKKU), Suwon, South Korea; Department of Biomedical Engineering, Sungkyunkwan University (SKKU), Suwon, South Korea; Institute of Quantum Biophysics (IQB), Sungkyunkwan University (SKKU), Suwon, South Korea.
    3D hanging spheroid plate for high-throughput CART cell cytotoxicity assay2022In: Journal of Nanobiotechnology, E-ISSN 1477-3155, Vol. 20, no 1, article id 30Article in journal (Refereed)
    Abstract [en]

    Background: Most high-throughput screening (HIS) systems studying the cytotoxic effect of chimeric antigen receptor (CAR) T cells on tumor cells rely on two-dimensional cell culture that does not recapitulate the tumor micro-environment (TME). Tumor spheroids, however, can recapitulate the TME and have been used for cytotoxicity assays of CART cells. But a major obstacle to the use of tumor spheroids for cytotoxicity assays is the difficulty in separating unbound CART and dead tumor cells from spheroids. Here, we present a three-dimensional hanging spheroid plate (3DHSP), which facilitates the formation of spheroids and the separation of unbound and dead cells from spheroids during cytotoxicity assays.

    Results: The 3DHSP is a 24-well plate, with each well composed of a hanging dripper, spheroid wells, and waste wells. In the dripper, a tumor spheroid was formed and mixed with CART cells. In the 3DHSP, droplets containing the spheroids were deposited into the spheroid separation well, where unbound and dead T and tumor cells were separated from the spheroid through a gap into the waste well by tilting the 3DHSP by more than 20 degrees. Human epidermal growth factor receptor 2 (HER2)-positive tumor cells (BT474 and SKOV3) formed spheroids of approximately 300-350 pm in diameter after 2 days in the 3DHSP. The cytotoxic effects ofT cells engineered to express CAR recognizing HER2 (HER2-CAR T cells) on these spheroids were directly measured by optical imaging, without the use of live/dead fluorescent staining of the cells. Our results suggest that the 3DHSP could be incorporated into a HTS system to screen for CARs that enable T cells to kill spheroids formed from a specific tumor type with high efficacy or for spheroids consisting of tumor types that can be killed efficiently by T cells bearing a specific CAR.

    Conclusions: The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CART cells on tumor spheroids.

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  • 26. Chlichlia, K.
    et al.
    Los, Marek Jan
    Department of Immunology and Cell Biology, University of Muenster, Roentgenstr. 21, D-48149 Muenster, Germany.
    Schulze-Osthoff, Klaus
    Department of Immunology and Cell Biology, University of Muenster, Roentgenstr. 21, D-48149 Muenster, Germany.
    Gazzolo, L.
    INSERM U412, Ecole Normale S périeure de Lyon, 69367 Lyon, Cedex 07, France.
    Schirrmacher, V.
    Division of Cellular Immunology (G0100), Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D69120 Heidelberg, Germany.
    Khazaie, K.
    Division of Cellular Immunology (G0100), Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D69120 Heidelberg, Germany; 4Department of Cancer Immunology and AIDS, Dana Farber Cancer Institute, Boston, MA 02115, U.S.A..
    Redox events in HTLV-1 tax-induced apoptotic T-cell death2002In: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 4, no 3, p. 471-477Article in journal (Refereed)
    Abstract [en]

    A number of studies implicate reactive oxygen intermediates in the induction of DNA damage and apoptosis. Recent studies suggest that the human T-cell leukemia virus type I (HTLV-1) Tax protein induces oxidative stress and apoptotic T-cell death. Activation of the T-cell receptor/CD3 pathway enhances the Tax-mediated oxidative and apoptotic effects. Tax-mediated apoptosis and oxidative stress as well as activation of nuclear factor-kappaB can be potently suppressed by antioxidants. This review focuses on Tax-dependent changes in the intracellular redox status and their role in Tax-mediated DNA damage and apoptosis. The relevance of these observations to HTLV-1 virus-mediated T-cell transformation and leukemogenesis are discussed.

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  • 27.
    Christoffersson, Jonas
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    Aronsson, Christopher
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Jury, Michael
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Selegård, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Mandenius, Carl-Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    Fabrication of modular hyaluronan-PEG hydrogels to support 3D cultures of hepatocytes in a perfused liver-on-a-chip device2019In: Biofabrication, ISSN 1758-5082, E-ISSN 1758-5090, Vol. 11, no 1, p. 1-13, article id 015013Article in journal (Refereed)
    Abstract [en]

    Liver cell culture models are attractive in both tissue engineering and for development of assays for drug toxicology research. To retain liver specific cell functions, the use of adequate cell types and culture conditions, such as a 3D orientation of the cells and a proper supply of nutrients and oxygen, are critical. In this article, we show how extracellular matrix mimetic hydrogels can support hepatocyte viability and functionality in a perfused liver-on-a-chip device. A modular hydrogel system based on hyaluronan and poly(ethylene glycol) (HA-PEG), modified with cyclooctyne moieties for bioorthogonal strain-promoted alkyne-azide 1, 3-dipolar cycloaddition (SPAAC), was developed, characterized, and compared for cell compatibility to hydrogels based on agarose and alginate. Hepatoma cells (HepG2) formed spheroids with viable cells in all hydrogels with the highest expression of albumin and urea in alginate hydrogels. By including an excess of cyclooctyne in the HA backbone, azide-modified cell adhesion motifs (linear and cyclic RGD peptides) could be introduced in order to enhance viability and functionality of human induced pluripotent stem cell derived hepatocytes (hiPS-HEPs). In the HA-PEG hydrogels modified with cyclic RGD peptides hiPS-HEPs migrated and grew in 3D and showed an increased viability and higher albumin production compared to when cultured in the other hydrogels. This flexible SPAAC crosslinked hydrogel system enabled fabrication of perfused 3D cell culture of hiPS-HEPs and is a promising material for further development and optimization of liver-on-a-chip devices.

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    Fabrication of modular hyaluronan-PEG hydrogels to support 3D cultures of hepatocytes in a perfused liver-on-a-chip device
  • 28.
    Clayton, Zoe E.
    et al.
    Heart Res Inst, Australia.
    Tan, Richard P.
    Heart Res Inst, Australia; Univ Sydney, Australia.
    Miravet, Maria M.
    Linköping University, Faculty of Medicine and Health Sciences. Heart Res Inst, Australia.
    Lennartsson, Katarina
    Linköping University, Faculty of Medicine and Health Sciences. Heart Res Inst, Australia.
    Cooke, John P.
    Houston Methodist Res Inst, TX 77030 USA.
    Bursill, Christina A.
    Heart Res Inst, Australia.
    Wise, Steven G.
    Heart Res Inst, Australia.
    Patel, Sanjay
    Heart Res Inst, Australia; Univ Sydney, Australia.
    Induced pluripotent stem cell-derived endothelial cells promote angiogenesis and accelerate wound closure in a murine excisional wound healing mode2018In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 38, article id BSR20180563Article in journal (Refereed)
    Abstract [en]

