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  • 1.
    Andraos, R.
    et al.
    Department of Clinical and Experimental medicine, Linköping University, Linköping, Sweden.
    Södergren, A.L.
    Department of Clinical and Experimental medicine, Linköping University, Linköping, Sweden.
    Öllinger, K.
    Department of Clinical and Experimental medicine, Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Department of Clinical and Experimental medicine, Linköping University, Linköping, Sweden.
    Reactive oxygen species enhance generation of subpopulations of procoagulant platelets2014Conference paper (Refereed)
  • 2.
    Andraos, R.
    et al.
    Linköping University, Linköping, Sweden.
    Södergren, A.L.
    Linköping University, Linköping, Sweden.
    Öllinger, K.
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Reactive oxygen species enhance generation of subpopulations of procoagulant platelets2014Conference paper (Refereed)
  • 3.
    Aronson, D.
    et al.
    Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.
    Wojtaszewski, J.
    Copenhagen Muscle Research Center, August Krogh Institute, University of Copenhagen, Copenhagen, Denmark.
    Thorell, A.
    Department of Surgery, Karolinska Hospital and Institute, Stockholm, Sweden.
    Nygren, J.
    Department of Surgery, Karolinska Hospital and Institute, Stockholm, Sweden.
    Zangen, A.
    Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.
    Richter, E. A.
    Copenhagen Muscle Research Center, August Krogh Institute, University of Copenhagen, Copenhagen, Denmark.
    Ljungqvist, Olle
    Department of Surgery, Karolinska Hospital and Institute, Stockholm, Sweden.
    Fielding, R. A.
    Department of Health Sciences, Sargent Coll. All. Hlth. Professions, Boston University, Boston, MA , United States.
    Goodyear, L. J.
    Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA; Joslin Diabetes Center, One Joslin Place, Boston, MA, United States.
    Extracellular-regulated protein kinase cascades are activated in response to injury in human skeletal muscle1998In: American Journal of Physiology, ISSN 0002-9513, E-ISSN 2163-5773, Vol. 275, no 2, p. C555-C561Article in journal (Refereed)
    Abstract [en]

    The mitogen-activated protein (MAP) kinase signaling pathways are believed to act as critical signal transducers between stress stimuli and transcriptional responses in mammalian cells. However, it is not known whether these signaling cascades also participate in the response to injury in human tissues. To determine whether injury to the vastus lateralis muscle activates MAP kinase signaling in human subjects, two needle biopsies or open muscle biopsies were taken from the same incision site 30-60 min apart. The muscle biopsy procedures resulted in striking increases in dual phosphorylation of the extracellular-regulated kinases (ERK1 and ERK2) and in activity of the downstream substrate, the p90 ribosomal S6 kinase. Raf-1 kinase and MAP kinase kinase, upstream activators of ERK, were also markedly stimulated in all subjects. In addition, c-Jun NH2-terminal kinase and p38 kinase, components of two parallel MAP kinase pathways, were activated following muscle injury. The stimulation of the three MAP kinase cascades was present only in the immediate vicinity of the injury, a finding consistent with a local rather than systemic activation of these signaling cascades in response to injury. These data demonstrate that muscle injury induces the stimulation of the three MAP kinase cascades in human skeletal muscle, suggesting a physiological relevance of these protein kinases in the immediate response to tissue injury and possibly in the initiation of wound healing.

  • 4.
    Basabe-Desmonts, L.
    et al.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland; Biomedical Diagnostics Institute Programme, Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Ramström, Sofia
    Biomedical Diagnostics Institute Programme, Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Lopez-Alonso, A.
    Biomedical Diagnostics Institute Programme, Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Somers, M.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Ricco, A. J.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Kenny, D.
    Biomedical Diagnostics Institute Programme, Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Disposable bioanalytical microdevice for monitoring the effect of anti-platelet drugs2010In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010, 2010, p. 1388-1390Conference paper (Refereed)
    Abstract [en]

    We report a disposable self-powered integrated microfluidic chip that enables a rapid and simple platelet-function assay from small samples of whole blood. The chip integrates a single-cell adhesion assay with a microfluidic platform; it enables accurate quantification of platelet adhesion, and it controls whole blood flow rate, shear stress, volume of sample, and assay time.

  • 5.
    Basabe-Desmonts, L.
    et al.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Ramström, Sofia
    BDI Programme, Molecular & Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Meade, G.
    BDI Programme, Molecular & Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    O'Neill, S.
    BDI Programme, Molecular & Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Riaz, A.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Lee, L. P.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland; Biomolecular Nanotechnology Center, Berkeley Sensor & Actuator Center, Department of Bioengineering, University of California, Berkeley CA, USA.
    Ricco, A. J.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Kenny, D.
    BDI Programme, Molecular & Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Single-Step Separation of Platelets from Whole Blood Coupled with Digital Quantification by Interfacial Platelet Cytometry (iPC)2010In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 26, no 18, p. 14700-14706Article in journal (Refereed)
    Abstract [en]

    We report the efficient single-step separation of individual platelets from unprocessed whole blood, enabling digital quantification of platelet function using interfacial platelet cytometry (iPC) on a chip iPC is accomplished by the precision micropatterning of platelet-specific protein surfaces on solid substrates By separating platelets From whole blood using specific binding to protein spots of a defined size. iPC implements a simple incubate-and-rinse approach, without sample preparation, that enables (I) the study of platelets in the physiological situation of interaction with a protein surface, (2) the choice of the number of platelets bound on each protein spot, from one to many, (3) control of the platelet platelet distance, including the possibility to study noninteracting single platelets, (4) digital quantification (counting) of platelet adhesion to selected protein matrices, enabling statistical characterization of platelet subpopuladons from meaningfully large numbers of single platelets, (5) the study of platelet receptor expression and spatial distribution, and (6) a detailed study of the morphology of isolated single platelets at activation levels that can be manipulated To date, we have demonstrated 1-4 of the above list Platelets were separated from whole blood using tPC with fibrinogen, von Willebrand factor (VWF), and anti-CD42b antibody printed "spots" ranging from a fraction of one to several platelet diameters (2-24 full) The number of platelets captured per spot depends strongly on the protein matrix and the surface area of the spot, together with the platelet volume, morphology, and activation state Blood samples from healthy donors, a May-Hegglin-anomaly patient, and a Glanzmann's Thrombasthenia patient were analyzed via iPC to confirm the specificity of the interaction between protein matrices and platelets For example, the results indicate that platelets interact with fibrinogen spots only through the fibrinogen receptor (aIlb beta 3) and, relevant to diagnostic applications, platelet adhesion correlates strongly with normal versus abnormal platelet function A critical function of platelets is to adhere to regions of damage on blood vessel walls, in contrast to conventional flow cytometry, where platelets are suspended in solution, iPC enables physiologically relevant platelet bioassays based on platelet/protein-matrix inter actions on surfaces. This technology should be inexpensive to implement in clinical assay format, is readily integrable into fluidic microdevices, and paves the way for high-throughput platelet assays from microliter volumes of whole blood.

  • 6. Batakis, P.
    et al.
    Lopez-Alonso, A.
    Claesson, K.
    Kenny, D.
    Basabe-Desmonts, L.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Microcontact printing as a tool to study platelet adhesion2013Conference paper (Refereed)
  • 7.
    Boknas, N.
    et al.
    Linköping University, Linköping, Sweden.
    Faxalv, L.
    Linköping University, Linköping, Sweden.
    Lindahl, T. L.
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Contact activation: important to consider when measuring the contribution of tissue factor-bearing microparticles to thrombin generation using phospholipid-containing reagents2014In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, E-ISSN 1538-7836, Vol. 12, no 4, p. 515-518Article in journal (Refereed)
    Abstract [en]

    Background: A commercial MP reagent containing phospholipids is used for thrombin generation (TG) measurements to estimate the procoagulant activity of microparticles (MPs). Previous reports have shown that contact activation affects TG when TF levels are low, and that addition of phospholipids might augment this effect.

    Objectives: To quantify the impact of contact activation on TG in the presence of phospholipids and low/no TF, as is the case using a commercially available MP-reagent. Methods Thrombin generation was analyzed using MP- or platelet-rich plasma (PRP)-reagent in the presence and absence of corn trypsin inhibitor and anti-TF antibodies, respectively. To quantify the impact of different experimental parameters on contact activation, microparticle-depleted plasma was analyzed in the presence of different concentrations of phospholipids, TF and/or contact activating agents (kaolin).

    Results: Even with low contact activating blood collection tubes, substantial thrombin generation was observed with the MP-reagent, but this was completely inhibited by addition of corn trypsin inhibitor. Control experiments illustrate that the phospholipids in the reagent play a major role in enhancing TG initiated by FXIIa. Even with the PRP-reagent, which is recommended for determining the content of phospholipids from MPs, TG was partly dependent on contact activation.

    Conclusions: Contact activation plays a major role in TG when using reagents/samples containing phospholipids but little or no tissue factor. This needs to be considered and accounted for in future clinical studies using TG to assess the procoagulant activity of MPs.

  • 8. Boknäs, N.
    et al.
    Faxälv, L.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Phospholipid-containing reagents for the detection of tissue factor on microparticles by thrombin generation cause analytical errors by amplifying the contact activation pathway2013Conference paper (Refereed)
  • 9.
    Boknäs, N.
    et al.
    Linköping University, Linköping, Sweden.
    Faxälv, L.
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    LLinköping University, Linköping, Sweden.
    Lindahl, T.
    Linköping University, Linköping, Sweden.
    Thrombin generation in plasma measured with a commercial reagent for the detection of microparticle-derived tissue factor is heavily influenced by contact activation2013In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, E-ISSN 1538-7836, Vol. 11, no S1, p. 401-402Article in journal (Refereed)
  • 10.
    Centellas, Daniel Sanchez
    et al.
    Linköping University, Linköping, Sweden.
    Gudlur, Sushanth
    Linköping University, Linköping, Sweden; Nanyang Technology University, Singapore, Singapore..
    Vicente-Carrillo, Alejandro
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Lindahl, Tomas L.
    Linköping University, Linköping, Sweden.
    A cluster of aspartic residues in the extracellular loop II of PAR 4 is important for thrombin interaction and activation of platelets2017In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 154, p. 84-92Article in journal (Refereed)
    Abstract [en]

    Thrombin activates platelets via proteolytic cleavage of protease-activated receptors (PARs) 1 and 4. The two PARs have distinct but complementary roles. The mechanisms responsible for PAR1 activation by thrombin have been extensively studied. However, much less is known regarding thrombin activation of PAR4, especially the potential involvement of regions of PAR4 other than the N-terminal, which is bound to the catalytic site of thrombin. We have studied PAR4 in S. cerevisiae strainMMY12, an expression system in which the GPCR receptors are connected to a Lac Z reporter gene resulting in increased beta-galactosidase activity. This approach was used to assess PAR4 mutants to evaluate the contribution of different aspartic residues in facilitating PAR4 activation. Furthermore, peptides mimicking parts of the PAR4 N-terminal and the second extracellular loop (ECLII) were tested for their ability to inhibit platelet activation by thrombin. Binding of these peptides to gamma-thrombin was studied by monitoring the decrease in tryptophan fluorescence intensity of thrombin. We conclude that not only the N-terminal but also the electronegative aspartic residues D224, D230 and D235 (located in ECLII) are be important for PAR4 binding to thrombin. We further suggest that they play a role for the tethered ligand binding to the receptor, as mutations also affected activation in response to a PAR4-activating peptide mimicking the new N-terminal formed after cleavage. This agrees with previous results on PAR1 and thrombin binding. We suggest that the ECLII of PAR4 could be a potential target for antithrombotic drug development.

