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  • 101.
    Petersson, Christoffer
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Assessing the interaction between Helicobacter pylori and human neutrophils by freeze-fracture replica labeling2005In: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 36, no 6, p. 558-562Article in journal (Refereed)
    Abstract [en]

    We recently introduced a freeze-fracture replica labeling method adapted to studies of bacterial envelopes. This report describes a further development of this detergent-digested freeze-fracture replica labeling technique, thus more exactly the conception of this explicit methodology for visualization of bacteria-host cell interactions. Our experimental model employs human neutrophils and the gastric pathogenic bacterium Helicobacter pylori. The phagocytic process performed by the neutrophils represents a crucial element of the host defense system against invading microorganisms, and by so doing, it allows direct observation of the interplay between bacteria and host cells at an ultrastructural level. The here launched methodology can be used as a tool to investigate the events taking place between pathogenic microbes and phagocytes, as well as for pinpoint targeting of other cell-cell communications in the field of cell biology. © 2005 Elsevier Ltd. All rights reserved.

  • 102.
    Pettersson, Jonas
    et al.
    Department of Cell and Molecular Biology, University of Umeå, Umeå, Sweden.
    Nordfelth, Roland
    Department of Cell and Molecular Biology, University of Umeå, Umeå, Sweden.
    Dubinina, Elena
    Department of Cell and Molecular Biology, University of Umeå, Umeå, Sweden.
    Bergman, Tomas
    Department of Cell and Molecular Biology, University of Umeå, Umeå, Sweden.
    Gustafsson, Mikael
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Wolf-Watz, Hans
    Department of Cell and Molecular Biology, University of Umeå, Umeå, Sweden.
    Modulation of Virulence Factor Expression by Pathogen Target Cell Contact1996In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 273, no 5279, p. 1231-1233Article in journal (Refereed)
    Abstract [en]

    Upon contact with the eukaryotic cell, Yersinia pseudotuberculosis increased the rate of transcription of virulence genes (yop), as determined by in situ monitoring of light emission from individual bacteria expressing luciferase under the control of the yopE promoter. The microbe-host interaction triggered export of LcrQ, a negative regulator of Yop expression, via the Yop-type III secretion system. The intracellular concentration of LcrQ was thereby lowered, resulting in increased expression of Yops. These results suggest a key role for the type III secretion system of pathogenic bacteria to coordinate secretion with expression of virulence factors after physical contact with the target cell.

  • 103.
    Pivoriunas, A.
    et al.
    Department of Experimental Research, Institute of Experimental and Clinical Medicine, Žygimantu 9, 01102 Vilnius, Lithuania.
    Savickiene, J.
    Department of Experimental Research, Institute of Experimental and Clinical Medicine, Žygimantu 9, 01102 Vilnius, Lithuania.
    Treigyte, G.
    Department of Developmental Biology, Institute of Biochemistry, Mokslininku 12, 08662 Vilnius, Lithuania.
    Tunaitis, V.
    Department of Experimental Research, Institute of Experimental and Clinical Medicine, Žygimantu 9, 01102 Vilnius, Lithuania.
    Navakauskiene, R.
    Department of Developmental Biology, Institute of Biochemistry, Mokslininku 12, 08662 Vilnius, Lithuania.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    PI 3-K signaling pathway suppresses PMA-induced expression of p21WAF1/ Cip1 in human leukemia cells2007In: Molecular and Cellular Biochemistry, ISSN 0300-8177, E-ISSN 1573-4919, Vol. 302, no 1-2Article in journal (Refereed)
    Abstract [en]

    Despite the understanding of the importance of phosphoinositide 3-kinase (PI 3-K) signaling pathway in the regulation of cellular proliferation, little is known about its role during phorbol 12-myristate 13-acetate (PMA)-induced differentiation in human leukemia cells. Here, we report a novel finding that PI 3-K inhibition by LY294002 significantly increases p21WAF1/Cip1 expression in PMA-stimulated human leukemia cells NB4 and THP1. LY294002 potentiated expression of p21WAF1/Cip1 via a p53-independent mechanism and did not affect mitogen activated protein kinase (MAPK) activation. Electrophoretic mobility shift (EMSA) experiments revealed that blocking of PI 3-K was associated with increased binding of transcription factor Sp1 to the PMA-responsive sites on the p21WAF1/Cip1 promoter. Pretreatment with rapamycin, an inhibitor of mTOR kinase, decreased the expression of p21WAF1/Cip1 protein in PMA-stimulated NB4 cells. The level of PMA-induced p21WAF1/ Cip1 protein expression was lower in NB4 cells overexpressing wild type protein kinase C ? (PKC ?) compared to those transfected with empty vector or with kinase inactive PKC ?. Sp1 binding to the p21WAF1/Cip1 promoter was completely lost in a wild type PKC ? overexpressing and PMA-stimulated NB4 cells. We demonstrate that PI 3-K signaling pathway suppresses PMA-induced expression of p21WAF1/Cip1 in human leukemia cells, and that this effect is partly mediated by PKC ?. © Springer Science+Business Media, LLC 2007.

  • 104.
    Pivoriunas, Augustas
    et al.
    Institute for Experimentat and Clinical Medicine, Vilnius.
    Surovas, Andrejus
    Institute for Experimentat and Clinical Medicine, Vilnius.
    Borutinskaite, Veronika
    Institute for Experimentat and Clinical Medicine, Vilnius.
    Matuzevicius, Dalius
    Institute for Experimentat and Clinical Medicine, Vilnius.
    Treigyte, Grazina
    Lithuania Academy of Science.
    Savickiene, Jurate
    Lithuania Academy of Science.
    Tunaitis, Virginijus
    Institute for Experimentat and Clinical Medicine, Vilnius.
    Aldonyte, Ruta
    Institute for Experimentat and Clinical Medicine, Vilnius.
    Jarmalaviciute, Akvile
    Institute for Experimentat and Clinical Medicine, Vilnius.
    Suriakaite, Kristina
    Institute for Experimentat and Clinical Medicine, Vilnius.
    Liutkevicius, Evaldas
    JSC Imunolita, Vilnius.
    Venalis, Algirdas
    Institute for Experimentat and Clinical Medicine, Vilnius.
    Navakauskas, Dalius
    Vilnius Gediminas Technical University.
    Navakauskiene, Ruta
    Lithuania Academy of Science.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Proteomic Analysis of Stromal Cells Derived from the Dental Pulp of Human Exfoliated Deciduous Teeth2010In: STEM CELLS AND DEVELOPMENT, ISSN 1547-3287, Vol. 19, no 7, p. 1081-1093Article in journal (Refereed)
    Abstract [en]

    Human dental pulp derived from exfoliated deciduous teeth has been described as a promising alternative source of multipotent stem cells. While these cells share certain similarities with mesenchymal stem-like cells (MSC) isolated from other tissues, basically they are still poorly characterized. In this study, for the first time, a proteomic map of abundantly expressed proteins in stromal cells derived from the dental pulp of human exfoliated deciduous teeth (SHED) was established. We also analyzed proteomic signatures of 2 clonal strains derived from SHEDs by single-cell cloning. The SHEDs were established from enzyme-disaggregated deciduous dental pulp from 6-year-old children. They had typical fibroblastoid morphology and high colony-forming efficiency index (16.4%). Cloning was performed at the second passage using limiting dilution in a 96-well plate (0.3 cell/well). Differentiation assessment revealed strong osteogenic but no adipogenic potential of the SHEDs in either clonal strain. The cells expressed characteristic antigens of MSC-like cells, including CD73, CD90, CD105, CD146, and did not express hematopoietic markers CD14, CD34, and CD45, as assessed with FACS analysis. For proteomic studies, cytosolic and nuclear proteins were analyzed with 2-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). All proteins were identified with high level of confidence (the lowest sequence coverage was 27%). Identification of highly expressed proteins in SHEDs revealed proteomic profiles very similar to that of MSC-like cells derived from other tissues. We also found a high degree of similarity between proteomic signatures of primary SHEDs and clonal cell strains. Thus, our data confirm a close resemblance between SHEDs and MSC-like cells from other tissues and may serve as starting point for creating-comprehensive proteomic maps.

  • 105.
    Ragnarsson, Eva Ge
    et al.
    Uppsala University.
    Schoultz, Ida
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Surgery .
    Gullberg, Elisabet
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Surgery .
    Carlsson, Anders
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Surgery .
    Tafazoli, Farideh
    Linköping University, Department of health and environment. Linköping University, Faculty of Health Sciences.
    Lerm, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Söderholm, Johan D
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Surgery . Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    Artursson, Per
    Uppsala University.
    Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis2008In: Laboratory Investigation, ISSN 0023-6837, E-ISSN 1530-0307, Vol. 88, no 11, p. 1215-1226Article in journal (Refereed)
    Abstract [en]

    Crohns disease is characterized by a defect in intestinal barrier function, where bacteria are considered the most important inflammation-driving factor. Enteric bacteria, including E. coli and Yersinia spp, affect tight junctions in enterocytes, but little is known about bacterial effects on the transcellular pathway. Our objective was to study the short-term effects of Y. pseudotuberculosis on uptake of nanoparticles across human villus epithelium. Monolayers of human colon epithelium-derived Caco-2 cells and biopsies of normal human ileum were studied after 2 h exposure to Y. pseudotuberculosis expressing (inv+) or lacking (inv-) the bacterial adhesion molecule, invasin. Transepithelial transport of fluorescent nanoparticles (markers of transcytosis) was quantified by flow cytometry, and mechanisms explored by using inhibitors of endocytosis. Epithelial expressions of beta 1-integrin and particle uptake pathways were studied by confocal microscopy. The paracellular pathway was assessed by electrical resistance (TER), mannitol flux, and expression of tight junction proteins occludin and caludin-4 by confocal microscopy. Inv + Y. pseudotuberculosis adhered to the apical surface of epithelial cells and induced transcytosis of exogenous nanoparticles across Caco-2 monolayers (30-fold increase, P < 0.01) and ileal mucosa (268 +/- 47% of control; P < 0.01), whereas inv-bacteria had no effect on transcytosis. The transcytosis was concentration-, particle size-and temperature-dependent, and possibly mediated via macropinocytosis. Y. pseudotuberculosis also induced apical expression of beta 1-integrin on epithelial cells. A slight drop in TER was seen after exposure to inv+ Y. pseudotuberculosis, whereas mannitol flux and tight junction protein expression was unchanged. In summary, Y. pseudotuberculosis induced apical expression of beta 1-integrin and stimulated uptake of nanoparticles via invasin-dependent transcytosis in human intestinal epithelium. Our findings suggest that bacterial factors may initiate transcytosis of luminal exogenous particles across human ileal mucosa, thus presenting a novel mechanism of intestinal barrier dysfunction.