    Chronic wounds are a major complication in patients with cardiovascular diseases. Cell therapies have shown potential to stimulate wound healing, but clinical trials using adult stem cells have been tempered by limited numbers of cells and invasive procurement procedures. Induced pluripotent stem cells (iPSCs) have several advantages of other cell types, for example they can be generated in abundance from patients somatic cells (autologous) or those from a matched donor. iPSCs can be efficiently differentiated to functional endothelial cells (iPSC-ECs). Here, we used a murine excisional wound model to test the pro-angiogenic properties of iPSC-ECs in wound healing. Two full-thickness wounds were made on the dorsum of NOD-SCID mice and splinted. iPSC-ECs (5 x 10(5)) were topically applied to one wound, with the other serving as a control. Treatment with iPSC-ECs significantly increased wound perfusion and accelerated wound closure. Expression of endothelial cell (EC) surface marker, platelet endothelial cell adhesion molecule (PECAM-1) (CD31), and pro-angiogenic EC receptor, Tie1, mRNA was up-regulated in iPSC-EC treated wounds at 7 days post-wounding. Histological analysis of wound sections showed increased capillary density in iPSC-EC wounds at days 7 and 14 post-wounding, and increased collagen content at day 14. Anti-GFP fluorescence confirmed presence of iPSC-ECs in the wounds. Bioluminescent imaging (BLI) showed progressive decline of iPSC-ECs over time, suggesting that iPSC-ECs are acting primarily through short-term paracrine effects. These results highlight the pro-regenerative effects of iPSC-ECs and demonstrate that they are a promising potential therapy for intractable wounds.

  • 29.
    Diaz, David
    et al.
    Department of Medicine, University of Alcalá, Madrid, Spain.
    Barcenilla, Hugo
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Children's and Women's Health. Linköping University, Faculty of Medicine and Health Sciences.
    Prieto, Alfredo
    Department of Medicine, University of Alcalá, Madrid, Spain.
    Monserrat, Jorge
    Department of Medicine, University of Alcalá, Madrid, Spain.
    Alvarez-Mon, Melchor
    Immune System Diseases and Oncology Service, University Hospital “Príncipe de Asturias”, Alcalá de Henares, Madrid, Spain.
    Accurate Enumeration of Apoptotic Cancer Cells Using Flow Cytometry2022In: Apoptosis and Cancer: Methods and Protocols / [ed] Hugo Barcenilla, David Diaz, New York, NY: Springer US , 2022, p. 35-44Chapter in book (Other academic)
    Abstract [en]

    The frequency of apoptotic cells in a given phenotypically defined population is usually calculated the apoptotic index (AI), i.e., the percentage of apoptotic cells displaying a specific linage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. Firstly, apoptotic cells fragment into apoptotic bodies that later disintegrate. Secondly, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. The present chapter will describe a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of lineage antigen expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures.

  • 30.
    Domert, Jakob
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Rao, Sahana Bhima
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Agholme, Lotta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Brorsson, Ann-Christin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Marcusson, Jan
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences.
    Hallbeck, Martin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Inflammation Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    Nath, Sangeeta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Spreading of Amyloid-β Peptides via Neuritic Cell-to-cell Transfer Is Dependent on Insufficient Cellular Clearance2014In: Neurobiology of Disease, ISSN 0969-9961, E-ISSN 1095-953X, Vol. 65, p. 82-92Article in journal (Refereed)
    Abstract [en]

    The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimer's disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aβ) residues 1-42 (oAβ1-42) using our donor-acceptor 3-D co-culture model. We now show that different Aβ-isoforms (fluorescently labeled 1-42, 3(pE)-40, 1-40 and 11-42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aβ-isoform. Although different Aβ isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aβ can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aβ1-42 isoform, which further promotes cell-to-cell transfer; thus, oAβ1-42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD.

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  • 31.
    Durrieu, Lucia
    et al.
    Univ Buenos Aires UBA, Argentina; Natl Council Sci & Tech Res IFIBYNE, Argentina.
    Bush, Alan
    Linköping University, Department of Biomedical Engineering, Division of Biomedical Engineering. Linköping University, Faculty of Science & Engineering. Univ Buenos Aires UBA, Argentina; Natl Council Sci & Tech Res IFIBYNE, Argentina.
    Grande, Alicia
    Univ Buenos Aires UBA, Argentina; Natl Council Sci & Tech Res IFIBYNE, Argentina.
    Johansson, Rickard
    Linköping University, Department of Biomedical Engineering, Division of Biomedical Engineering. Linköping University, Faculty of Science & Engineering.
    Janzén, David
    Linköping University, Department of Biomedical Engineering, Division of Biomedical Engineering. Linköping University, Faculty of Science & Engineering.
    Katz, Andrea
    Univ Buenos Aires UBA, Argentina.
    Cedersund, Gunnar
    Linköping University, Department of Biomedical Engineering, Division of Biomedical Engineering. Linköping University, Faculty of Science & Engineering.
    Colman-Lerner, Alejandro
    Univ Buenos Aires UBA, Argentina; Natl Council Sci & Tech Res IFIBYNE, Argentina.
    Characterization of cell-to-cell variation in nuclear transport rates and identification of its sources2023In: ISCIENCE, ISSN 2589-0042, Vol. 26, no 1, article id 105906Article in journal (Refereed)
    Abstract [en]

    Nuclear transport is an essential part of eukaryotic cell function. Here, we present scFRAP, a model-assisted fluorescent recovery after photobleaching (FRAP)-based method to determine nuclear import and export rates independently in individual live cells. To overcome the inherent noise of single-cell measurements, we performed sequential FRAPs on the same cell. We found large cell-to-cell variation in transport rates within isogenic yeast populations. For passive trans-port, the variability in NPC number might explain most of the variability. Using this approach, we studied mother-daughter cell asymmetry in the active nuclear shuttling of the transcription factor Ace2, which is specifically concentrated in daughter cell nuclei in early G1. Rather than reduced export in the daughter cell, as previously hypothesized, we found that this asymmetry is mainly due to an increased import in daughters. These results shed light on cell-to-cell variation in cellular dynamics and its sources.

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  • 32.
    Englund, Ulrika
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Brask, Johan
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Elinder, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Inhibition of SCN2A ortholog upregulation in Xenopus laevis oocytes prevents cell death2014Manuscript (preprint) (Other academic)
    Abstract [en]

    Transport of ions across the cell membrane is essential for the regulation of cell death and tissue homeostasis, and alterations in the function of voltage-gated ion channels and of the intracellular ionic compositions interfere with these processes. Opening of K, Na , or Cl channels have been linked to the apoptotic process and in many cases, opening of these channels precede caspase-3 activation and are thus early events in the apoptotic process. Consistent with the role of these channels in apoptosis, inhibition of these channels prevents or delays the apoptotic process. However, the role of ion channels during apoptosis has been difficult to explore, mainly due to unspecific/non-selective ion channe blockers. In the present investigation, the molecular identity of a  voltage-gated Na channel in oocytes from Xenopus laevis, which is crucial for the apoptotic response to mechanical stress, was identified. Specific down regulation of SCN2A Na channel expression by miRNA prevented apoptosis, suggesting that Na+ influx is essential for apoptosis in Xenopus oocytes.