  • 11.
    Chaillou, Thomas
    et al.
    Département Environnements Opérationnels, Institut de Recherche Biomédicale des Armées Antenne de la Tronche, La Tronche cedex, France.
    Koulmann, N.
    Département Environnements Opérationnels, Institut de Recherche Biomédicale des Armées Antenne de la Tronche, La Tronche cedex, France; Ecole du Val-de-Grâce, Paris, France.
    Meunier, A.
    Département Environnements Opérationnels, Institut de Recherche Biomédicale des Armées Antenne de la Tronche, La Tronche cedex, France.
    Chapot, R.
    Département Environnements Opérationnels, Institut de Recherche Biomédicale des Armées Antenne de la Tronche, La Tronche cedex, France.
    Serrurier, B.
    Département Environnements Opérationnels, Institut de Recherche Biomédicale des Armées Antenne de la Tronche, La Tronche cedex, France.
    Beaudry, M.
    Laboratoire Réponses Cellulaires et Fonctionnelles À l'Hypoxie, EA2363, Sorbonne-Paris-Cité, Université Paris, Bobigny Cedex, France.
    Bigard, X.
    Département Environnements Opérationnels, Institut de Recherche Biomédicale des Armées Antenne de la Tronche, La Tronche cedex, France.
    Effect of hypoxia exposure on the recovery of skeletal muscle phenotype during regeneration2014In: Molecular and Cellular Biochemistry, ISSN 0300-8177, E-ISSN 1573-4919, Vol. 390, no 1-2, p. 31-40Article in journal (Refereed)
    Abstract [en]

    Hypoxia impairs the muscle fibre-type shift from fast-to-slow during post-natal development; however, this adaptation could be a consequence of the reduced voluntary physical activity associated with hypoxia exposure rather than the result of hypoxia per se. Moreover, muscle oxidative capacity could be reduced in hypoxia, particularly when hypoxia is combined with additional stress. Here, we used a model of muscle regeneration to mimic the fast-to-slow fibre-type conversion observed during post-natal development. We hypothesised that hypoxia would impair the recovery of the myosin heavy chain (MHC) profile and oxidative capacity during muscle regeneration. To test this hypothesis, the soleus muscle of female rats was injured by notexin and allowed to recover for 3, 7, 14 and 28 days under normoxia or hypobaric hypoxia (5,500 m altitude) conditions. Ambient hypoxia did not impair the recovery of the slow MHC profile during muscle regeneration. However, hypoxia moderately decreased the oxidative capacity (assessed from the activity of citrate synthase) of intact muscle and delayed its recovery in regenerated muscle. Hypoxia transiently increased in both regenerated and intact muscles the content of phosphorylated AMPK and Pgc-1α mRNA, two regulators involved in mitochondrial biogenesis, while it transiently increased in intact muscle the mRNA level of the mitophagic factor BNIP3. In conclusion, hypoxia does not act to impair the fast-to-slow MHC isoform transition during regeneration. Hypoxia alters the oxidative capacity of intact muscle and delays its recovery in regenerated muscle; however, this adaptation to hypoxia was independent of the studied regulators of mitochondrial turn-over.

  • 12.
    Chaillou, Thomas
    et al.
    Operational environments, IRBA La Tronche, La Tronche, France.
    Malgoyre, A.
    Operational environments, IRBA La Tronche, La Tronche, France.
    Banzet, S.
    Operational environments, IRBA La Tronche, La Tronche, France.
    Chapot, R.
    Operational environments, IRBA La Tronche, La Tronche, France.
    Koulmann, N.
    Operational environments, IRBA La Tronche, La Tronche, France.
    Pugnière, P.
    Genomic Core Facility, IRBA La Tronche, La Tronche, France.
    Beaudry, M.
    Laboratoire Réponses Cellulaires et Fonctionnelles À l'Hypoxie, Université Paris, Bobigny, France.
    Bigard, X.
    Operational environments, IRBA La Tronche, La Tronche, France.
    Peinnequin, A.
    Genomic Core Facility, IRBA La Tronche, La Tronche, France.
    Pitfalls in target mRNA quantification for real-time quantitative RT-PCR in overload-induced skeletal muscle hypertrophy2011In: Physiological Genomics, ISSN 1094-8341, E-ISSN 1531-2267, Vol. 43, no 4, p. 228-235Article in journal (Refereed)
    Abstract [en]

    Quantifying target mRNA using real-time quantitative reverse transcription-polymerase chain reaction requires an accurate normalization method. Determination of normalization factors (NFs) based on validated reference genes according to their relative stability is currently the best standard method in most usual situations. This method controls for technical errors, but its physiological relevance requires constant NF values for a fixed weight of tissue. In the functional overload model, the increase in the total RNA concentration must be considered in determining the NF values. Here, we pointed out a limitation of the classical geNorm-derived normalization. geNorm software selected reference genes despite that the NF values extensively varied under experiment. Only the NF values calculated from four intentionally selected genes were constant between groups. However, a normalization based on these genes is questionable. Indeed, three out of four genes belong to the same functional class (negative regulator of muscle mass), and their use is physiological nonsense in a hypertrophic model. Thus, we proposed guidelines for optimizing target mRNA normalization and quantification, useful in models of muscle mass modulation. In our study, the normalization method by multiple reference genes was not appropriate to compare target mRNA levels between overloaded and control muscles. A solution should be to use an absolute quantification of target mRNAs per unit weight of tissue, without any internal normalization. Even if the technical variations will stay present as a part of the intergroup variations, leading to less statistical power, we consider this method acceptable because it will not generate misleading results.

  • 13.
    Coenen Schimke, J. M.
    et al.
    Endocrine Research Unit, Division of Endocrinology and Metabolism, Mayo Clinic and Mayo Foundation, Rochester, MN, USA.
    Ljungqvist, Olle
    Department of Medicine, University of Vermont, Burlington, VT, USA.
    Sarkar, G.
    Department of Medicine, University of Vermont, Burlington, VT, USA.
    Conover, C. A.
    Endocrine Research Unit, Division of Endocrinology and Metabolism, Mayo Clinic and Mayo Foundation, Rochester, MN, USA.
    Nair, K. S.
    Endocrine Research Unit, Division of Endocrinology and Metabolism, Mayo Clinic and Mayo Foundation, Rochester, MN, USA.
    A quantitative PCR measurement of messenger RNA expression of IGF-I, IGF-II and IGFBP-5 in human skeletal muscle1999In: Growth Hormone & IGF Research, ISSN 1096-6374, E-ISSN 1532-2238, Vol. 9, no 3, p. 179-186Article in journal (Refereed)
    Abstract [en]

    Insulin-like growth factor-I and -II (IGF-I and IGF-II) and their binding proteins are important components in growth promotion and tissue maintenance. We determined the presence of IGF-I, -II, and binding protein 5 (IGFBP-5) gene expression in human skeletal muscle and that mRNA abundance is not altered by nutrients and insulin. In the first protocol, (control) subjects were given water. In the second protocol, half of these subjects drank Polycose (carbohydrate) and the remaining subjects drank equal calories as a mixed meal. Quadriceps muscle biopsies were taken at 10 h. A semi-quantitative polymerase chain reaction was designed to measure gene expression. IGF-I, IGF-II and IGFBP-5 mRNA are present in adult human skeletal muscle, but no significant changes between meal groups were observed for IGF-I, IGF-II or IGFBP-5 mRNA levels, indicating that the expression of these genes are not altered acutely by nutrients and insulin.

  • 14.
    Connolly-Andersen, Anne-Marie
    et al.
    Umeå University, Umeå, Sweden.
    Sundberg, Erik
    Umeå University, Umeå, Sweden.
    Ahlm, Clas
    Umeå University, Umeå, Sweden.
    Hultdin, Johan
    Umeå University, Umeå, Sweden.
    Baudin, Maria
    Umeå University, Umeå, Sweden.
    Larsson, Johanna
    Umeå University, Umeå, Sweden.
    Dunne, Eimear
    Royal Coll Surgeons Ireland, Dublin, Ireland.
    Kenny, Dermot
    Royal Coll Surgeons Ireland, Dublin, Ireland.
    Lindahl, Tomas L.
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Nilsson, Sofie
    Umeå University, Umeå, Sweden.
    Increased Thrombopoiesis and Platelet Activation in Hantavirus-Infected Patients2015In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 212, no 7, p. 1061-1069Article in journal (Refereed)
    Abstract [en]

    Background: Thrombocytopenia is a common finding during viral hemorrhagic fever, which includes hemorrhagic fever with renal syndrome (HFRS). The 2 main causes for thrombocytopenia are impaired thrombopoiesis and/or increased peripheral destruction of platelets. In addition, there is an increased intravascular coagulation risk during HFRS, which could be due to platelet activation.

    Methods: Thrombopoiesis was determined by quantification of platelet counts, thrombopoietin, immature platelet fraction, and mean platelet volume during HFRS. The in vivo platelet activation was determined by quantification of soluble P-selectin (sP-selectin) and glycoprotein VI (sGPVI). The function of circulating platelets was determined by ex vivo stimulation followed by flow cytometry analysis of platelet surface-bound fibrinogen and P-selectin exposure. Intravascular coagulation during disease was determined by scoring for disseminated intravascular coagulation (DIC) and recording thromboembolic complications.

    Results: The levels of thrombopoietin, immature platelet fraction, and mean platelet volume all indicate increased thrombopoiesis during HFRS. Circulating platelets had reduced ex vivo function during disease compared to follow-up. Most interestingly, we observed significantly increased in vivo platelet activation in HFRS patients with intravascular coagulation (DIC and thromboembolic complications) as shown by sP-selectin and sGPVI levels. Conclusions. HFRS patients have increased thrombopoiesis and platelet activation, which contributes to intravascular coagulation.