  • 106.
    Rydell, Ewa
    et al.
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sjö, Anita
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Axelsson, Krister
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Protein kinase C and casein kinase II activities in two human colon carcinoma cell lines, HT-29 and CaCo-2: Possible correlation with differentiation1990In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 10, p. 293-299Article in journal (Refereed)
    Abstract [en]

    Protein kinase C (PK-C) and casein kinase II (CK-II) activities were studied in two human colon carcinoma cell lines (HT-29 and CaCO-2) undergoing differentiationin vitro resulting, in small-intestine-like cells. CaCo-2 cells, when grown under standard conditions, appear to undergo spontaneous differentiation. In these cells PK-C and CK-II activities were determined on day 5, 10 and 15. No significant differences in activities were seen either in PK-C or CK-II activity. HT-29 cells, when grown in glucose-free medium can be stimulated to undergo differentiation which is completed within 20 days. PK-C and CK-II activities were determined after 5, 10, 15, 20 and 25 days, respectively. PK-C activity rose from 7.9±3.5 pmole32P/mg protein/min at day 5 to 37.5±14.8 pmole32P/mg protein/min at day 20. After 25 days the activity was reduced to 20.0±7.8 pmole32P/mg protein/min. CK-II activity did not change significantly during day 5 to 20, but on day 25 there was a significant decrease in CK-II activity from 94.9±6.4 pmole32P/mg protein/min (day 20) to 62.6±3.9 pmole32P/mg protein/min (day 25) p=0.003. The results in this study indicate a role for PK-C and CK-II in cell growth and differentiation.

  • 107.
    Salim, Sa'ad
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences.
    Silva, Manuel A
    McMaster University.
    Keita, Åsa
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences.
    Larsson, Marie
    Linköping University, Department of Clinical and Experimental Medicine, Molecular Virology . Linköping University, Faculty of Health Sciences.
    Andersson, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Perdue, Mary H
    McMaster University.
    Söderholm, Johan D
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    CD83(+)CCR7(-) Dendritic Cells Accumulate in the Subepithelial Dome and Internalize Translocated Escherichia coli HB101 in the Peyers Patches of Heal Crohns Disease2009In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 174, no 1, p. 82-90Article in journal (Refereed)
    Abstract [en]

    Recurrent Crohns disease originates with small erosions in the follicle-associated epithelium overlying the Peyers patches. Animal studies have illustrated mucosal immune regulation by dendritic cells located in the subepithelial dome. The aim of this study was to characterize the dendritic cells at this specific site in patients with Crohns disease. Heal tissues were obtained after surgery performed on Crohns patients; ileal samples from noninflammatory bowel disease and ulcerative colitis served as standard and inflammatory controls, respectively. Flow cytometry of isolated intestinal mononuclear cells showed a larger subset of dendritic cells in Crohns samples compared with controls. This finding was corroborated by confocal microscopy, showing enhanced infiltrates of cells positive for the dendritic cell markers, DC-SIGN(+) and CD83(+), in the subepithelial dome. Moreover, the CD83(+) cells in Crohns tissues showed reduced expression of the lymph node migratory receptor, CCR7, possibly contributing to the high numbers of dendritic cells. After exposure to nonpathogenic Escherichia coli in Ussing chambers, dendritic cells in the subepithelial dome of Crohns disease demonstrated increased co-localization with translocated bacteria. Immunohistochemical results revealed that DC-SIGN(+) cells in Crohns tissues were found to express toll-like receptor 4 and produce tumor necrosis factor-a. In conclusion, nonmigrating dendritic cells that accumulate in the subepithelial dome and internalize nonpathogenic bacteria may be important for the onset and perpetuation of mucosal inflammation in Crohns disease.

  • 108.
    Savickiene, Jurate
    et al.
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Borutinskaite, Veronika-Viktorija
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Linköping University, Faculty of Health Sciences.
    Treigyte, Grazina
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Navakauskiene, Ruta
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    The novel histone deacetylase inhibitor BML-210 exerts growth inhibitory, proapoptotic and differentiation stimulating effects on the human leukemia cell lines2006In: European Journal of Pharmacology, ISSN 0014-2999, Vol. 549, no 1-3, p. 9-18Article in journal (Refereed)
    Abstract [en]

    Histone deacetylase inhibitors have a potent role in the strategy for the treatment of leukemias. BML-210 (N-(2-Aminophenyl)-N′ phenyloctanol diamine) is the novel histone deacetylase inhibitor, and its mechanism of action has not been characterized. In this study, we examined the in vitro effects of BML-210 on the human leukemia cell lines (NB4, HL-60, THP-1, and K562). We found that BML-210 inhibits the growth of all cell lines and promotes apoptosis in a dose- and time-dependent manner. BML-210 alone induces HL-60 and K562 cell differentiation (up to 30%) to granulocytes and erythrocytes, respectively, and in combination with differentiation agents — all-trans retinoic acid and hemin, markedly potentates it. Those treatments cause G1 arrest and histone H4 acetylation, affects transcription factor NF-κB and Sp1 binding activity to their consensus sequences, the p21 or the FasL promoters, and influences expression of Sp1, NF-κB, p21 and FasL. These findings suggest that BML-210 could be a promising antileukemic agent to induce apoptosis and to modulate differentiation through the modulation of histone acetylation and gene expression.

  • 109.
    Savickiene, Jurate
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine.
    Treigyte, G
    Pivoriunas, A
    Navakauskiene, Ruta
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Sp1 and NF-kappa B transcription factor activity in the regulation of the p21 and FasL promoters during promyelocytic leukemia cell monocytic differentiation and its associated apoptosis2004In: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 1030, p. 569-577Article in journal (Refereed)
    Abstract [en]

    Treatment of human acute promyelocytic leukemia cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest and differentiation toward monocytes, which subsequently die by apoptosis. However, the relationship between terminal differentiation and apoptosis remains unclear. Here we have studied Sp1 and nuclear factor B-K (NF-B-K) transcription factor activity in controlling promoters of cell cycle-regulating (p21/WAF1/CIP1) and cell death (FasL) genes during monocytic differentiation and apoptosis of the human acute promyelocytic leukemia cell lines NB4 and HL-60. Using the electrophoretic mobility shift assay, we observed that PMA treatment of NB4 cells caused an early response in Sp1 binding to the p21 and FasL promoters at 8 h. The firmly adherent cell phenotype, characteristic of differentiated cells, retained Sp1-binding activity to either promoter, but it was often lost completely in detached, apoptotic cells. The association of Sp1 with the p21 promoter during monocytic differentiation correlated with the levels of expressed p21 in the cytoplasmic fraction, as detected by immunoblotting. In HL-60 cells, very weak or no Sp1 binding to either promoter was observed. Low NF-B-K affinity for its consensus sites and to the FasL promoter was characteristic of apoptotic cells. The results of this study suggest a positive role of Sp1 and NF-B-K, as regulators of p21 and FasL genes, in leukemic cell survival and monocytic differentiation and a negative role in apoptotic cell death.

  • 110.
    Savickiene, Jurate
    et al.
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Treigyte, Grazina
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Borutinskaite, Veronika
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Linköping University, Faculty of Health Sciences.
    Navakauskiene, Ruta
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    The histone deacetylase inhibitor FK228 distinctly sensitizes the human leukemia cells to retinoic acid-induced differentiation2006In: Annals of the New York Academy of Sciences, ISSN 0077-8923, Vol. 1091, p. 368-384Article in journal (Refereed)
    Abstract [en]

    FK228 (depsipeptide) is a novel histone deacetylase inhibitor (HDACI) that has shown therapeutical efficacy in clinical trials for malignant lymphoma. In this article, we examined in vitro effects of FK228 on human leukemia cell lines, NB4 and HL-60. FK228 alone (0.2–1 ng/mL) inhibited leukemia cell growth in a dose-dependent manner and induced death by apoptosis. FK228 had selective differentiating effects on two cell lines when used for 6 h before induction of granulocytic differentiation by retinoic acid (RA) or in combination with RA. These effects were accompanied by a time- and dose-dependent histone H4 hyper-acetylation or histone H3 dephosphorylation and alterations in DNA binding of NF-κB in association with cell death and differentiation. Pifithrin-α (PFT), an inhibitor of p53 transcriptional activity, protected only NB4 cells with functional p53 from FK228-induced apoptosis and did not interfere with antiproliferative activity in p53-negative HL-60 cells. In NB4 cells, PFT inhibited p53 binding to the p21 (Waf1/Cip1) promotor and induced DNA binding of NF-κB leading to enhanced cell survival. Thus, beneficial effects of FK228 on human promyelocytic leukemia may be exerted through the induction of differentiation or apoptosis via histone modification and selective involvement of transcription factors, such as NF-κB and p53.