  • 33.
    Ericsson, Maria
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Henriksen, Rie
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Bélteky, Johan
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Sundman, Ann-Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Shionoya, Kiseko
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Jensen, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Long-Term and Transgenerational Effects of Stress Experienced during Different Life Phases in Chickens (Gallus gallus)2016In: PLOS ONE, E-ISSN 1932-6203, Vol. 11, no 4, article id e0153879Article in journal (Refereed)
    Abstract [en]

    Stress in animals causes not only immediate reactions, but may affect their biology for long periods, even across generations. Particular interest has been paid to perinatal stress, but also adolescence has been shown to be a sensitive period in mammals. So far, no systematic study has been performed of the relative importance of stress encountered during different life phases. In this study, groups of chickens were exposed to a six-day period of repeated stress during three different life phases: early (two weeks), early puberty (eight weeks) and late puberty (17 weeks), and the effects were compared to an unstressed control group. The short-term effects were assessed by behaviour, and the long-term and transgenerational effects were determined by effects on behavior and corticosterone secretion, as well as on hypothalamic gene expression. Short-term effects were strongest in the two week group and the eight week group, whereas long-term and transgenerational effects were detected in all three stress groups. However, stress at different ages affected different aspects of the biology of the chickens, and it was not possible to determine a particularly sensitive life phase. The results show that stress during puberty appears to be at least equally critical as the previously studied early life phase. These findings may have important implications for animal welfare in egg production, since laying hens are often exposed to stress during the three periods pinpointed here.

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  • 34.
    Eriksson, Emma S. E.
    et al.
    Department of Chemistry and Molecular Biology, University of Gothenburg.
    Erdtman, Edvin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Eriksson, Leif A.
    Department of Chemistry and Molecular Biology, University of Gothenburg.
    Permeability of 5-aminolevulinic acid oxime derivatives in lipid membranes2016In: Theoretical Chemistry accounts, ISSN 1432-881X, E-ISSN 1432-2234, Vol. 135, no 1, p. 1-9Article in journal (Refereed)
    Abstract [en]

    The endogenous molecule 5-aminolevulinic acid (5ALA) and its methyl ester (Me-5ALA) have been used as prodrugs in photodynamic treatment of actinic keratosis and superficial non-melanoma skin cancers for over a decade. Recently, a novel set of 5ALA derivatives based on introducing a hydrolyzable oxime functionality was proposed and shown to generate considerably stronger onset of the photoactive molecule protoporphyrin IX (PpIX) in the cells. In the current work, we employ molecular dynamics simulation techniques to explore whether the higher intercellular concentration of PpIX caused by the oxime derivatives is related to enhanced membrane permeability, or whether other factors contribute to this. It is concluded that the oximes show overall similar accumulation at the membrane headgroup regions as the conventional derivatives and that the transmembrane permeabilities are in general close to that of 5ALA. The highest permeability of all compounds explored is found for Me-5ALA, which correlates with a considerably lower fee energy barrier at the hydrophobic bilayer center. The high PpIX concentration must hence be sought in other factors, where slow hydrolysis of the oxime functionality is a plausible reason, enabling stronger buildup of PpIX over time.

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  • 35. Order onlineBuy this publication >>
    Eriksson, Ida
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Dealing with damaged lysosomes: Impact of lysosomal membrane stability in health and disease2022Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The lysosome is the main unit for degradation and plays important roles in various cellular processes, such as nutrient sensing, cholesterol regulation and cell death. Consequently, altered lysosomal function contributes to, or even causes, several diseases. Lysosomal membrane permeabilization (LMP) and release of lysosomal content to the cytosol can induce cell death, and is implicated in inflammation and neuronal decline in several neurodegenerative diseases. It has also emerged as a potential target in cancer therapy. Due to the detrimental effects of LMP, cells harbor several mechanisms to protect and prevent lysosomal membrane damage. The aim of this thesis was to elucidate how lysosomal membrane stability and repair mechanisms affect cell death and survival.  

    We find that lysosomal cholesterol is upregulated in response to an increased load of reactive oxygen species in a Parkinson’s disease cell model, and that augmented cholesterol protects from LMP. However, cholesterol also induces accumulation of α-synuclein and inhibits lysosome-mediated degradation, which can destabilize the lysosomal membrane and accelerate the course of disease. Further, we demonstrate that lysosomal membrane damage is counteracted by a calcium-dependent repair mechanism to prevent LMP. Lysosomes damaged beyond repair are instead sequestered in an autophagosome and degraded by intact lysosomes in a process called lysophagy. As a result, small vesicles containing lysosomal membrane proteins are generated, which we believe are used to restore lysosomal function. We show that malignant cells are more sensitive to LMP, and that they differ in their activation of damage-response mechanisms compared to normal cells. Moreover, in malignant cells, the intracellular position of the lysosomes determines the susceptibility to lysosomal damage. Peripherally located lysosomes are less sensitive, and by relocating lysosomes to the perinuclear area in the cell, we can sensitize lysosomes to LMP induction.  

    In summary, this thesis demonstrates the importance of damage-response mechanisms to protect from lysosomal membrane damage and maintain cellular function. It also indicates that targeting of lysosomal stability and repair is a potential therapeutic strategy in both neurodegenerative diseases and in cancer.

    List of papers
    1. Impact of high cholesterol in a Parkinsons disease model: Prevention of lysosomal leakage versus stimulation of alpha-synuclein aggregation
    Open this publication in new window or tab >>Impact of high cholesterol in a Parkinsons disease model: Prevention of lysosomal leakage versus stimulation of alpha-synuclein aggregation
    Show others...
    2017 (English)In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 96, no 2, p. 99-109Article in journal (Refereed) Published
    Abstract [en]

    Parkinsons disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated alpha-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), we found that MPP+-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of alpha-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP+-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect alpha-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinsons disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of alpha-synuclein accumulation. (C) 2017 Elsevier GmbH. All rights reserved.