  • 15. Deb, S.
    et al.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Intracellular signaling in platelet sub-populations: a flow cytometry-based approach2014Conference paper (Refereed)
  • 16.
    Deb, S.
    et al.
    Linköping University, Linköping, Sweden.
    Sjöström, C.
    Linköping University, Linköping, Sweden.
    Tharmakulanathan, A.
    Linköping University, Linköping, Sweden.
    Boknäs, N.
    Linköping University, Linköping, Sweden.
    Lotfi, K.
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Individual variation in hemostatic alterations caused by tyrosine kinase inhibitors: a way to improve personalized cancer therapy?2016In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 140, no Suppl. 1, p. S196-S197, article id PO-55Article in journal (Refereed)
    Abstract [en]

    INTRODUCTION: During the last two decades, Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukemia (CML), and are now considered standard treatment for this disease. However, TKIs can induce serious hemostatic side effects including cardiovascular disease and bleeding disorders. Blood platelet aggregation and formation of pro-coagulant platelets are important to allow a well-balanced hemostatic response. Therefore, a detailed understanding of what effect different TKIs exert on platelets and hemostasis could help to understand if there are differences of importance to minimize the risk of bleeding complications in treated patients.

    AIM: To investigate how TKIs used in CML (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) affect platelet activation and hemostasis.

    MATERIALS AND METHODS: We have developed a multi-parameter six color flow cytometry protocol to study different aspects of platelet function upon activation, e.g. formation of aggregatory (PAC-1-positive) and pro-coagulant (phosphatidylserine-exposing) platelets, exocytosis of alpha- and lysosomal granules and mitochondrial membrane potential.This protocol was performed in presence or absence of TKIs in blood from normal donors and in treated patients. Whole blood aggregometry (Multiplate®), thrombin generation in platelet-rich plasma and in vitro thrombus formation by free oscillation rheometry (ReoRox G2) was further evaluated in some situations.

    RESULTS: At clinically relevant concentrations, dasatinib significantly decreased the formation of procoagulant platelets. Ponatinib induced a slight decrease in formation of procoagulant platelets, whereas bosutinib and nilotinib showed opposite tendencies (n=7). Dasatinib also decreased platelet aggregation (n=4-6) and in vitro thrombus formation (n=3). Thrombin generation was not significantly affected by therapeutic levels of TKIs, whereas higher doses of dasatinib, bosutinib, ponatinib and imatinib significantly changed one or several of the thrombin generation parameters (n=7-8). Interestingly, large differences in response to the drugs were observed among the healthy donors, especially for dasatinib and bosutinib. Major inter-individual variations were also observed in dasatinib-treated patients, see Figure 1.

    CONCLUSIONS: Different TKIs show varying potency to affect platelet-based hemostasis. In addition, we found large inter-individual variations in how some drugs affected platelet function. Therefore, we suggest that development of a clinically useful protocol for platelet function testing could help to identify patients more susceptible to adverse drug reactions. Such a protocol could potentially help clinicians to gain insight into the risk of side effects, which could help to choose the most suitable drug for each individual patient.

  • 17.
    Donner, Lili
    et al.
    Department of Clinical and Experimental Hemostasis, Hemotherapy and Transfusion Medicine, Heinrich Heine University, Düsseldorf, Germany.
    Fälker, Knut
    Örebro University, School of Medical Sciences. Cardiovascular Research Centre, Örebro University Hospital, Örebro, Sweden.
    Gremer, Lothar
    Institute of Physical Biology, Heinrich Heine University, Düsseldorf, Germany; Institute of Structural Biochemistry (ICS-6), Research Centre Jülich, Jülich, Germany.
    Klinker, Stefan
    Institute of Physical Biology, Heinrich Heine University, Düsseldorf, Germany.
    Pagani, Giulia
    Institute for Pharmaceutical and Medicinal Chemistry, Department of Mathematics and Natural Sciences, Heinrich Heine University, Düsseldorf, Germany.
    Ljungberg, Liza U.
    Örebro University, School of Medical Sciences. Cardiovascular Research Centre, Örebro University Hospital, Örebro, Sweden.
    Lothmann, Kimberley
    Institute of Physical Biology, Heinrich Heine University, Düsseldorf, Germany.
    Rizzi, Federica
    Department of Biomedical, Biotechnological, and Translation Sciences, University of Parma, Parma, Italy; Centre for Molecular and Translational Oncology (COMT), University of Parma, Parma, Italy; National Institute of Biostructure and Biosystems (INBB), Rome, Italy.
    Schaller, Martin
    Department of Dermatology, University of Tübingen, Tübingen, Germany.
    Gohlke, Holger
    Institute for Pharmaceutical and Medicinal Chemistry, Department of Mathematics and Natural Sciences, Heinrich Heine University, Düsseldorf, Germany.
    Willbold, Dieter
    Institute of Physical Biology, Heinrich Heine University, Düsseldorf, Germany; Institute of Structural Biochemistry (ICS-6), Research Centre Jülich, Jülich, Germany.
    Grenegård, Magnus
    Örebro University, School of Medical Sciences. Cardiovascular Research Centre, Örebro University Hospital, Örebro, Sweden.
    Elvers, Margitta
    Department of Clinical and Experimental Hemostasis, Hemotherapy and Transfusion Medicine, Heinrich Heine University, Düsseldorf, Germany.
    Platelets contribute to amyloid-β aggregation in cerebral vessels through integrin αIIbβ3-induced outside-in signaling and clusterin release2016In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 9, no 429, article id ra52Article in journal (Refereed)
    Abstract [en]

    Cerebral amyloid angiopathy (CAA) is a vascular dysfunction disorder characterized by deposits of amyloid-β (Aβ) in the walls of cerebral vessels. CAA and Aβ deposition in the brain parenchyma contribute to dementia and Alzheimer's disease (AD). We investigated the contribution of platelets, which accumulate at vascular Aβ deposits, to CAA. We found that synthetic monomeric Aβ40 bound through its RHDS (Arg-His-Asp-Ser) sequence to integrin αIIbβ3, which is the receptor for the extracellular matrix protein fibrinogen, and stimulated the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin from platelets. Clusterin promoted the formation of fibrillar Aβ aggregates, and ADP acted through its receptors P2Y1 and P2Y12 on platelets to enhance integrin αIIbβ3 activation, further increasing the secretion of clusterin and Aβ40 binding to platelets. Platelets from patients with Glanzmann's thrombasthenia, a bleeding disorder in which platelets have little or dysfunctional αIIbβ3, indicated that the abundance of this integrin dictated Aβ-induced clusterin release and platelet-induced Aβ aggregation. The antiplatelet agent clopidogrel, which irreversibly inhibits P2Y12, inhibited Aβ aggregation in platelet cultures; in transgenic AD model mice, this drug reduced the amount of clusterin in the circulation and the incidence of CAA. Our findings indicate that activated platelets directly contribute to CAA by promoting the formation of Aβ aggregates and that Aβ, in turn, activates platelets, creating a feed-forward loop. Thus, antiplatelet therapy may alleviate fibril formation in cerebral vessels of AD patients.

  • 18.
    Elo, Laura L.
    et al.
    Biomathematics Research Group, Department of Mathematics, University of Turku, Turku, Finland; Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland.
    Järvenpää, Henna
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland; Turku Graduate School of Biomedical Sciences, Turku, Finland.
    Tuomela, Soile
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland; Turku Graduate School of Biomedical Sciences, Turku, Finland.
    Raghav, Sunil
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland.
    Ahlfors, Helena
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland; The National Graduate School in Informational and Structural Biology, Åbo Akademi University, Turku, Finland.
    Laurila, Kirsti
    Department of Signal Processing, Tampere University of Technology, Tampere, Finland.
    Gupta, Bhawna
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland.
    Lund, Riikka J.
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland; Department of Biological Science, University of Sheffield, Sheffield, United Kingdom.
    Tahvanainen, Johanna
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland; Drug Discovery Graduate School, University of Turku, Turku, Finland.
    Hawkins, R. David
    Ludwig Institute for Cancer Research, University of California, San Diego CA, United States.
    Oresic, Matej
    Örebro University, School of Medical Sciences. VTT Technical Research Centre of Finland, Espoo, Finland.
    Lähdesmäki, Harri
    Department of Signal Processing, Tampere University of Technology, Tampere, Finland; Department of Information and Computer Science, Helsinki University of Technology, Helsinki, Finland.
    Rasool, Omid
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland.
    Rao, Kanury V.
    Biomathematics Research Group, Department of Mathematics, University of Turku, Turku, Finland.
    Aittokallio, Tero
    International Centre for Genetic Engineering and Biotechnology, New Delhi, India; Immune Disease Institute, Harvard Medical School, Boston MA, United States.
    Lahesmaa, Riitta
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland; Immune Disease Institute, Harvard Medical School, Boston MA, United States.
    Genome-wide profiling of interleukin-4 and STAT6 transcription factor regulation of human Th2 cell programming2010In: Immunity, ISSN 1074-7613, E-ISSN 1097-4180, Vol. 32, no 6, p. 852-862Article in journal (Refereed)
    Abstract [en]

    Dissecting the molecular mechanisms by which T helper (Th) cells differentiate to effector Th2 cells is important for understanding the pathogenesis of immune-mediated diseases, such as asthma and allergy. Because the STAT6 transcription factor is an upstream mediator required for interleukin-4 (IL-4)-induced Th2 cell differentiation, its targets include genes important for this process. Using primary human CD4(+) T cells, and by blocking STAT6 with RNAi, we identified a number of direct and indirect targets of STAT6 with ChIP sequencing. The integration of these data sets with detailed kinetics of IL-4-driven transcriptional changes showed that STAT6 was predominantly needed for the activation of transcription leading to the Th2 cell phenotype. This integrated genome-wide data on IL-4- and STAT6-mediated transcription provide a unique resource for studies on Th cell differentiation and, in particular, for designing interventions of human Th2 cell responses.