  • 111. Savickiene, Jurate
    et al.
    Treigyte, Grazina
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Navakauskiene, Ruta
    p21 (Waf1/Cip1) and FasL gene activation via Sp1 and NFκB is required for leukemia cell survival but not for cell death induced by diverse stimuli2005In: International Journal of Biochemistry and Cell Biology, ISSN 1357-2725, E-ISSN 1878-5875, Vol. 37, no 4, p. 784-796Article in journal (Refereed)
    Abstract [en]

    The molecular mechanisms of the cellular response to different apoptotic effectors are only partially understood. Herein, the role of transcription factors, Sp1 and NFκB in differentiation-related and etoposide-induced apoptosis was examined in a number of human leukemia cell lines (HL-60, NB4, HEL, THP-1, K562). This was investigated with respect to the recruitment of one cell-cycle regulating gene, p21 and one cell death gene, FasL. Using electrophoretic mobility shift assay (EMSA), we consistently observed Sp1 and NFκB binding activity to the promoter of either gene during cell differentiation and the decrease associated with apoptosis upon long-term treatment with differentiation inducers in HL-60, NB4 and HEL cells. By contrast, Sp1 and NFκB binding capacities were lost in all myeloid cell lines undergoing etoposide-induced fast apoptosis. This effect was eliminated by the broad-spectrum caspase inhibitor, benzyloxycarbonyl-valinyl-alaninyl- aspartyl fluoromethylketone, thus restoring transcription factors' binding activity. However, sustained NFκB binding to the FasL promoter was noticed in apoptosis undergoing HEL cells treated by etoposide. Our results suggest that p21 and FasL gene activation is required for myeloid leukemia cell survival or maturation but not for cell death via Sp1 and NFκB as regulators of these genes. The findings also support the idea of a common mechanism for cellular responses to different apoptotic effectors in malignant hematopoietic cell lines. © 2004 Elsevier Ltd. All rights reserved.

  • 112.
    Savickiene, Jurate
    et al.
    Institute of Biochemistry, Vilnius.
    Treigyte, Grazina
    Institute of Biochemistry, Vilnius.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Navakauskiene, Ruta
    Institute of Biochemistry, Vilnius.
    Response of Retinoic Acid-Resistant KG1 Cells to Combination of Retinoic Acid with Diverse Histone Deacetylase Inhibitors2009In: NATURAL COMPOUNDS AND THEIR ROLE IN APOPTOTIC CELL SIGNALING PATHWAYS, ISSN 0077-8923, Vol. 1171, p. 321-333Article in journal (Refereed)
    Abstract [en]

    Acute promyelocytic leukemia KG1 cells with t(11;17) PLZF-RAR alpha respond poorly to the differentiation inducer all-trans retinoic acid (RA), and the reason for the RA resistance is the recruitment of histone deacetylase by PLZF-RAR alpha. Here, we demonstrate that histone deacetylase inhibitors (HDACIs), FK228, BML-210, phenyl butyrate, and vitamin B3, in different combinations with RA, act as KG1 cell growth inhibitors. Partial differentiation to granulocytes was induced by 3 mu mol/L RA, and its combination with HDAC inhibitors did not enhance RA-induced but potentiated apoptosis. HDACIs induced accumulation of hyperacetylated histone H4. Chromatin immunoprecipitation analysis has revealed phenyl butyrate and its combinations with RA and vitamin B3 cause histone H4 acetylation in the p21 promoter regions corresponding to p53 and/or Sp1 sites. This was coincident with the activation of the transcription factor p53-binding activity to the p21 promoter in electrophoretic mobility shift assay. The results indicate the possibility of using the combination of agents for therapeutic strategy in RA-resistant acute myeloid leukemia to produce both differentiation and apoptosis.

  • 113.
    Savickiene, Jurate
    et al.
    Institute for Biochemistry, Vilnius, Lithuania .
    Treigyte, Grazina
    Institute for Biochemistry, Vilnius, Lithuania .
    Vistartaite, Giedre
    Institute for Biochemistry, Vilnius, Lithuania .
    Tunaitis, Virginijus
    State Research Institute Centre Innovat Med, Vilnius, Lithuania .
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Navakauskiene, Ruta
    Institute for Biochemistry, Vilnius, Lithuania .
    C/EBP alpha and PU.1 are involved in distinct differentiation responses of acute promyelocytic leukemia HL-60 and NB4 cells via chromatin remodeling2011In: DIFFERENTIATION, ISSN 0301-4681, Vol. 81, no 1, p. 57-67Article in journal (Refereed)
    Abstract [en]

    C/EBP alpha and PU.1 are the basic transcription factors that control differentiation-related genes, including granulocyte- colony-stimulating factor (G-CSFR) and human neutrophil elastase (HNE). Here, we analyzed a role of C/EBP alpha and PU.1 in human acute leukemia cell lines, HL-60 and NB4, in association with a modified chromatin structure by histone deacetylase inhibitors, FK228, sodium phenyl butyrate and vitamin B3. We found that sodium phenyl butyrate alone and 6 h-pretreatment with phenyl butyrate or FK228 before the induction of differentiation with all-trans-retinoic acid in the presence of vitamin B3 effectively accelerated and enhanced differentiation to granulocytes in HL-60 but not in NB4 cells as detected by NBT test and the expression of CD11b and CD114 (G-CSFR) using flow cytometric analysis. HDACIs induced a time- and dose-dependent accumulation of hyper-acetylated histone H4 in both cell lines with the delay in NB4 cells. Time-dependent different induction of HL-60 and NB4 cell differentiation was paralleled by the activation of C/EBP alpha and PU.1 binding to the G-CSFR and the HNE promoters in electrophoretic mobility shift assay. Chromatin immunoprecipitation analysis revealed histone H4 acetylation in the G-CSF receptor promoter at the C/EBP alpha binding site in HL-60 but not in NB4 cells under the combined treatment. The results indicate that epigenetic events, such as histone acetylation, are involved in the activity modulation of the key transcription factors responsible for the induction of granulocytic differentiation in promyelocytic leukemia cells.

  • 114.
    Shleev, Sergey
    et al.
    Malmö University.
    Wetterö, Jonas
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology . Linköping University, Faculty of Health Sciences.
    Magnusson , Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Ruzgas, Tautgirdas
    Malmö University.
    Simultaneous use of electrochemistry and chemiluminescence to detect reactive oxygen species produced by human neutrophils2008In: Cell Biology International, ISSN 1065-6995, E-ISSN 1095-8355, Vol. 32, no 12, p. 1486-1496Article in journal (Refereed)
    Abstract [en]

    A novel approach for the simultaneous optical and electrochemical detection of biologically produced reactive oxygen species has been developed and applied. The set-up consists of a luminol-dependent chemiluminescence assay combined with two amperometric biosensors sensitive to superoxide anion radicals (O-2(center dot-))and hydrogen peroxide (H2O2), respectively. The method permits direct, real-time in vitro determination of both extra-and intracellular O-2(center dot-) and H2O2 produced by human neutrophil granulocytes. The rate of O-2(center dot-) production by stimulated neutrophils was calculated to about 10(-17) mol s(-1) per single cell. With inhibited NADPH oxidase, a distinct extracellular release of H2O2 instead of O-2(center dot-) was obtained from stimulated neutrophils with the rate of about 3 . 10(-18) mol s(-1) per single cell. When the H2O2 release was discontinued, fast H2O2 utilisation was observed. Direct interaction with and possibly attachment of neutrophils to redox protein-modified gold electrodes, resulted in a spontaneous respiratory burst in the population of cells closely associated to the electrode surface. Hence, further stimulation of human neutrophils with a potent receptor agonist (fMLF) did not significantly increase the O-2(center dot-) sensitive amperometric response. By contrast, the H2O2 sensitive biosensor, based on an HRP-modified graphite electrode, was able to reflect the bulk concentration of H2O2, produced by stimulated neutrophils and would be very useful in modestly equipped biomedical research laboratories. In summary, the system would also be appropriate for assessment of several other metabolites in different cell types, and tissues of varying complexity, with only minor electrode modifications.

  • 115.
    Shleev, Sergey
    et al.
    Malmö .
    Wetterö, Jonas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Rheumatology.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Ruzgas, Tautgirdas
    Malmö .
    Electrochemical characterization and application of azurin-modified gold electrodes for detection of superoxide2006In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 22, no 2, p. 213-219Article in journal (Refereed)
    Abstract [en]

    A novel biosensor for superoxide radical (O2{radical dot}-) detection based on Pseudomonas aeruginosa azurin immobilized on gold electrode was designed. The rate constant of azurin reduction by O2{radical dot}- was found to be 105 M-1 s-1 in solution and five times lower, i.e., 0.2 × 105 M-1 s-1, for azurin coupled to gold by 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP). The electron transfer rate between the protein and the electrode ranged from 2 to 6 s-1. The sensitivity of this biosensor to O2{radical dot}- was 6.8 × 102 A m-2 M-1. The response to the interference substances, such as uric acid, H2O2, and dimethylsulfoxide was negligible below 10 μM. The electrode was applied in three O2{radical dot}- generating systems: (i) xanthine oxidase (XOD), (ii) potassium superoxide (KO2), and (iii) stimulated neutrophil granulocytes. The latter was compared with luminol-amplified chemiluminescence. The biosensor responded to O2{radical dot}- in all three environments, and the signals were antagonized by superoxide dismutase. © 2006 Elsevier B.V. All rights reserved.