    Place, publisher, year, edition, pages
    ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2017
    Keywords
    Cholesterol; alpha-Synuclein; Parkinsons disease; Lovastatin; Lysosome; 1-Methyl-4-phenylpyridinium (MPP+); Reactive oxygen species (ROS)
    National Category
    Cell Biology
    Identifiers
    urn:nbn:se:liu:diva-142445 (URN)10.1016/j.ejcb.2017.01.002 (DOI)000412150200002 ()28109635 (PubMedID)
    Note

    Funding Agencies|Swedish Research Council; Parkinson Foundation at Linkoping University; Konung Gustaf V och Drottning Victorias Frimurarestiftelse

    Available from: 2017-10-31 Created: 2017-10-31 Last updated: 2022-04-04
    2. Interactions of the Lysosomotropic Detergent O-Methyl-Serine Dodecylamide Hydrochloride (MSDH) with Lipid Bilayer Membranes-Implications for Cell Toxicity
    Open this publication in new window or tab >>Interactions of the Lysosomotropic Detergent O-Methyl-Serine Dodecylamide Hydrochloride (MSDH) with Lipid Bilayer Membranes-Implications for Cell Toxicity
    Show others...
    2020 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 21, no 9, article id 3136Article in journal (Refereed) Published
    Abstract [en]

    O-methyl-serine dodecylamine hydrochloride (MSDH) is a detergent that accumulates selectively in lysosomes, a so-called lysosomotropic detergent, with unexpected chemical properties. At physiological pH, it spontaneously forms vesicles, which disassemble into small aggregates (probably micelles) below pH 6.4. In this study, we characterize the interaction between MSDH and liposomes at different pH and correlate the findings to toxicity in human fibroblasts. We find that the effect of MSDH on lipid membranes is highly pH-dependent. At neutral pH, the partitioning of MSDH into the liposome membrane is immediate and causes the leakage of small fluorophores, unless the ratio between MSDH and lipids is kept low. At pH 5, the partitioning of MSDH into the membrane is kinetically impeded since MSDH is charged and a high ratio between MSDH and the lipids is required to permeabilize the membrane. When transferred to cell culture conditions, the ratio between MSDH and plasma membrane lipids must therefore be low, at physiological pH, to maintain plasma membrane integrity. Transmission electron microscopy suggests that MSDH vesicles are taken up by endocytosis. As the pH of the endosomal compartment progressively drops, MSDH vesicles disassemble, leading to a high concentration of increasingly charged MSDH in small aggregates inside the lysosomes. At sufficiently high MSDH concentrations, the lysosome is permeabilized, the proteolytic content released to the cytosol and apoptotic cell death is induced.

    Place, publisher, year, edition, pages
    MDPI, 2020
    Keywords
    MSDH; liposome; lysosomal membrane permeabilization; lysosome; lysosomotropic detergent
    National Category
    Physical Chemistry
    Identifiers
    urn:nbn:se:liu:diva-174272 (URN)10.3390/ijms21093136 (DOI)000535581700107 ()32365555 (PubMedID)2-s2.0-85084276477 (Scopus ID)
    Note

    Funding agencies: The Swedish Cancer Society (KÖ), Konung Gustav V ochDrottning Victorias Frimurarestiftelse (KÖ) and Stiftelseförvaltningen vid Region Östergötland (AMVG and IE). 

    Available from: 2021-03-17 Created: 2021-03-17 Last updated: 2022-04-04Bibliographically approved
    3. Restoration of lysosomal function after damage is accompanied by recycling of lysosomal membrane proteins
    Open this publication in new window or tab >>Restoration of lysosomal function after damage is accompanied by recycling of lysosomal membrane proteins
    2020 (English)In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 11, no 5, article id 370Article in journal (Refereed) Published
    Abstract [en]

    Lysosomes are central organelles for cellular degradation and energy homeostasis. In addition, lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal content to the cytosol can initiate programmed cell death. The extent of LMP and available repair mechanisms determine the cell fate after lysosomal damage. In this study, we aimed to investigate the premises for lysosomal membrane repair after LMP and found that lysosomal membrane damage initiated by l-leucyl-l-leucine methyl ester (LLOMe) caused caspase-dependent apoptosis in almost 50% of the cells, while the rest recovered. Immediately after LLOMe addition, lysosomal proteases were detected in the cytosol and the ESCRT-components ALIX and CHMP4B were recruited to the lysosomal membrane. Next, lysophagic clearance of damaged lysosomes was evident and a concentration-dependent translocation of several lysosomal membrane proteins, including LAMP2, to the cytosol was found. LAMP2 was present in small vesicles with the N-terminal protein chain facing the lumen of the vesicle. We conclude that lysophagic clearance of damaged lysosomes results in generation of lysosomal membrane protein complexes, which constitute small membrane enclosed units, possibly for recycling of lysosomal membrane proteins. These lysosomal membrane complexes enable an efficient regeneration of lysosomes to regain cell functionality.

    Place, publisher, year, edition, pages
    NATURE PUBLISHING GROUP, 2020
    National Category
    Cell Biology
    Identifiers
    urn:nbn:se:liu:diva-166484 (URN)10.1038/s41419-020-2527-8 (DOI)000536536900006 ()32409651 (PubMedID)
    Note

    Funding Agencies|Swedish Cancer SocietySwedish Cancer Society; Stiftelseforvaltningen vid Region Ostergotland; Linkoping University

    Available from: 2020-06-20 Created: 2020-06-20 Last updated: 2022-04-04
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  • 36.
    Eriksson, Ida
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Nath, Sangeeta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Bornefall, Per
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
    Villamil Giraldo, Ana Maria
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Öllinger, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    Impact of high cholesterol in a Parkinsons disease model: Prevention of lysosomal leakage versus stimulation of alpha-synuclein aggregation2017In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 96, no 2, p. 99-109Article in journal (Refereed)
    Abstract [en]

    Parkinsons disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated alpha-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), we found that MPP+-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of alpha-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP+-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect alpha-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinsons disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of alpha-synuclein accumulation. (C) 2017 Elsevier GmbH. All rights reserved.

  • 37.
    Eriksson, Ida
    et al.
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Vainikka, Linda
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Persson, Lennart
    Linköping University, Department of Health, Medicine and Caring Sciences, Division of Diagnostics and Specialist Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Respiratory Medicine. Linköping University, Center for Medical Image Science and Visualization (CMIV).
    Öllinger, Karin
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange2023In: METHODS AND PROTOCOLS, ISSN 2409-9279, Vol. 6, no 4, article id 72Article in journal (Refereed)
    Abstract [en]

    Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.

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  • 38.
    Eriksson, Ida
    et al.
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Wäster, Petra
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Öllinger, Karin
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Restoration of lysosomal function after damage is accompanied by recycling of lysosomal membrane proteins2020In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 11, no 5, article id 370Article in journal (Refereed)
    Abstract [en]

    Lysosomes are central organelles for cellular degradation and energy homeostasis. In addition, lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal content to the cytosol can initiate programmed cell death. The extent of LMP and available repair mechanisms determine the cell fate after lysosomal damage. In this study, we aimed to investigate the premises for lysosomal membrane repair after LMP and found that lysosomal membrane damage initiated by l-leucyl-l-leucine methyl ester (LLOMe) caused caspase-dependent apoptosis in almost 50% of the cells, while the rest recovered. Immediately after LLOMe addition, lysosomal proteases were detected in the cytosol and the ESCRT-components ALIX and CHMP4B were recruited to the lysosomal membrane. Next, lysophagic clearance of damaged lysosomes was evident and a concentration-dependent translocation of several lysosomal membrane proteins, including LAMP2, to the cytosol was found. LAMP2 was present in small vesicles with the N-terminal protein chain facing the lumen of the vesicle. We conclude that lysophagic clearance of damaged lysosomes results in generation of lysosomal membrane protein complexes, which constitute small membrane enclosed units, possibly for recycling of lysosomal membrane proteins. These lysosomal membrane complexes enable an efficient regeneration of lysosomes to regain cell functionality.