  • 19. Faxälv, L.
    et al.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Soutukorva, K.
    Tengvall, P.
    Lindahl, T.L.
    Coagulation factor XII is not activated by platelets but facilitates platelet-driven propagation of coagulation 2012Conference paper (Refereed)
  • 20. Faxälv, L.
    et al.
    Wallstedt, M.
    Ramström, Sofia
    Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland; Dublin City University, Dublin, Ireland.
    A flow cytometric method to study platelet adhesion to protein-coated polystyrene beads2011Conference paper (Refereed)
  • 21.
    Fälker, Knut
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Biomedicine; Dept Clin & Expt Med, Linköping Univ, Linköping, Sweden.
    Klarström-Engström, Kristin
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Biomedicine.
    Bengtsson, Torbjörn
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Biomedicine.
    Lindahl, Tomas L.
    Dept Clin & Expt Med, Linköping Univ, Linköping, Sweden.
    Grenegård, Magnus
    Örebro University, School of Medicine, Örebro University, Sweden. Department of Biomedicine.
    The Toll-like receptor 2/1 (TLR2/1) complex initiates human platelet activation via the src/Syk/LAT/PLC gamma 2 signalling cascade2014In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 26, no 2, p. 279-286Article in journal (Refereed)
    Abstract [en]

    The specific TLR2/1 complex activator Pam3CSK4 has been shown to provoke prominent activation and aggregation of human non-nucleated platelets. As Pam3CSK4-evoked platelet activation does not employ the major signalling pathway established in nucleated immune cells, we investigated if the TLR2/1 complex on platelets may initiate signalling pathways known to be induced by physiological agonists such as collagen via GPVI or thrombin via PARs. We found that triggering TLR2/1 complex-signalling with Pam3CSK4, in common with that induced via GPVI, and in contrast to that provoked by PARS, involves tyrosine phosphorylation of the adaptor protein LAT as well as of PLC gamma 2 in a src- and Syk-dependent manner. In this respect, we provide evidence that Pam3CSK4 does not cross-activate GPVI. Further, by the use of platelets from a Glanzmann's thrombasthenia patient lacking beta(3), in contrast to findings in nucleated immune cells, we show that the initiation of platelet activation by Pam3CSK4 does not involve integrin beta(3) signalling; whereas the latter, subsequent to intermediate TXA2 synthesis and signalling, was found to be indispensable for proper dense granule secretion and full platelet aggregation. Together, our findings reveal that triggering the TLR2/1 complex with Pam3CSK4 initiates human platelet activation by engaging tyrosine kinases of the src family and Syk, the adaptor protein LAT, as well as the key mediator PLC gamma 2. (C) 2013 Elsevier Inc. All rights reserved.

  • 22.
    Hagemann, Urs B.
    et al.
    Affitech Research AS, Oslo, Norway; Algeta/Bayer AS, Oslo, Norway.
    Gunnarsson, Lavinia
    Örebro University, School of Science and Technology. Affitech Research AS, Oslo, Norway.
    Geraudie, Solene
    Affitech Research AS, Oslo, Norway; Radiumhospitalet, Oslo, Norway.
    Scheffler, Ulrike
    Affitech Research AS, Oslo, Norway; ProBioGen AG, Berlin, Germany.
    Griep, Remko A.
    Affitech Research AS, Oslo, Norway; AbCheck, Plzen, Czech Republic.
    Reiersen, Herald
    Affitech Research AS, Oslo, Norway; Jiffy International AS, Aas, Norway .
    Duncan, Alexander R.
    Affitech Research AS, Oslo, Norway; Actigen Ltd, Cambridge, United Kingdom.
    Kiprijanov, Sergej M.
    Affitech Research AS, Oslo, Norway; BerGenBio AS, Bergen, Norway.
    Fully Human Antagonistic Antibodies against CCR4 Potently Inhibit Cell Signaling and Chemotaxis2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 7, article id e103776Article in journal (Refereed)
    Abstract [en]

    Background: CC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis.

    Methodology: Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies.

    Significance: For the first time, successful generation of anti-G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer.

  • 23.
    Holm, Ann-Charlotte B. Svensson
    et al.
    Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden; Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
    Grenegård, Magnus
    Örebro University, School of Medicine, Örebro University, Sweden. Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Linköping, Sweden; Department of Clinical Pathology and Clinical Genetics, County Council of Östergötland, Linköping, Sweden.
    Ollinger, Karin
    Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden; Department of Clinical Pathology and Clinical Genetics, County Council of Östergötland, Linköping, Sweden.
    Lindström, Eva G.
    Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
    Inhibition of 12-lipoxygenase reduces platelet activation and prevents their mitogenic function2014In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 25, no 2, p. 111-117Article in journal (Refereed)
    Abstract [en]

    The aim of the present study was to investigate the role of 12-lipoxygenase (12-LOX) on platelet-induced airway smooth muscle cell (ASMC) proliferation. Co-incubation of platelets and ASMC caused platelet activation as determined by morphological changes. Simultaneously, reactive oxygen species (ROS)-generation was detected and ASMC proliferation (measured by using the MTS assay) increased significantly. Furthermore, we found that the 12-LOX inhibitors cinnamyl-3,4-dihydroxy-a-cyanocinnamate (CDC) and Baicalein prevented platelet activation in a co-cultures of platelets and ASMC. The inhibitory effect of CDC and Baicalein on platelets was also registered in a pure platelet preparation. Specifically, the 12-LOX inhibitors reduced collagen-induced platelet aggregation both in the presence and absence of external added fibrinogen. Importantly, platelet-induced ASMC proliferation and ROS production generated during the platelet/ASMC interaction was significantly inhibited in the presence of 12-LOX inhibitors. In conclusion, our findings reveal that 12-LOX is crucial for the observed enhancement of ASMC proliferation in co-cultures of platelets and ASMC. The present result suggests that 12-LOX activity is important in the initial step of platelet/ASMC interaction and platelet activation. Such action of 12-LOX represents a potential important mechanism that may contribute to platelet-induced airway remodelling.

  • 24.
    Huerta-García, Elizabeth
    et al.
    División Académica Multidisciplinaria de Comalcalco, Universidad Juárez Autónoma Tabasco, Comalcalco, Tabasco, México.
    Ramos-Godinez, María Del Pilar
    Departamento de Microscopía Electrónica, Ciudad de México, México .
    López-Saavedra, Alejandro
    Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, Ciudad de México, México.
    Alfaro-Moreno, Ernesto
    Örebro University, School of Science and Technology. of Environmental Health, Karolinska Institute, Stockholm, Sweden.
    Gómez-Crisóstomo, Nancy Patricia
    División Académica Multidisciplinaria de Comalcalco, Universidad Juárez Autónoma Tabasco, Comalcalco, Tabasco, México.
    Colín-Val, Zaira
    Departamento de Fisiología, Instituto Nacional de Cardiología “Ignacio Chávez”, Ciudad de México, México.
    Sánchez-Barrera, Helen
    Departamento de Fisiología, Instituto Nacional de Cardiología “Ignacio Chávez”, Ciudad de México, México.
    López-Marure, Rebeca
    Departamento de Fisiología, Instituto Nacional de Cardiología “Ignacio Chávez”, Ciudad de México, México.
    Internalization of Titanium Dioxide Nanoparticles Is Mediated by Actin-Dependent Reorganization and Clathrin- and Dynamin-Mediated Endocytosis in H9c2 Rat Cardiomyoblasts2019In: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 32, no 4, p. 578-588Article in journal (Refereed)
    Abstract [en]

    Titanium dioxide nanoparticles (TiO2 NPs) are widely used for industrial and commercial applications. Once inside the body, they translocate into the bloodstream and reach different areas of the cardiovascular system including the heart, increasing the risk of developing cardiovascular diseases; consequently, the investigation of their interaction with cardiac cells is required. We previously showed that TiO2 NPs are internalized by H9c2 rat cardiomyoblasts, and here, we examined the molecular mechanisms underlying this process. TiO2 NPs internalization was evaluated by transmission electron microscopy, time-lapse microscopy, and flow cytometry. Changes in the actin cytoskeleton were studied by phalloidin staining. Endocytic uptake mechanisms for nanoparticles were probed with chemical inhibitors, whereas clathrin and dynamin expression was measured by Western blot. Cellular uptake of TiO2 NPs occurred early after 30 min exposure, and large aggregates were observed after 1 h. Actin cytoskeleton reorganization included cell elongation plus lower density and stability of actin fibers. Cytochalasin-D inhibited TiO2 NPs uptake, indicating actin-mediated internalization. Dynamin and clathrin levels increased early after TiO2 NPs exposure, and their inhibition reduced nanoparticle uptake. Therefore, TiO2 NPs internalization by H9c2 rat cardiomyoblasts involves actin cytoskeleton reorganization and clathrin/dynamin-mediated endocytosis.

  • 25.
    Ignatova, A.
    et al.
    Dmitry Rogachev National Research Center of Pediatric Hematology Oncology and Immunology, Moscow, Russian Federation.
    Nikitin, E.
    Botkin City Clinical Hospital, Moscow, Russian Federation.
    Tharmakulanathan, A.
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Örebro University, School of Medical Sciences. Linköping University, Linköping, Sweden; Department of Clinical Medicine, School of Medical Sciences, Örebro University, Örebro,.
    Poletaev, A.
    Dmitry Rogachev National Research Center of Pediatric Hematology Oncology and Immunology, Moscow, Russian Federation.
    Polokhov, D.
    Dmitry Rogachev National Research Center of Pediatric Hematology Oncology and Immunology, Moscow, Russian Federation.
    Fedotov, A.
    Dmitry Rogachev National Research Center of Pediatric Hematology Oncology and Immunology, Moscow, Russian Federation.
    Seregina, E.
    Dmitry Rogachev National Research Center of Pediatric Hematology Oncology and Immunology, Moscow, Russian Federation.
    Pshonkin, A.
    Dmitry Rogachev National Research Center of Pediatric Hematology Oncology and Immunology, Moscow, Russian Federation.
    Vorobiev, V
    Botkin City Clinical Hospital, Moscow, Russian Federation.
    Ptushkin, V.
    Botkin City Clinical Hospital, Moscow, Russian Federation.
    Panteleev, M.
    Dmitry Rogachev National Research Center of Pediatric Hematology Oncology and Immunology, Moscow, Russian Federation.
    Platelet Function and Ibrutinib Treatment in Chronic Lymphocytic Leukaemia and Mantle Cell Lymphoma: Effects of Drug and of Disease Itself2017In: Research and Practice in Thrombosis and Haemostasis, ISSN 2475-0379, Vol. 1, no S1, p. 1344-1344Article in journal (Refereed)
  • 26.
    Jacobsen, Annette
    et al.
    School of Science and Technology, Örebro University, Örebro, Sweden; School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, Australia.
    Yemaneab, Bisrat
    School of Science and Technology, Örebro University, Örebro, Sweden.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Reference gene selection for qPCR Is dependent on cell type rather than treatment in colonic and vaginal human epithelial cell lines2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 12, p. e115592-Article in journal (Refereed)
    Abstract [en]

    The ability of commensal bacteria to influence gene expression in host cells under the influence of pathogenic bacteria has previously been demonstrated, however the extent of this interaction is important for understanding how bacteria can be used as probiotics. Real-time quantitative polymerase chain reaction is the most sensitive tool for evaluating relative changes to gene expression levels. However as a result of its sensitivity an appropriate method of normalisation should be used to account for any variation incurred in preparatory experimental procedures. These variations may result from differences in the amount of starting material, quality of extracted RNA, or in the efficiency of the reverse transcriptase or polymerase enzymes. Selection of an endogenous control gene is the preferred method of normalisation, and ideally a proper validation of the gene's appropriateness for the study in question should be performed. In this study we used quantitative polymerase chain reaction data and applied four different algorithms (geNorm, BestKeeper, NormFinder, and comparative ΔCq) to evaluate eleven different genes as to their suitability as endogenous controls for use in studies involving colonic (HT-29) and vaginal (VK2/E6E7) human mucosal epithelial cells treated with probiotic and pathogenic bacteria. We found phosphoglycerate kinase 1 to be most appropriate for HT-29 cells, and ribosomal protein large P0 to be the best choice for VK2/E6E7 cells. We also showed that use of less stable reference genes can lead to less accurate quantification of expression levels of gene of interest (GOI) and also can result in decreased statistical significance for GOI expression levels when compared to control. Additionally, we found the cell type being analysed had greater influence on reference gene selection than the treatment performed. This study provides recommendations for stable endogenous control genes for use in further studies involving colonic and vaginal cell lines after bacterial challenge.