  • 116.
    Sjö, Anita
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holmgren Peterson, Kajsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    A possible role for α-dystrobrevin in the reorganization of tight junctions in epithelial cellsManuscript (preprint) (Other academic)
    Abstract [en]

    Alpha-dystrobrevin (α -DB) has been described primarily as a cytoplasmic component of the dystrophin-glycoprotein complex (DGC) in skeletal muscle cells. Isoforms of α -DB show different localization in cells and tissues; at basolateral membranes in epithelial cells, dystrobrevins mediate contact with the extracellular matrix, peripheral and transmembrane proteins and the filamentous actin cytoskeleton. Beside their structural role, α -DBs are assumed to be important in cell signalling and cell differentiation. We have assessed the role of a-DB in two epithelial cell lines (MDCK I and HT 29) that are in different developmental stages and exhibit distinct permeability characteristics. Using a polyclonal anti-α-DB antibody, we have investigated its localization and association with tight junction (TJ)-associated proteins before and after protein kinase C (PKC) activation with phorbol myristate acetate (PMA). In both cell lines, there was submembranous localization of α -DB both apically and basolaterally, as assessed with confocal imaging. PKC caused a reorganization of TJ, which was parallel with increased localization of α -DB to TJ areas, especially in MDCK I cells. Moreover, the TJ-associated protein Z0-1 co-immunoprecipitated with α -DB, as displayed with SDS-PAGE and immunoblotting. Thus, α -DBs not only take part in the regulation of the contact between intra- and extracellular proteins at basolateral membranes, but possibly also in the regulation of tight junctions via ZO-1 and PKC-activation.

  • 117.
    Sjö, Anita
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Holmgren Peterson, Kajsa
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Association of a-dystrobrevin with reorganizing tight junctions2005In: Journal of Membrane Biology, ISSN 0022-2631, E-ISSN 1432-1424, Vol. 203, no 1, p. 21-30Article in journal (Refereed)
    Abstract [en]

    Alpha-dystrobrevin (a-DB) has been described primarily as a cytoplasmic component of the dystrophin-glycoprotein complex in skeletal muscle cells. Isoforms of a-DB show different localization in cells and tissues, at basolateral membranes in epithelial cells, dystrobrevins mediate contact with the extracellular matrix, peripheral and transmembrane proteins and the filamentous actin cytoskeleton. Beside their structural role, a-DBs are assumed to be important in cell signalling and cell differentiation. We have primarily assessed the role of a-DB in two epithelial cell lines (MDCK I, HT 29), which represent different developmental stages and exhibit distinct permeability characteristics. Using a polyclonal anti-a-DB antibody, we have investigated its expression, localization and association with tight junction (TJ)- associated proteins (ZO-1, occludin) before and after protein kinase C (PKC) activation with phorbol myristate acetate. Distinct subsets of a-DB isoforms were detected in the two cell lines by immunoblotting. In both cell lines there was submembranous localization of a-DB both apically and basolaterally, shown with confocal imaging. PKC activation caused a reorganization of TJ, which was parallel to increased localization of a-DB to TJ areas, most pronounced in MDCK I cells. Moreover, actin and ZO-1 co-immunoprecipitated with a-DB, as displayed with immunoblotting. Our findings suggest that a-dystrobrevin specifically is associated with the tight junctions during their reorganization. © Springer Science+Business Media, Inc. 2005.

  • 118.
    Sjö, Anita
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holmgren Peterson, Kajsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Distinct Effects of Protein Kinase C on the Barrier Function at Different Developmental Stages2003In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 23, no 2-3, p. 87-102Article in journal (Refereed)
    Abstract [en]

    We show here, that activation of protein kinase C by the phorbol ester PMA improves barrier function in colon carcinoma (HT 29) cells. By contrast, in canine kidney (MDCK I) cells it caused increased permeability and opening of tight junctions; the latter has also been noticed in other studies. Thus, with PMA confluent HT 29 cells responded with a reduced passage of 330 kDa sodium fluorescein, increased transepithelial electrical resistance, and a change in the cell shape of the HT 29 cells from an irregular to a regular, hexagonal form. Confocal imaging revealed parallel distinct changes in the staining of occludin and caludin-1, viz. a translocation from cytoplasmic clusters to apical cell–cell contacts. Interestingly, in both cell lines protein kinase A activation caused a decreased in the threonine phosphorylation of occludin that correlated with tight junction assembly in HT 29 cells and tight junction disassembly in MDCK I cells. We conclude that protein kinase C regulation of the epithelial barrier involves specific molecular mechanisms and achieves distinct effects at different developmental stages.

  • 119.
    Sjö, Anita
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Holmgren Peterson, Kajsa
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Protein kinase C activation has distinct effects on the localization, phosphorylation and detergent solubility of the claudin protein family in tight and leaky epithelial cells2010In: Journal of Membrane Biology, ISSN 0022-2631, E-ISSN 1432-1424, Vol. 236, no 2, p. 181-189Article in journal (Refereed)
    Abstract [en]

    We have previously shown that protein kinase C (PKC) activation has distinct effects on the structure and barrier properties of cultured epithelial cells (HT29 and MDCK I). Since the claudin family of tight junction (TJ)-associated proteins is considered to be crucial for the function of mature TJ, we assessed their expression patterns and cellular destination, detergent solubility and phosphorylation upon PKC stimulation for 2 or 18 h with phorbol myristate acetate (PMA). In HT29 cells, claudins 1, 3, 4 and 5 and possibly claudin 2 were redistributed to apical cell-cell contacts after PKC activation and the amounts of claudins 1, 3 and 5, but not of claudin 2, were increased in cell lysates. By contrast, in MDCK I cells, PMA treatment resulted in redistribution of claudins 1, 3, 4 and 5 from the TJ and in reorganization of the proteins into more insoluble complexes. Claudins 1 and 4 were phosphorylated in both MDCK I and HT29 cells, but PKC-induced changes in claudin phosphorylation state were detected only in MDCK I cells. A major difference between HT29 and MDCK I cells, which have low and high basal transepithelial electrical resistance, respectively, was the absence of claudin 2 in the latter. Our findings show that PKC activation targets in characteristic ways the expression patterns, destination, detergent solubility and phosphorylation state of claudins in epithelial cells with different capacities to form an epithelial barrier.

  • 120.
    Sjö, Anita
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Peterson, KH
    Protein kinase C activation improves the barrier of HT 29 epithelial cells and regulates translocation and phosphorylation of tight junction proteins2002In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 13, p. 2788-Conference paper (Other academic)
  • 121.
    Sjöberg, Veronika
    et al.
    Department of Clinical Microbiology, Immunology, Umeå University, Umeå, Sweden.
    Hollén, Elisabet
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Pietz, Grzegorz
    ology, Immunology, Umeå University, Umeå, Sweden.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Fälth-Magnusson, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Linköping.
    Sundström, Mia
    Department of Clinical Microbiology, Immunology, Umeå University, Umeå, Sweden.
    Holmgren Peterson, Kajsa
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Sandström, Olof
    Department of Clinical Sciences, Pediatrics, Umeå University, Umeå, Sweden.
    Hernell, Olle
    Department of Clinical Sciences, Pediatrics, Umeå University, Umeå, Sweden.
    Hammarström, Sten
    Department of Clinical Microbiology, Immunology, Umeå University, Umeå, Sweden.
    Högberg, Lotta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Norrköping.
    Hammarström, Marie-Louise
    Department of Clinical Microbiology, Immunology, Umeå University, Umeå, Sweden.
    Noncontaminated dietary oats may hamper normalization of the intestinal immune status in childhood celiac disease.2014In: Clinical and Translational Gastroenterology, ISSN 2155-384X, E-ISSN 2155-384X, Vol. 5, no e58Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: Life-long, strict gluten-free diet (GFD) is the only treatment for celiac disease (CD). Because there is still uncertainty regarding the safety of oats for CD patients, the aim was to investigate whether dietary oats influence the immune status of their intestinal mucosa.

    METHODS: Paired small intestinal biopsies, before and after >11 months on a GFD, were collected from children with CD who were enrolled in a randomized, double-blind intervention trial to either of two diets: standard GFD (GFD-std; n=13) and noncontaminated oat-containing GFD (GFD-oats; n=15). Expression levels of mRNAs for 22 different immune effector molecules and tight junction proteins were determined by quantitative reverse transcriptase (RT)-PCR.

    RESULTS: The number of mRNAs that remained elevated was higher in the GFD-oats group (P=0.05). In particular, mRNAs for the regulatory T cell (Treg) signature molecules interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1), the cytotoxicity-activating natural killer (NK) receptors KLRC2/NKG2C and KLRC3/NKG2E, and the tight junction protein claudin-4 remained elevated. Between the two groups, most significant differences were seen for claudin-4 (P=0.003) and KLRC3/NKG2E (P=0.04).

    CONCLUSIONS: A substantial fraction of pediatric CD patients seem to not tolerate oats. In these patients, dietary oats influence the immune status of the intestinal mucosa with an mRNA profile suggesting presence of activated cytotoxic lymphocytes and Tregs and a stressed epithelium with affected tight junctions. Assessment of changes in levels of mRNA for claudin-4 and KLC3/NKG2E from onset to after a year on oats containing GFD shows promise to identify these CD patients.

  • 122.
    Stenhammar, Lars
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Norrköping.
    Fälth-Magnusson, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Grodzinsky, Ewa
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Department of Health and Society, General Practice. Östergötlands Läns Landsting, Local Health Care Services in the West of Östergötland, Unit of Research and Development in Local Health Care, County of Östergötland.
    Hallert, Claes
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Welfare and Care (IVV), Self-Care and Learning. Östergötlands Läns Landsting, Local Health Care Services in the East of Östergötland, Department of Internal Medicine VHN.
    Högberg, Lotta
    Barn och ungdomsmed kliniken Vrinnevisjukhuset, Norrköping.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Från ax till limpa - några svenska bidrag till forskningen om celiaki2004In: Läkartidningen, ISSN 0023-7205, E-ISSN 1652-7518, Vol. 101, no 48, p. 3932-3937Article in journal (Other academic)
  • 123.
    Sundqvist, Tommy
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Laurin, Pia
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Fälth-Magnusson, Karin
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Stenhammar, Lars
    Department of Pediatrics, Central Hospital, Norrköping, Sweden.
    Significantly increased levels of nitric oxide products in urine of children with celiac disease1998In: Journal of Pediatric Gastroenterology and Nutrition - JPGN, ISSN 0277-2116, E-ISSN 1536-4801, Vol. 27, no 2, p. 196-198Article in journal (Refereed)
    Abstract [en]

    Background: Celiac disease is characterized by morphologic and functional aberrations of the small intestinal mucosa, i.e. crypt hyperplasia, villous atrophy, infiltration of intraepithelial lymphocytes, and alteration of permeability. Nitric oxide has been shown to affect mucosal permeability after ischemia-reperfusion, but little is known about the regulatory role of nitric oxide in celiac disease. The purpose of this study was to assess nitric oxide production in children with celiac disease and in control subjects.