  • 39.
    Eriksson, Ida
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Öllinger, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    Appelqvist, Hanna
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells2017In: Lysosomes: Methods and Protocols / [ed] Karin Öllinger;Hanna Appelqvist, Humana Press, 2017, Vol. 1594, p. 179-189Chapter in book (Refereed)
    Abstract [en]

    The acidic environment of the lysosomal lumen provides an optimal milieu for the acid hydrolases and is also essential for fusion/fission of endo-lysosomal compartments and sorting of cargo. Evidence suggests that maintaining lysosomal acidity is essential to avoid disease. In this chapter, we describe a protocol for analyzing the lysosomal pH in cultured cells using the fluorescent probe fluorescein isothiocyanate (FITC)-dextran together with a dual-emission ratiometric technique suitable for flow cytometry. Fluorescence-labeled dextran is endocytosed and accumulated in the lysosomal compartment. FITC shows a pH-dependent variation in fluorescence when analyzed at maximum emission wavelength and no variation when analyzing at the isosbestic point, thereby the ratio can be used to determine the lysosomal pH. A standard curve is obtained by equilibrating intralysosomal pH with extracellular pH using the ionophore nigericin. The protocol also includes information regarding procedures to induce lysosomal alkalinization and lysosomal membrane permeabilization.

  • 40.
    Everett, Jake
    et al.
    Department of Surgery, Texas Tech University Health Sciences Center, MS8312, 3601 4th Street, Lubbock, USA.
    Gabrilska, Rebecca
    Department of Surgery, Texas Tech University Health Sciences Center, MS8312, 3601 4th Street, Lubbock, USA.
    Rumbaugh, Kendra P.
    Department of Surgery, Texas Tech University Health Sciences Center, MS8312, 3601 4th Street, Lubbock, USA.
    Vikström, Elena
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Assessing Pseudomonas aeruginosa Autoinducer Effects on Mammalian Epithelial Cells2018In: Quorum Sensing: Methods and Protocols / [ed] Livia LeoniGiordano Rampioni, Humana Press, 2018, Vol. 1673, p. 213-225Chapter in book (Refereed)
    Abstract [en]

    The human mucosal environment in the gut is rich with interactions between microbiota and mammalian epithelia. Microbes such as the Gram-negative bacterium Pseudomonas aeruginosa may use quorum sensing to communicate with other microorganisms and mammalian cells to alter gene expression. Here, we present methodologies to evaluate the effects of P. aeruginosa N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-C12-HSL) on Caco-2 cell monolayers. First, we describe a method for assessing barrier function and permeability of epithelial cells when exposed to 3O-C12-HSL by measuring transepithelial electrical resistance (TER) and paracellular flow using fluorescently labeled dextran. Secondly, we detail methods to investigate the effect of 3O-C12-HSL on protein-protein interactions of epithelial junction proteins. Lastly, we will detail imaging techniques to visualize Caco-2 barrier disruption following exposure to 3O-C12-HSL through the use of confocal laser scanning microscopy (CLSM) and a super resolution technique, stimulated emission depletion (STED) microscopy, to achieve nanoscale visualization of Caco-2 monolayers.

  • 41.
    Fernández-Gago, Rocío
    et al.
    Department of Medicine, Surgery and Veterinary Anatomy, University of León, 24071 León, Spain.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Alonso, Marta E
    Department of Animal Production, University of León, 24071 León, Spain.
    González, J Ramiro
    Department of Medicine, Surgery and Veterinary Anatomy, University of León, 24071 León, Spain.
    Alegre, Beatriz
    Department of Medicine, Surgery and Veterinary Anatomy, University of León, 24071 León, Spain.
    Domínguez, Juan C
    Department of Medicine, Surgery and Veterinary Anatomy, University of León, 24071 León, Spain.
    Martínez-Pastor, Felipe
    INDEGSAL (Institute for Animal Health and Cattle Development), University of León, 24071 León, Spain /Molecular Biology (Cell Biology), University of León, 24071 León, Spain/ .
    Thawing boar semen in the presence of seminal plasma improves motility, modifies subpopulation patterns and reduces chromatin alterations.2017In: Reproduction, fertility, and development, ISSN 1031-3613, Vol. 29, no 8, p. 1576-1584Article in journal (Refereed)
    Abstract [en]

    Seminal plasma could have positive effects on boar semen after thawing. In the present study we investigated changes in the motility and chromatin structure in spermatozoa over 4h incubation (37°C) of boar semen thawed in the presence of 0%, 10% or 50% seminal plasma from good-fertility boars. Cryopreserved doses were used from seven males, three of which were identified as susceptible to post-thawing chromatin alterations. Motility was analysed by computer-aided sperm analysis every hour, and data were used in a two-step clustering, yielding three subpopulations of spermatozoa (slow non-linear, fast non-linear, fast linear). Chromatin structure was analysed using a sperm chromatin structure assay and flow cytometry to determine the DNA fragmentation index (%DFI) as a percentage, the standard deviation of the DFI (SD-DFI) and the percentage of high DNA stainability (%HDS), indicating chromatin compaction. Thawing without seminal plasma resulted in a rapid loss of motility, whereas seminal plasma helped maintain motility throughout the incubation period and preserved the subpopulation comprising fast and linear spermatozoa. The incidence of chromatin alterations was very low in samples from non-susceptible males, whereas samples from males susceptible to post-thawing chromatin alterations exhibited marked alterations in%DFI and%HDS. Seminal plasma partly prevented these alterations in samples from susceptible males. Overall, 50% seminal plasma was the most efficient concentration to protect motility and chromatin. Some changes were concomitant with physiological events reported previously (e.g., semen thawed with 50% seminal plasma increased the production of reactive oxygen species and yielded higher fertility after AI). Thawing in the presence of seminal plasma could be particularly useful in the case of samples susceptible to post-thawing chromatin damage.