  • 27.
    Kharlyngdoh, Joubert Banjop
    et al.
    Örebro University, School of Science and Technology.
    Asnake, Solomon
    Örebro University, School of Science and Technology.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    TBECH, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane, alters androgen receptor regulation in response to mutations associated with prostate cancer2016In: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333, Vol. 307, p. 91-101Article in journal (Refereed)
    Abstract [en]

    Point mutations in the AR ligand-binding domain (LBD) can result in altered AR structures leading to changes of ligand specificity and functions. AR mutations associated to prostate cancer (PCa) have been shown to result in receptor activation by non-androgenic substances and anti-androgenic drugs. Two AR mutations known to alter the function of anti-androgens are the ART877A mutation, which is frequently detected mutation in PCa tumors and the ARW741C that is rare and has been derived in vitro following exposure of cells to the anti-androgen bicalutamide. AR activation by non-androgenic environmental substances has been suggested to affect PCa progression. In the present study we investigated the effect of AR mutations (ARW741C and ART877A) on the transcriptional activation following exposure of cells to an androgenic brominated flame retardant, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH, also named DBE-DBCH). The AR mutations resulted in higher interaction energies and increased transcriptional activation in response to TBECH diastereomer exposures. The ART877A mutation rendered AR highly responsive to low levels of DHT and TBECH and led to increased AR nuclear translocation. Gene expression analysis showed a stronger induction of AR target genes in LNCaP cells (ART877A) compared to T-47D cells (ARWT) following TBECH exposure. Furthermore, AR knockdown experiments confirmed the AR dependency of these responses. The higher sensitivity of ART877A and ARW741C to low levels of TBECH suggests that cells with these AR mutations are more susceptible to androgenic endocrine disrupters.

  • 28.
    Koskela von Sydow, Anita
    et al.
    Department of Clinical Research Laboratory, Örebro University Hospital, Örebro, Sweden.
    Janbaz, Chris
    Faculty of Medicine and Health, Örebro University, Örebro, Sweden; Department of Plastic and Reconstructive Surgery, Örebro University Hospital, Örebro, Sweden .
    Kardeby, Caroline
    Örebro University, School of Medical Sciences.
    Repsilber, Dirk
    Örebro University, School of Medical Sciences.
    Ivarsson, Mikael
    Örebro University, School of Health Sciences.
    IL-1α Counteract TGF-β Regulated Genes and Pathways in Human Fibroblasts2016In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 117, no 7, p. 1622-1632Article in journal (Refereed)
    Abstract [en]

    Dysregulated wound healing is commonly associated with excessive fibrosis. Connective tissue growth factor (CTGF/CCN2) is characteristically overexpressed in fibrotic diseases and stimulated by transforming growth factor-β (TGF-β) in dermal fibroblasts. We previously showed that interleukin-1 (IL-1α) counteracts TGF-β-stimulated CTGF mRNA and protein expression in these cells. The aim of this study was to explore the effects of IL-1α on further genes and pathways in TGF-β regulated fibroblasts. Transcriptional microarray and multiple comparison analysis showed that the antagonizing effects of IL-1α was much more prominent than the synergistic effects, both with respect to number of genes and extent of changes in gene expression. Moreover, comparing canonical pathways by gene set enrichment analysis and the Ingenuity Pathway Analysis tool revealed that IL-1α counteracted TGF-β in the top six most confident pathways regulated by both cytokines. Interferon and IL-1 signaling, as well as two pathways involved in apoptosis signaling were suppressed by TGF-β and activated by IL-1α. Pathways involving actin remodeling and focal adhesion dynamics were activated by TGF-β and suppressed by IL-1α. Analyzing upstream regulators in part corroborate the comparison of canonical pathways and added cell cycle regulators as another functional group regulated by IL-1α. Finally, gene set enrichment analysis of fibrosis-related genes indicated that IL-1 moderately counteracts the collective effect of TGF-β on these genes. Microarray results were validated by qPCR. Taken together, the results indicate prominent antagonistic effects of IL-1α on TGF-β regulated interferon signaling, as well as on a wide variety of other genes and pathways in fibroblasts. This article is protected by copyright. All rights reserved.

  • 29.
    König, Julia
    et al.
    Institute of Cell Biology, Histology, and Embryology, Medical University of Graz, Graz, Austria.
    Huppertz, Berthold
    Institute of Cell Biology, Histology, and Embryology, Medical University of Graz, Graz, Austria.
    Desoye, Gernot
    Clinic of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria.
    Parolini, Ornella
    Centro di Ricerca E. Menni, Fondazione Poliambulanza, Istituto Ospedaliero, Brescia, Italy.
    Fröhlich, Julia D
    Institute of Cell Biology, Histology, and Embryology, Medical University of Graz, Graz, Austria; Clinic of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria.
    Weiss, Gregor
    Institute of Cell Biology, Histology, and Embryology, Medical University of Graz, Graz, Austria.
    Dohr, Gottfried
    Institute of Cell Biology, Histology, and Embryology, Medical University of Graz, Graz, Austria.
    Sedlmayr, Peter
    Institute of Cell Biology, Histology, and Embryology, Medical University of Graz, Graz, Austria.
    Lang, Ingrid
    Institute of Cell Biology, Histology, and Embryology, Medical University of Graz, Graz, Austria.
    Amnion-derived mesenchymal stromal cells show angiogenic properties but resist differentiation into mature endothelial cells2012In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 21, no 8, p. 1309-1320Article in journal (Refereed)
    Abstract [en]

    Mesenchymal stromal cells derived from the human amnion (hAMSC) currently play an important role in stem cell research, as they are multipotent cells that can be isolated using noninvasive methods and are immunologically tolerated in vivo. The objective of this study was to evaluate their endothelial differentiation potential with regard to a possible therapeutic use in vascular diseases. hAMSC were isolated from human term placentas and cultured in Dulbecco's modified Eagle's medium (DMEM) (non-induced hAMSC) or endothelial growth medium (EGM-2) (induced hAMSC). Induced hAMSC changed their fibroblast-like toward an endothelial-like morphology, and were able to take up acetylated low-density lipoprotein and form endothelial-like networks in the Matrigel assay. However, they did not express the mature endothelial cell markers von Willebrand factor and vascular endothelial-cadherin. Gene expression analysis revealed that induced hAMSC significantly downregulated pro-angiogenic genes such as tenascin C, Tie-2, vascular endothelial growth factor A (VEGF-A), CD146, and fibroblast growth factor 2 (FGF-2), whereas they significantly upregulated anti-angiogenic genes such as serpinF1, sprouty1, and angioarrestin. Analysis of protein expression confirmed the downregulation of FGF-2 and Tie-2 (27%±8% and 13%±1% of non-induced cells, respectively) and upregulation of the anti-angiogenic protein endostatin (226%±4%). Conditioned media collected from hAMSC enhanced viability of endothelial cells and had a stabilizing effect on endothelial network formation as shown by lactate dehydrogenase and Matrigel assay, respectively. In summary, endothelial induced hAMSC acquired some angiogenic properties but resisted undergoing a complete differentiation into mature endothelial cells by upregulation of anti-angiogenic factors. Nevertheless, they had a survival-enhancing effect on endothelial cells that might be useful in a variety of cell therapy or tissue-engineering approaches.

  • 30.
    Lindahl, T. L.
    et al.
    Linköping University, Linköping, Sweden.
    Macwan, A. S.
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Protease-activated receptor 4 is more important than protease-activated receptor 1 for the thrombin-induced procoagulant effect on platelets2016In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, E-ISSN 1538-7836, Vol. 14, no 8, p. 1639-1641Article in journal (Refereed)
  • 31.
    Lindahl, Tomas L.
    et al.
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Boknas, Niklas
    Linköping University, Linköping, Sweden; Region Östergötland, Linköping, Sweden..
    Faxalv, Lars
    Linköping University, Linköping, Sweden.
    Caveats in studies of the physiological role of polyphosphates in coagulation2016In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 44, no 1, p. 35-39Article in journal (Refereed)
    Abstract [en]

    Platelet-derived polyphosphates (polyP), stored in dense granule and released upon platelet activation, have been claimed to enhance thrombin activation of coagulation factor XI (FXI) and to activate FXII directly. The latter claim is controversial and principal results leading to these conclusions are probably influenced by methodological problems. It is important to consider that low-grade contact activation is initiated by all surfaces and is greatly amplified by the presence of phospholipids simulating the procoagulant membranes of activated platelets. Thus, proper use of inhibitors of the contact pathway and a careful choice of materials for plates and tubes is important to avoid artefacts. The use of phosphatases used to degrade polyP has an important drawback as it also degrades the secondary activators ADP and ATP, which are released from activated platelets. In addition, the use of positively charged inhibitors, such as polymyxin B, to inhibit polyP in platelet-rich plasma and blood is problematic, as polymyxin B also slows coagulation in the absence of polyP. In conclusion we hope awareness of the above caveats may improve research on the physiological roles of polyP in coagulation.