    Methods: The sum of nitrite and nitrate in the urine was measured with a colorimetric method in 137 children with a median age of 3 years, 84 patients and 53 reference children, all of whom underwent a small intestinal biopsy to confirm or overrule suspicion of celiac disease.

    Results: Median urinary nitrite-nitrate concentration in celiac children was 3323µM (4147 ± 1102; mean ± SEM) at first clinical examination and 2501 µM (2939 ± 386) after gluten challenge, which was significantly higher than concentrations in reference children(1029 µM; 1174 ± 116) and in children with celiac disease on a gluten-free diet (882 µM; 1369 ± 360) (p< 0.0001).

    Conclusions: A gluten-containing diet is associated with an increased nitrite-nitrate secretion in the urine in children with celiac disease, presumably as a result of nitric oxide synthase activation and nitric oxide production in the diseased small intestinal mucosa.

  • 124.
    Surapureddi, Sailesh
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Svartz, Jesper
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Colocalization of leukotriene C synthase and microsomal glutathione S-transferase elucidated by indirect immunofluorescence analysis2000In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 480, no 2-3, p. 239-243Article in journal (Refereed)
    Abstract [en]

    We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S-transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well-known sources of leukotriene C synthase, both expressed microsomal glutathione S-transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S-transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S-transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein–protein interaction may contribute to the regulation of LTC4 production.

  • 125.
    Söderholm, Johan D
    et al.
    Linköping University, Department of Biomedicine and Surgery, Surgery. Linköping University, Faculty of Health Sciences.
    Holmgren Peterson, Kajsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Olaison, Gunnar
    Linköping University, Department of Biomedicine and Surgery, Surgery. Linköping University, Faculty of Health Sciences.
    Franzén, Lennart E.
    Department of Pathology, Lund University–MAS, Malmö.
    Weström, Björn
    Department of Animal Physiology, Lund University, Lund, Sweden.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sjödahl, Rune
    Linköping University, Department of Biomedicine and Surgery, Surgery. Linköping University, Faculty of Health Sciences.
    Epithelial permeability to proteins in the noninflamed ileum of Crohn's disease?1999In: Gastroenterology, ISSN 0016-5085, E-ISSN 1528-0012, Vol. 117, no 1, p. 65-72Article in journal (Refereed)
    Abstract [en]

    Background & Aims: Crohn's disease (CD) is associated with a disturbed intestinal barrier. Permeability studies have focused on inert molecules, but little is known about transepithelial transport of macromolecules with antigenic potential in humans. The aim of this study was to quantify permeation and to characterize passage routes for macromolecules in ileal mucosa in CD.

    Methods: Noninflamed and inflamed ileal mucosa specimens from patients with CD (n = 12) and ileal specimens from patients with colon cancer (n = 7) were studied regarding transmucosal permeation of ovalbumin, dextran (mol wt, 40,000), and 51Cr-EDTA for 90 minutes in vitro in Ussing chambers. Transepithelial passage routes for fluorescent ovalbumin and dextran 40,000 were investigated by confocal microscopy.

    Results: Noninflamed ileum from CD patients showed increased permeation of ovalbumin compared with ileum from colon cancer patients (P < 0.05). Dextran permeation was equal in the three groups, whereas 51Cr-EDTA permeability was increased in inflamed ileum. Ovalbumin passed both transcellularly and paracellularly, but dextran followed a strictly paracellular route. Both markers were subsequently endocytosed by cells of the lamina propria.

    Conclusions: Noninflamed ileal mucosa from patients with CD shows increased epithelial permeability to ovalbumin, probably by augmented transcytosis. This increase in antigen load to the lamina propria could be an initiating pathogenic event in CD.

  • 126.
    Tafazoli, Farideh
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holmström, Ann
    Department of Microbiology, National Defence Research Establishment, Umeå, Sweden.
    Forsberg, Åke
    Department of Microbiology, National Defence Research Establishment, Umeå, Sweden.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Apically Exposed, Tight Junction-Associated β1-Integrins Allow Binding and YopE-Mediated Perturbation of Epithelial Barriers by Wild-Type Yersinia Bacteri2000In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 68, no 9, p. 5335-5343Article in journal (Refereed)
    Abstract [en]

    Using polarized epithelial cells, primarily MDCK-1, we assessed the mode of binding and effects on epithelial cell structure and permeability of Yersinia pseudotuberculosis yadA-deficient mutants. Initially, all bacteria except the invasin-deficient (inv) mutant adhered apically to the tight junction areas. These contact points of adjacent cells displayed β1-integrins together with tight junction-associated ZO-1 and occludin proteins. Indeed, β1-integrin expression was maximal in the tight junction area and then gradually decreased along the basolateral membranes. Wild-type bacteria also opened gradually the tight junction to paracellular permeation of different-sized markers, viz., 20-, 40-, and 70-kDa dextrans and 45-kDa ovalbumin, as well as to their own translocation between adjacent cells in intimate contact with β1-integrins. The effects on the epithelial cells and their barrier properties could primarily be attributed to expression of the Yersinia outer membrane protein YopE, as the yopE mutant bound but caused no cytotoxicity. Moreover, the apical structure of filamentous actin (F-actin) was disturbed and tight junction-associated proteins (ZO-1 and occludin) were dispersed along the basolateral membranes. It is concluded that the Yersinia bacteria attach to β1-integrins at tight junctions. Via this localized injection of YopE, they perturb the F-actin structure and distribution of proteins forming and regulating tight junctions. Thereby they promote paracellular translocation of bacteria and soluble compounds.

  • 127.
    Tafazoli, Farideh
    et al.
    Linköping University, Faculty of Arts and Sciences. Linköping University, Department of health and environment.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Ultrastructural characteristics of the perturbation of the barrier properties of epithelial cells by pathogenic Yersiniae pseudotuberculosis bacteria2000In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 11, p. 2653-Conference paper (Other academic)
  • 128.
    Tafazoli, Farideh
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Zheng, Limin
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Disruption of Epithelial Barrier Integrity by Salmonella enterica Serovar Typhimurium Requires Geranylgeranylated Proteins2003In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 71, no 2, p. 872-881Article in journal (Refereed)
    Abstract [en]

    Epithelial cells that line the human intestinal mucosa constitute the initial sites of host invasion by bacterial pathogens. A number of bacteria, such as Salmonella and Yersinia spp., have been shown to disrupt the integrity of the epithelial barrier, although little is known about the mechanisms underlying that effect. We found that polarized MDCK-1 epithelial cells infected with invasive Salmonella enterica serovar Typhimurium SL1344 exhibited marked changes in F-actin organization, an increase in the paracellular flux of dextran, and a rapid decrease in transepithelial electrical resistance (TER). In contrast, infection with an isogenic noninvasive mutant (hilA) increased the TER in these cells. Pretreating MDCK-1 cells with the inhibitors for tyrosine kinase (genistein) or phosphatidylinositol 3-kinase (wortmannin) did not affect invasion and subsequent perturbation of the epithelial barrier by serovar Typhimurium. Instead, the geranylgeranyltransferase 1 inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, clearly reversed the capacity of serovar Typhimurium to disrupt the epithelial barrier. The substrates for GGTI-298 include Rho family GTPases, as indicated by inhibiting prenylation of Rac1 and Cdc42. Infection with wild-type serovar Typhimurium increased the level of activated Rac1 and Cdc42 and caused these proteins to accumulate apically in MDCK-1 cells. This Salmonella-induced accumulation of Rac1 and Cdc42 and alteration of the junction-associated proteins ZO-1, occludin, and E-cadherin in MDCK-1 cells were markedly inhibited by GGTI-298. These results suggest that activation of geranylgeranylated proteins, including Rac1 and Cdc42, is critical for disruption of barrier integrity by serovar Typhimurium in polarized MDCK-1 cells.

  • 129.
    Tafazoli, Farideh
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Zheng, Liming
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Disruption of Barrier Integrity by Salmonella typhimurium Requires Activation of Cdc42 and Rac1 in Epithelial CellsManuscript (preprint) (Other academic)
    Abstract [en]

    Epithelial cells that line the human intestinal mucosa constitute the initial sites of host invasion by bacterial pathogens. A number of bacteria, such as Salmonella and Yersinia, have been shown to disrupt the integrity of the epithelial barrier, although little is known about the mechanisms underlying that effect. We found that polarized MDCK-1 epithelial cells infected with invasive Salmonella typhimurium SLI344 exhibited marked changes in F-actin organization and a rapid decrease in transepithelial electrical resistance (TER). In contrast, infection with isogenic noninvasive mutants (hilA, prgH, and sipC) increased the TER in these cells. Pretreating MDCK-1 cells with the tyrosine kinase inhibitor genistein or the PI-3 kinase inhibitor wortmannin did not affect invasion and subsequent perturbation of the epithelial barrier by S. typhimurium. Instead, the specific geranylgeranyltransferase-1 inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, clearly reversed the capacity of S. typhimurium to disrupt the epithelial barrier. The best-known substrates for GGTI-298 include Rho family GTPases, Racl and Cdc42. Infection with wild-type S. typhimurium increased the level of activated Racl and Cdc42 and caused these proteins to accumulate apically in MDCK-1 cells. GGTI-298-induced inactivation of Racl and Cdc42 prevented alteration of the tight and adherens junction-associated proteins Z0-1, occludin, and E-cadherin in MDCK-1 cells infected with invasive Salmonella. These results indicate that activation of Racl and Cdc42, but not tyrosine kinase or PI-3 kinase, is essential for disruption of barrier integrity by S. typhimurium in polarized MDCK-1 cells.