  • 42.
    Ferrari, D.
    et al.
    Department of Internal Medicine I, Eberhard-Karls University, D-72076 Tübingen, Germany.
    Stepczynska, A.
    Department of Internal Medicine I, Eberhard-Karls University, D-72076 Tübingen, Germany.
    Los, Marek Jan
    Department of Internal Medicine I, Eberhard-Karls University, D-72076 Tübingen, Germany.
    Wesselborg, Sebastian
    Department of Internal Medicine I, Eberhard-Karls University, D-72076 Tübingen, Germany.
    Schulze-Osthoff, Klaus
    Department of Internal Medicine I, Eberhard-Karls University, D-72076 Tübingen, Germany.
    Differential regulation and ATP requirement for caspase-8 and caspase-3 activation during CD95- and anticancer drug-induced apoptosis1998In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 188, no 5, p. 979-984Article in journal (Refereed)
    Abstract [en]

    Apoptosis is induced by different stimuli, among them triggering of the death receptor CD95, staurosporine, and chemotherapeutic drugs. In all cases, apoptosis is mediated by caspases, although it is unclear how these diverse apoptotic stimuli cause protease activation. Two regulatory pathways have been recently identified, but it remains unknown whether they are functionally independent or linked to each other. One is mediated by recruitment of the proximal regulator caspase-8 to the death receptor complex. The other pathway is controlled by the release of cytochrome c from mitochondria and the subsequent ATP-dependent activation of the death regulator apoptotic protease-activating factor 1 (Apaf-1). Here, we report that both pathways can be dissected by depletion of intracellular ATP. Prevention of ATP production completely inhibited caspase activation and apoptosis in response to chemotherapeutic drugs and staurosporine. Interestingly, caspase-8, whose function appeared to be restricted to death receptors, was also activated by these drugs under normal conditions, but not after ATP depletion. In contrast, inhibition of ATP production did not affect caspase activation after triggering of CD95. These results suggest that chemotherapeutic drug-induced caspase activation is entirely controlled by a receptor-independent mitochondrial pathway, whereas CD95-induced apoptosis can be regulated by a separate pathway not requiring Apaf-1 function.

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  • 43.
    Franz, F
    et al.
    Clinic of Endocrinology and Nephrology, Department of Internal Medicine, Neurology and Dermatology, University of Leipzig, Leipzig, Germany.
    Weidinger, C
    Clinic of Endocrinology and Nephrology, Department of Internal Medicine, Neurology and Dermatology, University of Leipzig, Leipzig, Germany.
    Krause, K
    Clinic of Endocrinology and Nephrology, Department of Internal Medicine, Neurology and Dermatology, University of Leipzig, Leipzig, Germany.
    Gimm, Oliver
    Department of General, Visceral, and Vascular Surgery, Martin-Luther-University of Halle-Wittenberg, Halle/Saale, Germany.
    Dralle, H
    Department of General, Visceral, and Vascular Surgery, Martin-Luther-University of Halle-Wittenberg, Halle/Saale, Germany.
    Führer, D
    Department of Endocrinology & Metabolism and Division of Laboratory Research, University Hospital Essen, Essen, Germany.
    The Transcriptional Regulation of FOXO Genes in Thyrocytes.2016In: Hormone and Metabolic Research, ISSN 0018-5043, E-ISSN 1439-4286, Vol. 48, no 9, p. 601-6Article in journal (Refereed)
    Abstract [en]

    FOXO transcription factors are key regulators of DNA damage repair, proliferation and apoptosis in thyrocytes. Thyroid malignancies show impaired FOXO function. In this study, we investigated the transcriptional regulation of FOXO isoforms in thyroid epithelial cells. mRNA expression of FOXO isoforms (FOXO1, 3 and 4) was determined in FRTL-5 cells stimulated with different growth factors and H2O2. Furthermore, the impact of PI3K/AKT signalling on FOXO transcription was investigated in PI3K p110α mutant FRTL-5 cells and regulatory dependence of FOXO transcription on FOXO was studied in FRTL-5 cells with hFOXO3 overexpression. Finally, mRNA expression levels of FOXO isoforms were determined in human epithelial thyroid tumours. Growth factor deprivation induced transcription of FOXO1, 3 and 4, whereas insulin stimulation decreased FOXO1 and FOXO4 transcription in FRTL-5 cells. Inhibition of the PI3K/AKT cascade amplified FOXO1 and FOXO4 expression. In contrast, H2O2 and TSH did not influence FOXO transcription in thyrocytes. Overexpression of PI3K p110α inhibited FOXO3 and induced FOXO4 transcription. In human thyroid tumours, FOXO1 and FOXO3 mRNA levels were significantly downregulated in papillary thyroid carcinoma when compared to normal tissues. In contrast, follicular thyroid carcinomas showed significant upregulation of FOXO4 mRNA.In this paper, we demonstrate an influence of PI3K signalling on FOXO transcription in thyrocytes. Moreover, we show that thyroid cancers exhibit alterations in FOXO transcription besides the previously reported alterations in posttranslational FOXO3 regulation. These findings may add to the concept of targeting the PI3K pathway in advanced thyroid cancers.

  • 44.
    Gardela, Jaume
    et al.
    Univ Autonoma Barcelona, Spain.
    Ruiz-Conca, Mateo
    Univ Autonoma Barcelona, Spain.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences. Univ Autonoma Barcelona, Spain.
    Mogas, Teresa
    Univ Autonoma Barcelona, Spain.
    Lopez-Bejar, Manel
    Univ Autonoma Barcelona, Spain.
    Induction of CIRBP expression by cold shock on bovine cumulus-oocyte complexes2019In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 54, p. 82-85Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to induce the cold-inducible RNA-binding protein (CIRBP) expression on cumulus-oocyte complexes (COCs) through exposure to a sub-lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold-inducible RNA-binding protein (CIRBP) were assessed after 24 hr of in vitro maturation of control (38.5 degrees C) and cold-stressed oocytes (33.5 degrees C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold-stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold-stressed groups, cold-stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes.

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  • 45.
    Gerdin, Linda
    et al.
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Surgery in Linköping. Surg Clin Jonkoping Cty, Sweden.
    Gonzalez-Castro, Ana M.
    Univ Autonoma Barcelona, Spain; Univ Autonoma Barcelona, Spain.
    Ericson, Ann-Charlott
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Molecular Medicine and Virology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Surgery in Linköping.
    Persborn, Mats
    Surg Clin Jonkoping Cty, Sweden.
    Santos, Javier
    Univ Autonoma Barcelona, Spain; Univ Autonoma Barcelona, Spain; Ctr Invest Biomed Red Enfermedades Hepat & Digest, Spain.
    Walter, Susanna
    Linköping University, Department of Health, Medicine and Caring Sciences, Division of Diagnostics and Specialist Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Mag- tarmmedicinska kliniken. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Surgery in Linköping.
    Keita, Åsa
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Surgery in Linköping.
    Vicario, Maria
    Univ Autonoma Barcelona, Spain; Univ Autonoma Barcelona, Spain; Ctr Invest Biomed Red Enfermedades Hepat & Digest, Spain; Nestle Res Soc Prod Nestle SA, Switzerland.
    Söderholm, Johan D
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Surgery in Linköping.
    Acute psychological stress increases paracellular permeability and modulates immune activity in rectal mucosa of healthy volunteers2023In: United European Gastroenterology journal, ISSN 2050-6406, E-ISSN 2050-6414, Vol. 11, no 1, p. 31-41Article in journal (Refereed)
    Abstract [en]