  • 32.
    Lindberg, Erika
    et al.
    Sahlgrenska Acad, Univ Gothenburg, Gothenburg, Sweden.
    Andersson, Bert
    Sahlgrenska Acad, Univ Gothenburg, Gothenburg, Sweden.
    Hultgren Hörnquist, Elisabeth
    Örebro University, School of Health and Medical Sciences.
    Magnusson, Yvonne
    Sahlgrenska Acad, Univ Gothenburg, Gothenburg, Sweden.
    Impaired activation of IFN-gamma+CD4+ T cells in peripheral blood of patients with dilated cardiomyopathy2010In: Cellular Immunology, ISSN 0008-8749, E-ISSN 1090-2163, Vol. 263, no 2, p. 224-229Article in journal (Refereed)
    Abstract [en]

    Viral persistence and autoantibodies are pathogenic components in patients with idiopathic dilated cardiomyopathy (DCM). The aim was to evaluate T-cell function in DCM using different flow cytometry based detection techniques. Following stimulation, the frequency of IFN-gamma-producing CD4+ T cells was significantly lower in patients compared with controls. In contrast, the frequency of IL-4 producing CD4+ T cells was no different. In supernatants of cultured PBMC, IFN-gamma and IL-10 were significantly lower in patients. In addition, lymphocyte proliferation was significantly lower in patients compared with controls, whereas major lymphocyte subsets were not different. IFN-gamma and IL-10 are key cytokines in the ability to mount protective immune responses and to maintain self-tolerance. A reduced activation of T-helper 1 (IFN-gamma producing) cells and a decreased capacity to produce IL-10, found in the present study, could explain parts of the autoimmune features seen in patients with DCM.

  • 33.
    Lindfors, Erno
    et al.
    VTT Technical Research Centre of Finland, Espoo, Finland; LifeGlimmer GmbH, Berlin, Germany; Chemistry Building, Wageningen, Netherlands.
    Jouhten, Paula
    VTT Technical Research Centre of Finland, Espoo, Finland.
    Oja, Merja
    VTT Technical Research Centre of Finland, Espoo, Finland.
    Rintala, Eija
    VTT Technical Research Centre of Finland, Espoo, Finland.
    Oresic, Matej
    Örebro University, School of Medical Sciences. VTT Technical Research Centre of Finland, Espoo, Finland.
    Penttilä, Merja
    VTT Technical Research Centre of Finland, Espoo, Finland.
    Integration of transcription and flux data reveals molecular paths associated with differences in oxygen-dependent phenotypes of Saccharomyces cerevisiae2014In: BMC Systems Biology, ISSN 1752-0509, E-ISSN 1752-0509, Vol. 8, article id 16Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Saccharomyces cerevisiae is able to adapt to a wide range of external oxygen conditions. Previously, oxygen-dependent phenotypes have been studied individually at the transcriptional, metabolite, and flux level. However, the regulation of cell phenotype occurs across the different levels of cell function. Integrative analysis of data from multiple levels of cell function in the context of a network of several known biochemical interaction types could enable identification of active regulatory paths not limited to a single level of cell function.

    RESULTS: The graph theoretical method called Enriched Molecular Path detection (EMPath) was extended to enable integrative utilization of transcription and flux data. The utility of the method was demonstrated by detecting paths associated with phenotype differences of S. cerevisiae under three different conditions of oxygen provision: 20.9%, 2.8% and 0.5%. The detection of molecular paths was performed in an integrated genome-scale metabolic and protein-protein interaction network.

    CONCLUSIONS: The molecular paths associated with the phenotype differences of S. cerevisiae under conditions of different oxygen provisions revealed paths of molecular interactions that could potentially mediate information transfer between processes that respond to the particular oxygen availabilities.

  • 34.
    Lindgren, M.
    et al.
    Dilaforette AB, Solna, Sweden.
    Meijers, J. C. M.
    University of Amsterdam, Amsterdam, Netherlands.
    Biemond, B. J.
    University of Amsterdam, Amsterdam, Netherlands.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Lindahl, T. L.
    Linköping University, Linköping, Sweden.
    Eriksson, P-O
    Dilaforette AB, Solna, Sweden.
    Leitgeb, A. M.
    Dilaforette AB, Solna, Sweden.
    Wahlgren, M.
    Karolinska Institute, Stockholm, Sweden.
    Hogwood, J.
    The National Institute for Biological Standards and Control (NIBSC), South Mimms, England.
    Gray, E.
    The National Institute for Biological Standards and Control (NIBSC), South Mimms, England.
    Holmer, E.
    Dilaforette AB, Solna, Sweden.
    Sevuparin: effects on hemostasis of a novel polysaccharide drug derived from heparin2015In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, E-ISSN 1538-7836, Vol. 13, no S2, p. 369-369Article in journal (Refereed)
  • 35.
    Ljungberg, Liza U.
    et al.
    Division of Drug Research/Pharmacology, Department of Medical and Health Sciences, Linköping University, Linköping.
    Persson, Karin
    Division of Drug Research/Pharmacology, Department of Medical and Health Sciences, Linköping University, Linköping.
    Effect of nicotine and nicotine metabolites on angiotensin-converting enzyme in human endothelial cells2008In: Endothelium, ISSN 1062-3329, E-ISSN 1029-2373, Vol. 15, no 5-6, p. 239-45Article in journal (Refereed)
    Abstract [en]

    Nicotine has been shown to induce endothelial dysfunction, which is an early marker of atherosclerosis. Nicotine undergoes extensive metabolism in the liver, forming a number of major and minor metabolites. There are very limited data on the effect of nicotine metabolites on the cardiovascular system. This study investigates the effects of nicotine and the nicotine metabolites, cotinine, cotinine-N-oxide, nicotine-1'-N-oxide, norcotinine, trans-3'-hydroxycotinine, on angiotensin-converting enzyme (ACE) in human endothelial cells. Cultured endothelial cells obtained from human umbilical cord vein (HUVECs) were stimulated with nicotine or nicotine metabolites in concentrations similar to those observed in plasma during smoking. ACE activity and expression were analyzed using commercial kits. The results showed that nicotine and nicotine metabolites can increase both activity and expression of ACE. However, a marked individual variation in the response to the drugs was observed. This variation was not associated with the ACE insertion/deletion polymorphism. Tobacco contains numerous chemical compounds, and the underlying cause for development of atherosclerosis in smokers is probably multifactorial. The results from this study could explain one cellular mechanism by which smoking exerts negative effect on the vascular system.

  • 36.
    Lopez, A.
    et al.
    Molecular and Cellular Therapeutics (MCT), Royal College of Surgeons, Dublin, Ireland.
    Kenny, D.
    Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Basabe-Desmonts, L.
    Molecular and Cellular Therapeutics (MCT), Royal College of Surgeons, Dublin, Ireland.
    Ramström, Sofia
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Jose, B.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Somers, M.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    A novel platelet assay that measures the effect of clopidogrel in vivo2013In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, E-ISSN 1538-7836, Vol. 11, no S2, p. 73-73Article in journal (Refereed)
  • 37.
    Lopez-Alonso, A.
    et al.
    NBIPI Program, Molecular & Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Basabe-Desmonts, L.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Ramström, Sofia
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Jose, B.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Sommers, M.
    Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    Redahan, L.
    Nephrology & Renal Transplant Medicine, Beaumont Hospital, Dublin, Ireland.
    Conlon, P.
    Nephrology & Renal Transplant Medicine, Beaumont Hospital, Dublin, Ireland.
    Kenny, D.
    NBIPI Program, Molecular & Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland; Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland.
    A novel platelet function assay that identifies bleeding risk and quantifies anti-platelet drug effect2012Conference paper (Refereed)
  • 38.
    Lopez-Alonso, Ana
    et al.
    Royal College of Surgeons in Ireland, Dublin, Ireland.
    Jose, Bincy
    Biomedical Diagnostics Institute, Dublin City University, Dublin, Ireland.
    Somers, Martin
    Biomedical Diagnostics Institute, Dublin City University, Dublin, Ireland.
    Egan, Karl
    Royal College of Surgeons in Ireland, Dublin, Ireland.
    Foley, David P.
    Division of Cardiology, Beaumont Hospital, Dublin, Ireland.
    Ricco, Antonio J.
    Biomedical Diagnostics Institute, Dublin City University, Dublin, Ireland.
    Ramström, Sofia
    Royal College of Surgeons in Ireland, Dublin, Ireland; Department of Clinical and Experimental Medicine, Clinical Chemistry, Linköping University, Linköping, Sweden.
    Basabe-Desmonts, Lourdes
    Biomedical Diagnostics Institute, Dublin City University, Dublin, Ireland; CIC microGUNE, Polo Innovación Garaia, Arrasate-Mondragón, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.
    Kenny, Dermot
    Royal Coll Surgeons Ireland, Dublin, Ireland.
    Individual Platelet Adhesion Assay: Measuring Platelet Function and Antiplatelet Therapies in Whole Blood via Digital Quantification of Cell Adhesion2013In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 85, no 13, p. 6497-6504Article in journal (Refereed)
    Abstract [en]

    Widespread monitoring of platelet function and the effect of antiplatelet drugs will improve outcomes in cardiovascular patients, but platelet function testing is not routine in clinical practice. We report a rapid, accurate methodology to quantify platelet-protein interactions: a microarray of contact-printed 6-mu m fibrinogen dots on a transparent substrate binds platelets from whole blood, one platelet per dot. The fractional occupancy of an array of fibrinogen dots after a predefined incubation time quantitatively assays platelet adhesion to the protein matrix. We demonstrate this technique by measurement of platelet adhesion to fibrinogen as a means to quantify the effect of the P2Y(12) and alpha IIb beta 3 receptor inhibitors cangrelor and abciximab, respectively, both in vitro-by incubating the drug with a freshly drawn blood sample-and in blood from patients treated with antiplatelet agents. The effects of single- and dual-antiplatelet therapy are also assessed. Results from this platelet-binding assay are well correlated with standard techniques including flow cytometry and light transmission aggregometry. This assay technology, readily integrated with microfluidic platforms, is generally applicable to the assay of cell-protein interactions and promises more effective, rapid assay of drug effects in cardiovascular disease patients.