  • 130.
    Tafazoli, Farideh
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Zeng, Carl Q.
    Division of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas.
    Estes, Mary K.
    Division of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Svensson, Lennart
    Department of Virology, Swedish Institute for Infectious Disease Control (SMI), Karolinska Institute, Solna.
    NSP4 Enterotoxin of Rotavirus Induces Paracellular Leakage in Polarized Epithelial Cells2001In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 75, no 3, p. 1540-1546Article in journal (Refereed)
    Abstract [en]

    The nonstructural NSP4 protein of rotavirus has been described as the first viral enterotoxin. In this study we have examined the effect of NSP4 on polarized epithelial cells (MDCK-1) grown on permeable filters. Apical but not basolateral administration of NSP4 was found to cause a reduction in the transepithelial electrical resistance, redistribution of filamentous actin, and an increase in paracellular passage of fluorescein isothiocyanate-dextran. Significant effects on transepithelial electrical resistance were noted after a 20- to 30-h incubation with 1 nmol of NSP4. Most surprisingly, the epithelium recovered its original integrity and electrical resistance upon removal of NSP4. Preincubation of nonconfluent MDCK-1 cells with NSP4 prevented not only development of a permeability barrier but also lateral targeting of the tight-junction-associated Zonula Occludens-1 (ZO-1) protein. Taken together, these data indicate new and specific effects of NSP4 on tight-junction biogenesis and show a novel effect of NSP4 on polarized epithelia.

  • 131.
    Tejle, Katarina
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Lindroth, Margaretha
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Rasmusson, Birgitta
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Wild-type Leishmania donovani promastigotes block maturation, increase integrin expression and inhibit detachment of human monocyte-derived dendritic cells - The influence of phosphoglycans2008In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 279, no 1, p. 92-102Article in journal (Refereed)
    Abstract [en]

    The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs. © 2007 Federation of European Microbiological Societies.

  • 132.
    Tejle, Katarina
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Rasmusson, Birgitta
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Phagocytosis and phagosome maturation are regulated by calcium in J774 macrophages interacting with un-opsonized prey2002In: Bioscience Reports, ISSN 0144-8463, Vol. 22, no 5-6, p. 529-540Article in journal (Refereed)
    Abstract [en]

    Phagocytosis by neutrophils, macrophages, and other professional phagocytes requires rapid remodeling of actin. Early phagosomes are surrounded by a rim of F-actin that is disassembled during phagosomoal maturation. Breakdown of periphagosomal F-actin and phagolysosome fusion are calcium dependent processes in neutrophils interacting with serum-opsonized prey, but appears to be calcium independent in macrophages interacting with serum- or IgG-opsonized prey. In the present study, we found that calcium was necessary for phagocytosis, breakdown of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey. We also observed that lipophosphoglycan (LPG) from Leishmania donovani promastigotes required calcium to exert its inhibitory effect on macrophage phagocytosis and periphagosomal F-actin breakdown. We conclude that calcium is essential for phagocytosis, depolymerization of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey, as well as for proper functioning of LPG.

  • 133.
    Tjellstrom, B.
    et al.
    Karolinska Institute, Sweden Norrkoping Hospital, Sweden .
    Stenhammar, Lars
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Norrköping.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
    Fälth-Magnusson, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Linköping.
    Hollén, Elisabet
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Norin, E.
    Karolinska Institute, Sweden .
    Midtvedt, T.
    Karolinska Institute, Sweden .
    Högberg, Lotta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Norrköping.
    The effects of oats on the function of gut microflora in children with coeliac disease2014In: Alimentary Pharmacology and Therapeutics, ISSN 0269-2813, E-ISSN 1365-2036, Vol. 39, no 10, p. 1156-1160Article in journal (Refereed)
    Abstract [en]

    Background Faecal short chain fatty acids (SCFAs) are produced by the gut microflora. We have previously reported high faecal SCFA levels in children with coeliac disease (CD), indicating alteration in gut microfloral metabolism. Data accumulated over recent decades by us and others suggest that wheat-free oats can safely be included in a gluten-free diet (GFD). However, concerns have been raised with respect to the safety of oats in a subset of coeliacs. Aim To describe faecal SCFA patterns in children with newly diagnosed CD treated for 1year with a GFD with or without oats. Methods This report is part of a randomised, double-blind study on the effect of a GFD containing oats (GFD-oats) vs. a standard GFD (GFD-std). Faecal samples were received from 34 children in the GFD-oats group and 37 in the GFD-std group at initial diagnosis and/or after 1year on a GFD. Faecal SCFAs were analysed. Results The GFD-std group had a significantly lower total faecal SCFA concentration at 12months compared with 0months (Pless than0.05). In contrast, total SCFA in the GFD-oats group remained high after 1year on the GFD. The children in the GFD-oats group had significantly higher acetic acid (Pless than0.05), n-butyric acid (Pless than0.05) and total SCFA concentration (Pless than0.01) after 1-year diet treatment compared to the GFD-std group. Conclusions Our results indicate that oats do affect the gut microflora function, and that some coeliac children receiving oats may develop gut mucosal inflammation, that may present a risk for future complications.

  • 134.
    Tjellstrom, Bo
    et al.
    Karolinska Institute, Sweden Norrkoping Hospital, Sweden .
    Högberg, Lotta
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Norrköping.
    Stenhammar, Lars
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Norrköping.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Midtvedt, Tore
    Karolinska Institute, Sweden .
    Norin, Elisabeth
    Karolinska Institute, Sweden .
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Effect of exclusive enteral nutrition on gut microflora function in children with Crohns disease2012In: Scandinavian Journal of Gastroenterology, ISSN 0036-5521, E-ISSN 1502-7708, Vol. 47, no 12, p. 1454-1459Article in journal (Refereed)
    Abstract [en]

    Objective. Exclusive enteral nutrition (EEN) is a first-line treatment in children with active Crohns disease (CD) but is seldom used in adults with active disease. The mode of action of EEN in suppressing mucosa] inflammation is not fully understood, but modulation of intestinal microflora activity is one possible explanation. The aim of this study was to investigate the effect of 6-week EEN in children with active CD, with special reference to intestinal microflora function. Materials and methods. Fecal samples from 18 children (11 boys, 7 girls; median age 13.5 years) with active CD (13 children with small bowel/colonic and 5 with perianal disease) were analyzed for short chain fatty acid (SCFA) pattern as marker of gut microflora function. The children were studied before and after EEN treatment. Results from 12 healthy teenagers were used for comparison. Results. Eleven (79%) of the children with small bowel/colonic CD responded clinically positively to EEN treatment showing decreased levels of pro-inflammatory acetic acid as well as increased concentrations of anti-inflammatory butyric acids and also of valeric acids, similar to the levels in healthy age-matched children. In children with active perianal CD, however, EEN had no positive effect on clinical status or inflammatory parameters. Conclusions. The authors present new data supporting the hypothesis that the well-documented anti-inflammatory effect of EEN in children with active small bowel/colonic CD is brought about by modulation of gut microflora activity, resulting in an anti-inflammatory SCFA pattern. By contrast, none of the children with perianal disease showed clinical or biochemical improvement after EEN treatment.

  • 135.
    Tjellstrom, Bo
    et al.
    Karolinska Institute, Sweden.
    Högberg, Lotta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Region Östergötland, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Norrköping. Linköping University, Faculty of Medicine and Health Sciences. County Council Ostergotland, Sweden.
    Stenhammar, Lars
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Region Östergötland, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Norrköping. Linköping University, Faculty of Medicine and Health Sciences. County Council Ostergotland, Sweden.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Midtvedt, Tore
    Karolinska Institute, Sweden.
    Norin, Elisabeth
    Karolinska Institute, Sweden.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Letter: A Role for Bacteria in Celiac Disease? in DIGESTIVE DISEASES AND SCIENCES, vol 61, issue 7, pp 2140-21402016In: Digestive Diseases and Sciences, ISSN 0163-2116, E-ISSN 1573-2568, Vol. 61, no 7, p. 2140-2140Article in journal (Other academic)
    Abstract [en]

    n/a

  • 136.
    Tjellström, Bo
    et al.
    Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Norrköping. Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.
    Högberg, Lotta
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Norrköping.
    Stenhammar, Lars
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Norrköping.
    Fälth-Magnusson, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Linköping.
    Magnusson, Karl-Erik
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Norin, Elisabeth
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Midtvedt, Tore
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.
    Faecal short-chain fatty acid pattern in childhood coeliac disease is normalised after more than one year's gluten-free diet2013In: Microbiological Ecology in Health and Disease, ISSN 0891-060X, E-ISSN 1651-2235, Vol. 24Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: Recent work indicates that the gut microflora is altered in patients with coeliac disease (CD). Faecal short-chain fatty acids (SCFAs) are produced by the gut microflora. We have previously reported a high SCFA output in children with symptomatic and asymptomatic CD at presentation, as well as in CD children on a gluten-free diet (GFD) for less than 1 year, indicating deviant gut microfloral function. In this report, we focus on faecal SCFA production in coeliacs on GFD for more than 1 year.

    MATERIALS AND METHODS: Faecal samples were collected from 53 children with CD at presentation, 74 coeliac children on GFD for less than 1 year, and 25 individuals diagnosed with CD in childhood and on GFD for more than 1 year. The control group comprised 54 healthy children (HC). The faecal samples were analysed to show the SCFA pattern taken as a marker of gut microflora function. We applied a new fermentation index, reflecting the inflammatory activity of the SCFAs (amount of acetic acid minus propionic acid and n-butyric acid, together divided by the total amount of SCFAs).