    Background Psychological stress and increased permeability are implicated as contributing factors in the initiation and worsening of gastrointestinal diseases. A link between stress and intestinal permeability has been shown in animal models as well as in human small intestine, but stress effects on the human colorectal mucosal barrier has not been reported. Objective To investigate the potential effects of acute psychological stress on colorectal mucosal barrier function and to explore stress-induced molecular events in the rectal mucosa under healthy conditions. Methods Endoscopic biopsies were taken from the rectosigmoid region of healthy volunteers, who had been subjected to dichotomous listening stress and after a control session, respectively. Paracellular and transcellular permeability were assessed in modified Ussing chambers. RNA expression (microarray technology confirmed by quantitative real-time polymerase chain reaction) and biological pathway analysis were used to investigate the local mucosal response to acute stress. Results Dichotomous listening stress induced a subjective and objective stress response, and significantly increased paracellular but not transcellular permeability. We also identified a stress-induced reduction in RNA expression of genes related to immune cell activation and maturation (CR2, CD20, TCLA1, BANK1, CD22, FDCSP), signaling molecules of homing of immune cells to the gut (chemokines: CCL21, CXCL13, and CCL19, and receptors: CCR7, CXCR5), and innate immunity (DUOX2). Eight of the 10 top down-regulated genes are directly involved in B cell activation, signaling and migration. The systemic stress response correlated positively with paracellular permeability and negatively with DUOX2 expression. Conclusion Dichotomous listening stress increases paracellular permeability and modulates immune cell activity in the rectal mucosa. Further studies are warranted to identify the primary mechanisms of stress-mediated reduction of mucosal defensive activity and barrier dysfunction, and their potential implications for gastrointestinal disorders.

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  • 46.
    Ghavami, Saeid
    et al.
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Asoodeh, Ahmad
    Department of Biophysics and Biochemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran; Department of Chemistry Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran .
    Klonisch, Thomas
    Department of Human Anatomy and Cell Sciences, and Manitoba Institute of Child Health, Winnipeg, Canada.
    Halayko, Andrew J.
    Department of Physiology, University of Manitoba, Winnipeg, MB, Canada; Manitoba Institute of Child Health, Winnipeg, MB, Canada.
    Kadkhoda, Kamran
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Kroczak, Tadeusz J.
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Gibson, Spencer B.
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada; BioApplications Enterprises, Winnipeg, Canada.
    Booy, Evan P.
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Naderi-Manesh, Hossein
    Department of Biophysics and Biochemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran.
    Los, Marek Jan
    BioApplications Enterprises, Winnipeg, MB, Canada.
    Brevinin-2R semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway2008In: Journal of Cellular and Molecular Medicine (Print), ISSN 1582-1838, E-ISSN 1582-4934, Vol. 12, no 3, p. 1005-1022Article in journal (Refereed)
    Abstract [en]

    Brevinin-2R is a novel non-hemolytic defensin that was isolated from the skin of the frog Rana ridibunda. It exhibits preferential cytotoxicity towards malignant cells, including Jurkat (T-cell leukemia), BJAB (B-cell lymphoma), HT29/219, SW742 (colon carcinomas), L929 (fibrosarcoma), MCF-7 (breast adenocarcinoma), A549 (lung carcinoma), as compared to primary cells including peripheral blood mononuclear cells (PBMC), T cells and human lung fibroblasts. Jurkat and MCF-7 cells overexpressing Bcl2, and L929 and MCF-7 over-expressing a dominant-negative mutant of a pro-apoptotic BNIP3 (ΔTM-BNIP3) were largely resistant towards Brevinin-2R treatment. The decrease in mitochondrial membrane potential (ΔΨm), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin-2R-triggered death. Brevinin-2R interacts with both early and late endosomes. Lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin-B and cathepsin-L prevented Brevinin-2R-induced cell death. Autophagosomes have been detected upon Brevinin-2R treatment. Our results show that Brevinin-2R activates the lysosomalmitochondrial death pathway, and involves autophagy-like cell death.

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  • 47.
    Ghavami, Saeid
    et al.
    Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, University of Manitoba, Canada.
    Eshraghi, Mehdi
    Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, University of Manitoba, Canada.
    Kadkhoda, Kamran
    Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, University of Manitoba, Canada.
    Mutawe, Mark M.
    Department of Physiology, University of Manitoba, Canada; Manitoba Institute of Child's Health, University of Manitoba, Canada.
    Maddika, Subbareddy
    Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, University of Manitoba, Canada.
    Bay, Graham H.
    Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, University of Manitoba, Canada.
    Wesselborg, Sebastian
    Department of Internal Medicine I, University of Tübingen, Tübingen, Germany.
    Halayko, Andrew J.
    Department of Physiology, University of Manitoba, Winnipeg, MB, Canada; Manitoba Institute of Child Health, Winnipeg, MB, Canada; Department of Internal Medicine, University of Manitoba, Winnipeg, MB, Canada .
    Klonisch, Thomas
    Department of Human Anatomy and Cell Sciences, and Manitoba Institute of Child Health, Winnipeg, Canada.
    Los, Marek Jan
    Interfaculty Institute for Biochemistry, University of Tübingen, Germany; BioApplications Enterprises, Winnipeg, MB, Canada.
    Role of BNIP3 in TNF-induced cell death - TNF upregulates BNIP3 expression2009In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1793, no 3, p. 546-560Article in journal (Refereed)
    Abstract [en]

    Tumor necrosis factor alpha (TNF) is a cytokine that induces caspase-dependent (apoptotic) and caspase-independent (necrosis-like) cell death in different cells. We used the murine fibrosarcoma cell line model L929 and a stable L929 transfectant over-expressing a mutated dominant-negative form of BNIP3 lacking the C-terminal transmembrane (TM) domain (L929-ΔTM-BNIP3) to test if TNF-induced cell death involved pro-apoptotic Bcl2 protein BNIP3. Treatment of cells with TNF in the absence of actinomycin D caused a rapid fall in the mitochondrial membrane potential (ΔΨm) and a prompt increase in reactive oxygen species (ROS) production, which was significantly less pronounced in L929-ΔTM-BNIP3. TNF did not cause the mitochondrial release of apoptosis inducing factor (AIF) and Endonuclease G (Endo-G) but provoked the release of cytochrome c, Smac/Diablo, and Omi/HtrA2 at similar levels in both L929 and in L929-ΔTM-BNIP3 cells. We observed TNF-associated increase in the expression of BNIP3 in L929 that was mediated by nitric oxide and significantly inhibited by nitric oxide synthase inhibitor N5-(methylamidino)-l-ornithine acetate. In L929, lysosomal swelling and activation were markedly increased as compared to L929-ΔTM-BNIP3 and could be inhibited by treatment with inhibitors to vacuolar H+-ATPase and cathepsins −B/−L. Together, these data indicate that TNF-induced cell death involves BNIP3, ROS production, and activation of the lysosomal death pathway.