  • 39.
    Macwan, A.
    et al.
    Linköping University, Linköping, Sweden.
    Boknäs, N.
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Berg, S.
    Linköping University, Linköping, Sweden.
    Faxälv, L.
    Linköping University, Linköping, Sweden.
    Lindahl, T.
    Linköping University, Linköping, Sweden.
    Gradient-dependent Modulation of Platelet Stimulatory G-protein Coupled Receptors (GPCR) Signaling2017In: Research and Practice in Thrombosis and Haemostasis, ISSN 2475-0379, Vol. 1, no S1, p. 1223-1223, article id PB568Article in journal (Refereed)
  • 40.
    Melin, Petter
    et al.
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Hakansson, Sebastian
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Schnürer, Johan
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Optimisation and comparison of liquid and dry formulations of the biocontrol yeast Pichia anomala J1212007In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 73, no 5, p. 1008-1016Article, review/survey (Refereed)
    Abstract [en]

    The biocontrol yeast Pichia anomala J121 can effectively reduce mould growth on moist cereal grains during airtight storage. Practical use of microorganisms requires formulated products that meet a number of criteria. In this study we compared different formulations of P. anomala. The best way to formulate P. anomala was freeze-drying. The initial viability was as high as 80%, with trehalose previously added to the yeast. Freeze-dried products could be stored at temperatures as high as 30 degrees C for a year, with only a minor decrease in viability. Vacuum-drying also resulted in products with high storage potential, but the products were not as easily rehydrated as freeze-dried samples. Upon desiccating the cells using fluidised-bed drying or as liquid formulations, a storage temperature of 10 degrees C was required to maintain viability. Dependent on the type of formulation, harvesting of cells at different nutritional stresses affected the initial viabilities, e.g. the initial viability for fluidised-bed-dried cells was higher when the culture was fed with excess glucose, but for freeze-drying it was superior when cells were harvested after depletion of carbon. Using micro-silos we found that the biocontrol activity remained intact after drying, storage and rehydration for all formulations.

  • 41.
    Morales, Luis Orlando
    et al.
    Department of Biosciences, Division of Plant Biology, University of Helsinki, Helsinki, Finland.
    Tegelberg, Riita
    Department of Biosciences, Division of Plant Biology, University of Helsinki, Helsinki, Finland.
    Brosché, Mikael
    Department of Biosciences, Division of Plant Biology, University of Helsinki, Helsinki, Finland.
    Keinänen, Markku
    Faculty of Biosciences, University of Eastern Finland, Joensuu, Finland.
    Lindfors, Anders
    School of Geosciences, University of Edinburgh, Edinburgh, UK.
    Aphalo, Pedro J.
    Department of Biosciences, Division of Plant Biology, University of Helsinki, Helsinki, Finland.
    Effects of solar UV-A and UV-B radiation on gene expression and phenolic accumulation in Betula pendula leaves2010In: Tree Physiology, ISSN 0829-318X, E-ISSN 1758-4469, Vol. 30, no 7, p. 923-934Article in journal (Refereed)
    Abstract [en]

    Ultraviolet (UV) radiation is an important environmental factor for plant communities; however, plant responses to solar UV are not fully understood. Here, we report differential effects of solar UV-A and UV-B radiation on the expression of flavonoid pathway genes and phenolic accumulation in leaves of Betula pendula Roth (silver birch) seedlings grown outdoors. Plants were exposed for 30 days to six UV treatments created using three types of plastic film. Epidermal flavonoids measured in vivo decreased when UV-B was excluded. In addition, the concentrations of six flavonoids determined by high-performance liquid chromatography-mass spectrometry declined linearly with UV-B exclusion, and transcripts of PAL and HYH measured by quantitative real-time polymerase chain reaction were expressed at lower levels. UV-A linearly regulated the accumulation of quercetin-3-galactoside and quercetin-3-arabinopyranoside and had a quadratic effect on HYH expression. Furthermore, there were strong positive correlations between PAL expression and accumulation of four flavonols under the UV treatments. Our findings in silver birch contribute to a more detailed understanding of plant responses to solar UV radiation at both molecular and metabolite levels.

  • 42.
    Nowinski, D.
    et al.
    Dept Surg Sci, Plast Surg Unit, Uppsala Univ, Uppsala, Sweden.
    Koskela, Anita
    Örebro University, School of Health and Medical Sciences. Örebro University Hospital, Örebro, Sweden.
    Kiwanuka, E.
    Dept Surg Sci, Plast Surg Unit, Uppsala Univ, Uppsala, Sweden.
    Boström, M.
    Dept Surg Sci, Plast Surg Unit, Uppsala Univ, Uppsala, Sweden.
    Gerdin, B.
    Dept Surg Sci, Plast Surg Unit,Uppsala Univ, Uppsala, Sweden.
    Ivarsson, Mikael
    Örebro University, School of Health and Medical Sciences. Örebro University Hospital, Örebro, Sweden.
    Inhibition of Connective Tissue Growth Factor/CCN2 Expression in Human Dermal Fibroblasts by Interleukin-1 alpha and beta2010In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 110, no 5, p. 1226-1233Article in journal (Refereed)
    Abstract [en]

    Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)-beta and intimately involved with tissue repair and overexpressed in various fibrotic conditions We previously showed that keratmocytes in vitro downregulate TGF-beta-induced expression of CTGF in fibroblasts by an interleukin (IL)-1 alpha-dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL-1 alpha and beta Human dermal fibroblasts and NIH 3T3 cells were treated with IL-1 alpha or beta in presence or absence of TGF-beta 1. IL-1 suppressed basal and TGF-beta-induced CTGF mRNA and protein expression. IL-1 alpha and beta inhibited TGF-beta-stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3-binding CAGA elements Furthermore. IL-1 alpha and beta inhibited TGF-beta-stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition. RNA interference suggested that TGF-beta activated kinase1 (TAK1) is necessary for IL-1 inhibition of TGF-beta-stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell Biochem. 110: 1226-1233, 2010. (C) 2010 Wiley-Liss. Inc

  • 43.
    Nylander, Martina
    et al.
    Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Osman, Abdimajid
    Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Aklint, Emma
    Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Larsson, Anders
    Department of Medical Sciences, University Hospital, Uppsala, Sweden.
    Lindahl, Tomas L.
    Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    The role of thrombin receptors PAR1 and PAR4 for PAI-1 storage, synthesis and secretion by human platelets2012In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 129, no 4, p. E51-E58Article in journal (Refereed)
    Abstract [en]

    Introduction: Arterial thrombi contain more platelets than venous thrombi and are more resistant to fibrinolysis. This resistance could partly be due to plasminogen activator inhibitor 1 (PAI-1) secreted by platelets. The aim of this study was to elucidate differences between thrombin receptors protease-activated receptor (PAR) 1 and 4 and platelet storage, secretion and synthesis of platelet PAI-1, as compared to other platelet alpha-granule proteins such as VEGF and endostatin.

    Materials and methods: Human isolated platelets were incubated with thrombin (0.5 U/ml), PAR1-activating peptide (AP) (0.4-30 mu M) or PAR4-AP (1.5-300 mu M) for up to 24 hours. ELISA, western blot and fluorescence microscopy were used to measure secretion, contents and localization of PAI-1, VEGF and endostatin. Results: Our results show that PAI-1 and VEGF might be co-localized and that endostatin does not co-localize with either PAI-1 or VEGF. PAI-1 and VEGF show a similar secretion pattern, being more sensitive to low grade PAR1 activation, but secretion was also observed with higher concentrations of PAR4-APs. PAI-1 is secreted in an active form. PAI-1 mRNA was found in platelets, and elevated levels of PAI-1 were detected after 24 hours incubation of platelets.

    Conclusions: PAI-1 and VEGF, but not endostatin, might be stored in the same alpha-granule in human platelets. PAI-1 and VEGF also show a similar secretion pattern, being more sensitive to PAR1 than to PAR4 activation, but the secretion is not exclusively selective. Our results also show that platelet PAI-1 is increased if incubated for 24 hours, both with addition of PAR1-activating peptide and without activation, which could indicate de novo synthesis.

  • 44.
    Oliynyk, Igor
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Örebro University Hospital, Örebro, Sweden.
    Hussain, Rashida
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Örebro University Hospital, Örebro, Sweden.
    Amin, Ahmad
    School of Health and Medical Sciences, University of Örebro, Örebro University Hospital, Örebro, Sweden.
    Johannesson, Marie
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Örebro University Hospital, Örebro, Sweden; Karolinska Institutet, Solna, Sweden.
    Roomans, Godfried M.
    Örebro University, School of Medicine, Örebro University, Sweden. Örebro University Hospital, Örebro, Sweden.
    The effect of NO-donors on chloride efflux, intracellular Ca2+ concentration and mRNA expression of CFTR and ENaC in cystic fibrosis airway epithelial cells2013In: Experimental and molecular pathology (Print), ISSN 0014-4800, E-ISSN 1096-0945, Vol. 94, no 3, p. 474-480Article in journal (Refereed)
    Abstract [en]

    Since previous studies showed that the endogenous bronchodilator, S nitrosglutathione (GSNO), caused amarked increase in CFTR-mediated chloride (Cl−) efflux and improved the trafficking of CFTR to the plasmamembrane, and that also the nitric oxide (NO)-donor GEA3162 had a similar, but smaller, effect on Cl− efflux, itwas investigatedwhether the NO-donor properties of GSNOwere relevant for its effect on Cl− efflux fromairwayepithelial cells. Hence, the effect of a number of other NO-donors, sodium nitroprusside (SNP), S-nitroso-Nacetyl-DL-penicillamine (SNAP), diethylenetriamine/nitric oxide adduct (DETA-NO), and diethylenetriamine/nitricoxide adduct (DEA-NONOate) on Cl− efflux from CFBE (ΔF508/ΔF508-CFTR) airway epithelial cells was tested.Cl− efflux was determined using the fluorescent N-(ethoxycarbonylmethyl)-6-methoxyquinoliniu bromide(MQAE)-technique. Possible changes in the intracellular Ca2+ concentration were tested by the fluorescent fluo-4method in a confocal microscope system. Like previously with GSNO, after 4 h incubation with the NO-donor, anincreased Cl− efflux was found (in the order SNAP > DETA-NO > SNP). The effect of DEA-NONOate on Cl− effluxwas not significant, and the compound may have (unspecific) deleterious effects on the cells. Again, as withGSNO, after a short (5 min) incubation, SNP had no significant effect on Cl− efflux. None of the NO-donors thathad a significant effect on Cl− efflux caused significant changes in the intracellular Ca2+ concentration. After 4 hpreincubation, SNP caused a significant increase in the mRNA expression of CFTR. SNAP and DEA-NONOatedecreased the mRNA expression of all ENaC subunits significantly. DETA-NO caused a significant decrease only inα-ENaC expression. After a short preincubation, none of the NO-donors had a significant effect, neither on theexpression of CFTR, nor on that of the ENaC subunits in the presence and absence of L-cysteine. It can be concludedthat the effect of GSNO on Cl−efflux is, at least in part, due to its properties as an NO-donor, and the effect is likely tobe mediated by CFTR, not by Ca2+-activated Cl− channels.