    RESULTS: In coeliacs on GFD for more than 1 year, the individual SCFAs, total SCFA, and fermentation index did not differ significantly from the findings in controls. In contrast, the faecal SCFA level was clearly higher in coeliacs treated with GFD for less than 1 year compared to those more than 1 year.

    CONCLUSIONS: This is the first study on SCFA patterns in faecal samples from individuals with CD on GFD for more than 1 year. Our study indicates that the disturbed gut microflora function in children with CD at presentation and after less than 1 year of GFD, previously demonstrated by us, is normalised on GFD for more than 1 year.

  • 137.
    Tjellström, Bo
    et al.
    Dept of Microbiology KS, Stockholm.
    Stenhammar, Lars
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Norrköping.
    Högberg, Lotta
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Norrköping.
    Fälth-Magnusson, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Midtvedt, Tore
    Dept of Microbiology KS, Stockholm.
    Sundqvist, Tommy
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Houlston, Richard
    Section of Cancer Genetics Inst of Cancer Research, Sutton, UK.
    Popat, Sanjay
    Section of Cancer Genetics Inst of Cancer Research, Sutton, UK.
    Norin, Elisabeth
    Dept of Microbiology KS, Stockholm.
    Gut microflora associated characteristics in first-degree relatives of children with celiac disease2007In: Scandinavian Journal of Gastroenterology, ISSN 0036-5521, E-ISSN 1502-7708, Vol. 42, no 10, p. 1204-1208Article in journal (Refereed)
    Abstract [en]

    Objective. In celiac disease (CD), enteropathy of the small bowel results from a T-cell-mediated reaction to gluten in the diet. In addition to gluten, other environmental and genetic factors participate in the disease pathogenesis. We have recently reported the finding of a significantly different short-chain fatty acid (SCFA) profile in fecal samples from children with CD compared to healthy controls reflecting an aberrant gut microflora. The aim of the present study was to make a functional evaluation of the gut microflora status in non-celiac 1st degree relatives of children with CD. Material and methods. Fecal samples from 76 symptom-free, non-celiac, 1st degree CD relatives and from 91 aged-matched healthy controls were analyzed for fecal tryptic activity (FTA) and a number of SCFAs. Results. There was a significantly lower level of acetic acid and total SCFAs as well as a significantly increased level of i-butyric acid and FTA in relatives compared to healthy controls. Conclusions. The FTA and the SCFA profiles in fecal samples from 1st degree relatives of children with CD are different from those of healthy individuals. The implication of this observation provides insight into the pathogenesis of CD and opens up the possibility of future new diagnostic, therapeutic and prophylactic strategies. © 2007 Taylor & Francis.

  • 138.
    Tjellström, Bo
    et al.
    Microbiology and Tumour biology center, Karolinska Institutet, Stockholm.
    Stenhammar, Lars
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Högberg, Lotta
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Norrköping.
    Fälth-Magnusson, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Midtvedt, Tore
    Microbiology and Tumour biology center, Karolinska Institutet, Stockholm.
    Sundqvist, Tommy
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Norin, E
    Microbiology and Tumour biology center, Karolinska Institutet, Stockholm.
    Gut microflora associated characteristics in children with celiac disease2005In: American Journal of Gastroenterology, ISSN 0002-9270, E-ISSN 1572-0241, Vol. 100, no 12, p. 2784-2788Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: The aim of the study was to investigate the metabolic function of intestinal microflora in children with celiac disease (CD) in order to find out if there is a deviant gut flora in CD patients compared to healthy controls. METHODS: The study group comprised children with CD, consecutively diagnosed according to current criteria given by the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition. Thirty-six children were studied at presentation, i.e., on a normal gluten-containing diet, with clinical symptoms and signs indicative of CD, positive celiac serology markers, and a small bowel biopsy showing severe enteropathy. Forty-seven patients were studied when they had been on a gluten-free diet (GFD) for at least 3 months. For comparison, a group of 42 healthy controls (HC) were studied. The functional status of the intestinal microflora was evaluated by gas-liquid chromatography of short chain fatty acids (SCFAs) in fecal samples. RESULTS: There was a significant difference between untreated CD children and HC as well as between treated CD children and HC regarding acetic, i-butyric, i-valeric acid, and total SCFAs. The propionic and n-valeric acids differed significantly between CD children on GFD and HC. Moreover, there was a strong correlation between i-butyric and i-valeric acids in all study groups. CONCLUSIONS: This is the first study of the SCFA pattern in fecal samples from children with CD. The results indicate that there is a difference in the metabolic activity of intestinal microbial flora in children with CD compared to that in HC. The finding of a different pattern of some SCFAs in celiacs both at presentation and during treatment with GFD indicates that it is a genuine phenomenon of CD not affected by either the diet, the inflammation, or the autoimmune status of the patient. © 2005 by Am. Coll. of Gastroenterology Published by Blackwell Publishing.

  • 139.
    Tjellström, Bo
    et al.
    Karolinska Institute.
    Stenhammar, Lars
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Norrköping.
    Högberg, Lotta
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Norrköping.
    Fälth-Magnusson, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Midtvedt, Tore
    Karolinska Institute.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Norin, Elisabeth
    Karolinska Institute.
    Screening-detected and symptomatic untreated celiac children show similar gut microflora-associated characteristics2010In: SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY, ISSN 0036-5521, Vol. 45, no 9, p. 1059-1062Article in journal (Refereed)
    Abstract [en]

    Objective. The aim of this study was to investigate the metabolic function of intestinal microflora in children with screening-detected celiac disease (CD) to see if there is an aberrant gut flora in screening-detected CD similar to symptomatic CD and contrary to healthy controls. Materials and methods. As part of a Swedish multicenter screening for CD, 912 12-year-old children were screened with serum anti-human tissue transglutaminase-IgA. Small bowel biopsy specimens from children with positive serology revealed 17 individuals with CD. The functional status of the intestinal microflora was evaluated by gas liquid chromatography of short chain fatty acids (SCFAs) in fecal samples. Our previously published findings in children with symptomatic CD and healthy controls were used as comparison. Results. The children with screening-detected CD had a similar fecal SCFA profile to children with symptomatic CD, but differed significantly from that in healthy children. Conclusions. This is the first study on SCFA patterns in fecal samples from children with screening-detected CD. The similarity of the fecal SCFA profile in screening-detected and symptomatic CD indicates common pathogenic mechanisms. This could open the way for new therapeutic or prophylactic measures based on novel biological principles.

  • 140.
    Tjellström, Bo
    et al.
    Karolinska Institute, Sweden.
    Stenhammar, Lars
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Norrköping.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Midtvedt, Tore
    Karolinska Institute, Sweden.
    Norin, Elisabeth
    Karolinska Institute, Sweden.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Högberg, Lotta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Norrköping.
    Letter: Exclusive Enteral Nutrition Does Not Normalize Gut Microflora Function in Pediatric Perianal Crohn Disease in JOURNAL OF PEDIATRIC GASTROENTEROLOGY AND NUTRITION, vol 61, issue 1, pp E4-E42015In: Journal of Pediatric Gastroenterology and Nutrition - JPGN, ISSN 0277-2116, E-ISSN 1536-4801, Vol. 61, no 1, p. E4-E4Article in journal (Other academic)
    Abstract [en]

    n/a

  • 141.
    Tjomsland, Vegard
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Molecular Virology . Linköping University, Faculty of Health Sciences.
    Sandström, Per
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    Spangeus, Anna
    Linköping University, Department of Medicine and Health Sciences, Internal Medicine . Linköping University, Faculty of Health Sciences.
    Messmer, Davorka
    University of California.
    Emilsson, Johan
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Falkmer, Ursula
    Jonköping Hospital.
    Falkmer, Sture
    Jonköping Hospital.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Borch, Kurt
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    Larsson, Marie
    Linköping University, Department of Clinical and Experimental Medicine, Molecular Virology . Linköping University, Faculty of Health Sciences.
    Pancreatic adenocarcinoma exerts systemic effects on the peripheral blood myeloid and plasmacytoid dendritic cells: an indicator of disease severity?2010In: BMC CANCER, ISSN 1471-2407, Vol. 10, no 87Article in journal (Refereed)
    Abstract [en]

    Background: Dendritic cells (DCs) isolated from tumor bearing animals or from individuals with solid tumors display functional abnormalities and the DC impairment has emerged as one mechanism for tumor evasion from the control of the immune system. Ductal pancreatic adenocarcinoma (PDAC), the most common pancreatic cancer, is recognized as a very aggressive cancer type with a mortality that almost matches the rate of incidence. Methods: We examined the systemic influence ductal pancreatic adenocarcinoma ( PDAC) exerted on levels of peripheral blood DCs and inflammatory mediators in comparison to the effects exerted by other pancreatic tumors, chronic pancreatitis, and age-matched controls. Results: All groups examined, including PDAC, had decreased levels of myeloid DCs (MDC) and plasmacytoid DCs (PDC) and enhanced apoptosis in these cells as compared to controls. We found elevated levels of PGE2 and CXCL8 in subjects with PDAC, and chronic pancreatitis. Levels of these inflammatory factors were in part restored in PDAC after tumor resection, whereas the levels of DCs were impaired in the majority of these patients similar to 12 weeks after tumor removal. Our results prove that solid pancreatic tumors, including PDAC, systemically affect blood DCs. The impairments do not seem to be tumor-specific, since similar results were obtained in subjects with chronic pancreatitis. Furthermore, we found that PDAC patients with a survival over 2 years had significant higher levels of blood DCs compared to patients with less than one year survival. Conclusions: Our findings points to the involvement of inflammation in the destruction of the blood MDCs and PDCs. Furthermore, the preservation of the blood DCs compartment in PDAC patients seems to benefit their ability to control the disease and survival.