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  • 48.
    Ghavami, Saeid
    et al.
    Department of Physiology, University of Manitoba, Winnipeg, MB, Canada; National Training Program in Allergy and Asthma, University of Manitoba, Winnipeg, MB, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, MB, Canada.
    Eshragi, Mehdi
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Ande, Sudharsana R
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Chazin, Walter J
    Department of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, USA.
    Klonisch, Thomas
    Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada.
    Halayko, Andrew J.
    Department of Physiology, University of Manitoba, Winnipeg, MB, Canada; Manitoba Institute of Child Health, Winnipeg, MB, Canada; Department of Internal Medicine, University of Manitoba, Winnipeg, MB, Canada .
    Mcneill, Karol D
    Manitoba Institute of Child Health, University of Manitoba, Winnipeg, Canada.
    Hashemi, Mohammad
    Department of Clinical Biochemistry, Zahedan University of Medical Sciences, Zahedan, Iran.
    Kerkhoff, Claus
    Institute of Immunology, University of Muenster, Roentgenstr. 21, Muenster, Germany.
    Los, Marek Jan
    Interfaculty Institute for Biochemistry, University of Tübingen, Germany.
    S100A8/A9 induces autophagy and apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes that involves BNIP32010In: Cell Research, ISSN 1001-0602, E-ISSN 1748-7838, Vol. 20, no 3, p. 314-331Article in journal (Refereed)
    Abstract [en]

    The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosis-inducing activity in various cells of different origins. Here, we present evidence that the underlying molecular mechanisms involve both programmed cell death I (PCD I, apoptosis) and PCD II (autophagy)-like death. Treatment of cells with S100A8/A9 caused the increase of Beclin-1 expression as well as Atg12-Atg5 formation. S100A8/A9-induced cell death was partially inhibited by the specific PI3-kinase class III inhibitor, 3-methyladenine (3-MA), and by the vacuole H+-ATPase inhibitor, bafilomycin-A1 (Baf-A1). S100A8/A9 provoked the translocation of BNIP3, a BH3 only pro-apoptotic Bcl2 family member, to mitochondria. Consistent with this finding, ΔTM-BNIP3 overexpression partially inhibited S100A8/A9-induced cell death, decreased reactive oxygen species (ROS) generation, and partially protected against the decrease in mitochondrial transmembrane potential in S100A8/A9-treated cells. In addition, either ΔTM-BNIP3 overexpression or N-acetyl-L-cysteine co-treatment decreased lysosomal activation in cells treated with S100A8/A9. Our data indicate that S100A8/A9-promoted cell death occurs through the cross-talk of mitochondria and lysosomes via ROS and the process involves BNIP3.

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  • 49.
    Ghavami, Saeid
    et al.
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Hashemi, M.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Kadkhoda, K.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Alavian, S. M.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Bay, G. H.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Apoptosis in liver diseases - detection and therapeutic applications2005In: Medical Science Monitor, ISSN 1234-1010, E-ISSN 1643-3750, Vol. 11, no 11, p. RA337-RA345Article, review/survey (Refereed)
    Abstract [en]

    The liver is continuously exposed to a large antigenic load that includes pathogens, toxins, tumor cells and dietary antigens. Amongst the hepatitis viruses, only hepatitis B virus (HBV) and hepatitis C virus (HCV) cause chronic hepatitis, which can progress to cirrhosis and hepatocellular carcinoma. Of the different antiviral defense systems employed by the tissue, apoptosis significantly contributes to the prevention of viral replication, dissemination, and persistence. Loss of tolerance to the liver autoantigens may result in autoimmune hepatitis (AIH). This review outlines the recent findings that highlight the role and mechanisms of apoptotic processes in the course of liver diseases. Among factors that contribute to liver pathology, we discuss the role of tumor necrosis factor (TNF)-alpha, HBx, ds-PKR, TRAIL, FasL, and IL-1 alpha. Since TNF and FasL-induced hepatocyte apoptosis is implicated in a wide range of liver diseases, including viral hepatitis, alcoholic hepatitis, ischemia/reperfusion liver injury, and fulminant hepatic failure, these items will be discussed in greater detail in this review. We also highlight some recent discoveries that pave the way for the development of new therapeutic strategies by protecting hepatocytes (for example by employing Bcl-2, Bcl-X-L or A1/Bfl-1, IAPs, or synthetic caspase inhibitors), or by the induction of apoptosis in stellate cells. The assessment of the severity of liver disease, as well as monitoring of patients with chronic liver disease, remains a major challenge in clinical hepatology practice. Therefore, a separate chapter is devoted to a novel cytochrome c - based method useful for the diagnosis and monitoring of fulminant hepatitis.

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  • 50.
    Ghavami, Saeid
    et al.
    Tarbiat Modarres University, Tehran, Iran.
    Kerkhoff, C.
    Institute of Experimental Dermatology, Münster, Germany .
    Los, Marek Jan
    Institute of Experimental Dermatology, Münster, Germany .
    Hashemi, M.
    Tarbiat Modarres University, Tehran, Iran.
    Sorg, C.
    Institute of Experimental Dermatology, Münster, Germany .
    Karami-Tehrani, F.
    Tarbiat Modarres University, Tehran, Iran.
    Mechanism of apoptosis induced by S100A8/A9 in colon cancer cell lines: the role of ROS and the effect of metal ions2004In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 76, no 1, p. 169-175Article in journal (Refereed)
    Abstract [en]

    The protein complex S100A8/A9, abundant in the cytosol of neutrophils, is secreted from the cells upon cellular activation and induces apoptosis in tumor cell lines and normal fibroblasts in a zinc-reversible manner. In the present study, we present evidence that the S100A8/A9 also exerts its apoptotic effect by a zinc-independent mechanism. Treatment of the colon carcinoma cells with different concentrations of human SI00A8/A9 or the metal ion chelator diethylenetriaminepentacetic acid (DTPA) resulted in a significant increase of cell death. Annexin V/phosphatidylinositol and Hoechst 33258 staining revealed that cell death was mainly of the apoptotic type. A significant increase in the activity of caspase-3 and -9 was observed in both cell lines after treatment. Caspase-8 activation was negligible in both cell lines. The cytotoxicity/apoptotic effect of human SI00A8/A9 and DTPA was inhibited significantly 2 2 (P<0.05) by Zn+2 and Cu+2, more effectively than by Ca2+ and Mg2+. The antioxidant N-acetyl-L-cysteine inhibited the cytotoxicity/apoptotic effect of SI00A8/A9 and DTPA. However, as a result of the different time-courses of both agents and that the S100A8/A9-induced apoptosis was not completely reversed, we conclude that S100A8/A9 exerts its apoptotic effect on two colon carcinoma cell lines through a dual mechanism: one via zinc exclusion from the target cells and the other through a yet-undefined mechanism, probably relaying on the cell-surface receptor(s).

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