  • 45.
    Olsson, A.
    et al.
    Blekinge Institute of Technology, Karlskrona, Sweden; Linköping University, Linköping, Sweden; Blekinge Hospital, Karlskrona, Sweden.
    Alfredsson, J.
    Linköping University, Linköping, Sweden.
    Ramström, Sofia
    Linköping University, Linköping, Sweden.
    Svedjeholm, R.
    Linköping University, Linköping, Sweden.
    Kenny, D.
    Clinical Research Centre, Royal College of Surgeons in Ireland, Dublin, Ireland.
    Håkansson, E.
    Linköping University, Linköping, Sweden.
    Berglund, J. S.
    Blekinge Institute of Technology, Karlskrona, Sweden.
    Berg, S.
    Linköping University, Linköping, Sweden.
    Better platelet function, less fibrinolysis and less hemolysis in re-transfused residual pump blood with the Ringer’s chase technique: a randomized pilot study2018In: Perfusion, ISSN 0267-6591, E-ISSN 1477-111X, Vol. 33, no 3, p. 185-193Article in journal (Refereed)
    Abstract [en]

    Introduction: Residual pump blood from the cardiopulmonary bypass (CPB) circuit is often collected into an infusion bag (IB) and re-transfused. An alternative is to chase the residual blood into the circulation through the arterial cannula with Ringer’s acetate. Our aim was to assess possible differences in hemostatic blood quality between these two techniques.

    Methods: Forty adult patients undergoing elective coronary artery bypass graft surgery with CPB were randomized to receive the residual pump blood by either an IB or through the Ringer’s chase (RC) technique. Platelet activation and function (impedance aggregometry), coagulation and hemolysis variables were assessed in the re-transfused blood and in the patients before, during and after surgery. Results are presented as median (25-75 quartiles).

    Results: Total hemoglobin and platelet levels in the re-transfused blood were comparable with the two methods, as were soluble platelet activation markers P-selectin and soluble glycoprotein VI (GPVI). Platelet aggregation (U) in the IB blood was significantly lower compared to the RC blood, with the agonists adenosine diphosphate (ADP) 24 (10-32) vs 46 (33-65), p<0.01, thrombin receptor activating peptide (TRAP) 50 (29-73) vs 69 (51-92), p=0.04 and collagen 24 (17-28) vs 34 (26-59), p<0.01. The IB blood had higher amounts of free hemoglobin (mg/L) (1086 (891-1717) vs 591(517-646), p<0.01) and D-dimer 0.60 (0.33-0.98) vs 0.3 (0.3-0.48), p<0.01. Other coagulation variables showed no difference between the groups. Conclusions: The handling of blood after CPB increases hemolysis, impairs platelet function and activates coagulation and fibrinolysis. The RC technique preserved the blood better than the commonly used IB technique.

  • 46.
    Ramström, Sofia
    Linkoping University, Linköping, Sweden.
    Experiences with “spontaneous” contact activation and Microvesicles in Thrombin generation.: Invited presentation at a round table discussion, “Pre-analytical problems and solutions for assays sensitive to contact activation“2014Conference paper (Other academic)
  • 47.
    Ramström, Sofia
    et al.
    Biomedical Diagnostics Institute (BDI) Programme, Molecular & Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    O'Neill, Sarah
    Biomedical Diagnostics Institute (BDI) Programme, Molecular & Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Dunne, Eimear
    Biomedical Diagnostics Institute (BDI) Programme, Molecular & Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Kenny, Dermot
    Biomedical Diagnostics Institute (BDI) Programme, Molecular & Cellular Therapeutics, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
    Annexin V binding to platelets is agonist, time and temperature dependent2010In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 21, no 4, p. 289-296Article in journal (Refereed)
    Abstract [en]

    Platelets bind annexin V when stimulated with combinations of platelet agonists such as collagen and thrombin. Previous studies have demonstrated significant heterogeneity of platelets binding annexin V. The relative role of the thrombin protease-activated receptors (PARs), PAR1 and PAR4, together with different methods of platelet preparation on annexin V binding to platelets is unclear. We therefore investigated the role of PAR1- and PAR4-activating peptides in combination with collagen-related peptide on annexin V binding. In diluted whole blood, PAR1- and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding. However, in washed platelets, PAR-activating peptides were less potent than thrombin at inducing annexin V binding. This difference was more pronounced when experiments were performed at 37 degrees C compared to room temperature. In studies of diluted whole blood, platelet rich plasma and washed platelets, platelets incubated at room temperature bound more annexin V than platelets incubated at 37 degrees C. We also saw a significant effect of time on annexin V binding, in that more annexin V bound to platelets with longer incubation times. In conclusion, PAR1 and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding in combination with collagen-related peptide in diluted whole blood and platelet rich plasma, but not in washed platelets. In addition, incubation temperature and time has a strong influence on annexin V binding to platelets. Thus variations in these conditions may explain the differences observed between previous studies.

  • 48.
    Ramström, Sofia
    et al.
    Linköping University, Linköping, Sweden.
    Södergren, Anna L.
    Linköping University, Linköping, Sweden.
    Tynngård, Nahreen
    Linköping University, Linköping, Sweden.
    Lindahl, Tomas L.
    Linköping University, Linköping, Sweden.
    Platelet Function Determined by Flow Cytometry: New Perspectives?2016In: Seminars in Thrombosis and Hemostasis, ISSN 0094-6176, E-ISSN 1098-9064, Vol. 42, no 3, p. 268-281Article, review/survey (Refereed)
    Abstract [en]

    Flow cytometry enables studies of several different aspects of platelet function in response to a variety of platelet agonists. This can be done using only a small volume of whole blood, and also in blood with low platelet counts. These properties, together with the increasing number of flow cytometers available in hospitals worldwide, make flow cytometry an interesting option for laboratories interested in studies of platelet function in different clinical settings. This review focuses on practical issues regarding the use of flow cytometry for platelet function testing. It provides an overview of available activation markers, platelet agonists, and experimental setup issues. The review summarizes previous experience and factors important to consider to perform high-quality platelet function testing by flow cytometry. It also discusses its current use and possibilities and challenges for future use of flow cytometry in clinical settings.

  • 49.
    Rodriguez-Cuenca, Sergio
    et al.
    Department of Clinical Biochemistry, Metabolic Research Laboratories, Institute of Metabolic Science, University of Cambridge, Cambridge, United Kingdom.
    Carobbio, Stefania
    Department of Clinical Biochemistry, Metabolic Research Laboratories, Institute of Metabolic Science, University of Cambridge, Cambridge, United Kingdom.
    Velagapudi, Vidya R.
    VTT Technical Research Centre of Finland, Espoo, Finland.
    Barbarroja, Nuria
    VTT Technical Research Centre of Finland, Espoo, Finland; Hospital Virgen de la Victoria, CIBERobn Fisiopatología de la Obesidad y Nutrición, Malaga, Spain.
    Moreno-Navarrete, Jose Maria
    Department of Diabetes, Endocrinology and Nutrition, Institut d'Investigació Biomédica de Girona, Girona, Spain; CIBERobn Fisiopatología de la Obesidad y Nutrición, Girona, Spain.
    Tinahones, Francisco Jose
    Hospital Virgen de la Victoria, CIBERobn Fisiopatología de la Obesidad y Nutrición, Malaga, Spain.
    Fernandez-Real, Jose Manuel
    Department of Diabetes, Endocrinology and Nutrition, Institut d'Investigació Biomédica de Girona, Girona, CIBERobn Fisiopatología de la Obesidad y Nutrición, Girona, Spain.
    Oresic, Matej
    Örebro University, School of Medical Sciences. VTT Technical Research Centre of Finland, Espoo, Finland.
    Vidal-Puig, Antonio
    Department of Clinical Biochemistry, Metabolic Research Laboratories, Institute of Metabolic Science, University of Cambridge, Cambridge, United Kingdom.
    Peroxisome proliferator-activated receptor γ-dependent regulation of lipolytic nodes and metabolic flexibility2012In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 32, no 8, p. 1555-1565Article in journal (Refereed)
    Abstract [en]

    Optimal lipid storage and mobilization are essential for efficient adipose tissue. Nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) regulates adipocyte differentiation and lipid deposition, but its role in lipolysis and dysregulation in obesity is not well defined. This investigation aimed to understand the molecular impact of dysfunctional PPARγ on the lipolytic axis and to explore whether these defects are also confirmed in common forms of human obesity. For this purpose, we used the P465L PPARγ mouse as a model of dysfunctional PPARγ that recapitulates the human pparγ mutation (P467L). We demonstrated that defective PPARγ impairs catecholamine-induced lipolysis. This abnormal lipolytic response is exacerbated by a state of positive energy balance in leptin-deficient ob/ob mice. We identified the protein kinase A (PKA) network as a PPARγ-dependent regulatory node of the lipolytic response. Specifically, defective PPARγ is associated with decreased basal expression of prkaca (PKAcatα) and d-akap1, the lipase genes Pnplaz (ATGL) and Lipe (HSL), and lipid droplet protein genes fsp27 and adrp in vivo and in vitro. Our data indicate that PPARγ is required for activation of the lipolytic regulatory network, dysregulation of which is an important feature of obesity-induced insulin resistance in humans.

  • 50.
    Saju, Jolly M.
    et al.
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore.
    Hossain, Mohammad Sorowar
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore; Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
    Liew, Woei Chang
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore.
    Pradhan, Ajay
    Örebro University, School of Science and Technology. Biology, Örebro Life Science Center.
    Thevasagayam, Natascha May
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore.
    Tan, Lydia Shun En
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore.
    Anand, Amit
    Bioimaging and Biocomputing, Temasek Life Sciences Laboratory, Singapore, Singapore.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology. Biology, Örebro Life Science Center.
    Orban, Laszlo
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore; Frontline Fish Genomics Research Group, Department of Animal Sciences, Georgikon Faculty, University of Pannonia, Keszthely, Hungary; Centre for Comparative Genomics, Murdoch University, Murdoch, Australia.
    Heat Shock Factor 5 Is Essential for Spermatogenesis in Zebrafish2018In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 25, no 12, p. 3252-3261Article in journal (Refereed)
    Abstract [en]

    Heat shock factors (Hsfs) are transcription factors that regulate responses to heat shock and other environmental stimuli. Four heat shock factors (Hsf1-4) have been characterized from vertebrates to date. In addition to stress response, they also play important roles in development and gametogenesis. Here, we study the fifth member of heat shock factor family, Hsf5, using zebrafish as a model organism. Mutant hsf5(-/-) males, generated by CRISPR/Cas9 technique, were infertile with drastically reduced sperm count, increased sperm head size, and abnormal tail architecture, whereas females remained fertile. We show that Hsf5 is required for progression through meiotic prophase 1 during spermatogenesis as suggested by the accumulation of cells in the leptotene and zygotene-pachytene stages and increased apoptosis in post-meiotic cells. hsf5(-/-) mutants show gonadal misregulation of a substantial number of genes with roles in cell cycle, apoptosis, protein modifications, and signal transduction, indicating an important role of Hsf5 in early stages of spermatogenesis.

12 1 - 50 of 66
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