  • 142.
    Treigyté, G.
    et al.
    aLaboratory of Developmental Biology, Institute of Biochemistry, Vilnius.
    Navakauskiene, R.
    aLaboratory of Developmental Biology, Institute of Biochemistry, Vilnius.
    Kulyté, Agné
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Gineitis, A.
    aLaboratory of Developmental Biology, Institute of Biochemistry, Vilnius.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils2000In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 57, no 13-14, p. 1997-2008Article in journal (Refereed)
    Abstract [en]

    Two-dimensional electrophoretic analysis was used to assess quantitative and qualitative changes in the expression and tyrosine phosphorylation of cytoplasmic proteins of proliferating, differentiating HL-60 cells and mature human blood neutrophils. The total tyrosine phosphorylation level of cytoplasmic proteins appeared approximately constant during the pre-commitment period, i.e., 6-24 h after induction of differentiation by 700 nM all-trans retinoic acid. At the time of granulocytic phenotype formation (48-120 h), the total level of tyrosine phosphorylation of cytoplasmic proteins increased significantly. Tyrosine phosphorylation of cytoplasmic proteins in matured blood neutrophils was significantly lower than that of cytoplasmic proteins of HL-60 cells differentiated for 96 h with retinoic acid. Immunoblotting with anti-Erk2 and anti-phosphotyrosine monoclonal IgG2bk antibodies showed that Erk2 was expressed and tyrosine-phosphorylated at different levels in HL-60 proliferating cells and in cells at all stages of differentiation. Our data showed that tyrosine phosphorylation of cytoplasmic proteins in differentiating HL-60 cells changes dramatically during the period of phenotype formation and is accompanied by increasing activity of Erk2. An increasing number of apoptotic cells appeared in the differentiating HL-60 cell population during the granulocyte maturation stage (48-120 h of differentiation). The appearance at this time of differentiation of a new set of tyrosine-phosphorylated cytoplasmic proteins (also distinctive for apoptotic HL-60 cells mediated by etoposide) together with an increasing number of apoptotic cells in the differentiating population strongly suggests that these proteins are associated with the apoptotic process.

  • 143.
    Tunaitis, Virginijus
    et al.
    State Research Institute Centre Innovat Med, Lithuania .
    Borutinskaite, Veronika
    Institute Biochem, Lithuania .
    Navakauskiene, Ruta
    Institute Biochem, Lithuania .
    Treigyte, Grazina
    Institute Biochem, Lithuania .
    Unguryte, Ausra
    State Research Institute Centre Innovat Med, Lithuania .
    Aldonyte, Ruta
    State Research Institute Centre Innovat Med, Lithuania.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Pivoriunas, Augustas
    State Research Institute Centre Innovat Med, Lithuania.
    Effects of different sera on adipose tissue-derived mesenchymal stromal cells2011In: JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, ISSN 1932-6254, Vol. 5, no 9, p. 733-746Article in journal (Refereed)
    Abstract [en]

    Current cell therapy protocols require considerable numbers of mesenchymal stromal cells (MSCs), which can be obtained only by in vitro expansion. The most important issue is a choice of optimal growth supplements for cell culture. Ideally, these should be of known composition, free of animal components and allow production of large homogenic populations of MSCs in a considerably short period of time. Since this standard has not been achieved to date, we aimed to assess the molecular responses of MSCs to different growth supplements commonly in use. MSCs were isolated from breast or abdominal adipose tissue and plated into DMEM supplemented with one of four different sera: fetal calf serum (FCS), pretested fetal calf serum (FCS-Sp), human allogeneic serum (HS) or artificial serum substitute (AS). MSCs cultivated with different serum supplements demonstrated distinct morphologies, high adipogenic and osteogenic differentiation potential and expressed characteristic antigens. Using real-time PCR, we found a large increase in PPAR gamma and Msx2 gene expression in both lines of proliferating MSCs cultivated with AS. We found that MSCs cultivated in the presence of different sera had similar global proteomic expression patterns, but comparisons of identified proteins revealed most differences in the MSCs cultivated with AS. Our results indicate that MSCs cultivated in the presence of FCS and HS display similar growth, differentiation, immunophenotypic and proteomic properties, while AS induces more profound changes in the physiology of MSCs, suggesting that further fundamental studies should be done before its introduction into clinical practice.

  • 144.
    Turkina, Maria
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Olofsson, Annelie
    Umeå University, Sweden.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Arnqvist, Anna
    Umeå University, Sweden.
    Vikström, Elena
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Helicobacter pylori vesicles carrying CagA localize in the vicinity of cell-cell contacts and induce histone H1 binding to ATP in epithelial cells2015In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 362, no 11, p. fnv076-Article in journal (Refereed)
    Abstract [en]

    Helicobacter pylori produces outer membrane vesicles (OMV), delivering bacterial substances including the oncogenic cytotoxin-associated CagA protein to their surroundings. We investigated the effects of H. pylori OMV carrying CagA (OMV-CagA) on cell junctions and ATP-binding proteome of epithelial monolayers, using proteomics, mass spectrometry and imaging. OMV-CagA localized in close vicinity of ZO-1 tight junction protein and induced histone H1 binding to ATP. We suggest the expression of novel events in the interactions between H. pylori OMV and epithelia, which may have an influence on host gene transcription and lead to different outcomes of an infection and development of cancer.

  • 145.
    Turoverova, L.V.
    et al.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Khotin, M.G.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Yudintseva, N.M.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Blinova, M.I.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Pinaev, G.P.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Tentler, D.G.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Analysis of extracellular matrix proteins produced by cultured cells2009In: Cell and Tissue Biology, ISSN 1990-519X, Vol. 3, no 5, p. 497-502Article in journal (Refereed)
    Abstract [en]

    The extracellular matrix (ECM) is a highly organized multimolecular structure essential for the vital functions of any organism. Although much of the data of extracellular matrix components has been accumulated, the isolation of an entire set of these proteins remains a complex procedure due to the high content of fibrillar proteins and proteoglycans, which form multidomain, netlike structures. In the study presented, we developed a method for isolating ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membranes. Subsequent treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibers significantly improved the fractioning of ECM proteins. The extraction of remaining proteins from the surface of the culture plate was preformed by a buffer developed based on Laemmli probe buffer. Using this method, we isolated ECM proteins synthesized by cultured cells, and the extracted proteins were suitable for future analysis by SDS PAGE and two-dimentional electrophoresis, as well as for identifying individual proteins by mass spectrometry. This study may allow us to compare assortments of ECM proteins isolated from different sources, and elucidate impact of various proteins on structure and property of extracellular matrix of investigated cells.

  • 146.
    Turoverova, L.V.
    et al.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Khotin, M.G.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Yudintseva, N.M.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Blinova, M.I.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Pinaev, G.P.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Tentler, D.G.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Analysis of extracellular matrix proteins produced by cultured cells2009In: Tsitologiya, ISSN 0041-3771, Vol. 51, no 8, p. 691-696Article in journal (Refereed)
    Abstract [en]

    Extracellular matrix (ECM) is a highly organized multimolecular structure essential for vital function of any organism. Although a lot of data on the extracellular matrix components has been accumulated, an isolation of the entire set of these proteins still remains to be a complex procedure since ECM contains fibrillar proteins and proteoglycans, which form multidomain net-like structures. In the presented study, we developed a method for isolation of ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membrane. Following treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibrils significantly improved fractioning of ECM proteins. Extraction of the remained proteins from culture plate surface was preformed using a buffer developed on the basis of Laemmli probe buffer. With this method, we isolated ECM proteins synthesized by culturing cells and suitable for a future analysis by SDS PAGE and two-dimentional electrophoresis as well as for identification of individual proteins by mass-spectrometry. This study may allow comparing protein contents of ECMs isolated from different sources, and elucidate influences of various proteins on the protein and the properties of extracellular matrix of investigated cells.

  • 147.
    Vicente Carrillo, Alejandro
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Edebert, I.
    Karlbergsvägen 83 B, Stockholm, Sweden.
    Garside, H.
    AstraZeneca Research and Dev, England.
    Cotgreave, I.
    Karolinska Institute, Sweden; Karolinska Institute, Sweden.
    Rigler, R.
    Karolinska Institute, Sweden.
    Loitto, Vesa
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Boar spermatozoa successfully predict mitochondrial modes of toxicity: Implications for drug toxicity testing and the 3R principles2015In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 29, no 3, p. 582-591Article in journal (Refereed)
    Abstract [en]

    Replacement of animal testing by in vitro methods (3-R principles) requires validation of suitable cell models, preferably obtained non-invasively, defying traditional use of explants. Ejaculated spermatozoa are highly dependent on mitochondrial production and consumption of ATP for their metabolism, including motility display, thus becoming a suitable model for capturing multiple modes of action of drugs and other chemicals acting via mitochondrial disturbance. In this study, a hypothesis was tested that the boar spermatozoon is a suitable cell type for toxicity assessment, providing a protocol for 3R-replacement of animals for research and drug-testing. Boar sperm kinetics was challenged with a wide variety of known frank mito-toxic chemicals with previously shown mitochondrial effects, using a semi-automated motility analyser allied with real-time fluorescent probing of mitochondrial potential (MitoTracker and JC-1). Output of this sperm assay (obtained after 30 min) was compared to cell viability (ATP-content, data obtained after 24-48 h) of a hepatome-cell line (HepG2). Results of compound effects significantly correlated (P less than 0.01) for all sperm variables and for most variables in (HepG2). Dose-dependent decreases of relative ATP content in HepG2 cells correlated to sperm speed (r= 0.559) and proportions of motile (r = 0.55) or progressively motile (r = 0.53) spermatozoa. The significance of the study relies on the objectivity of computerized testing of sperm motility inhibition which is comparable albeit of faster output than somatic cell culture models. Sperm suspensions, easily and painlessly obtained from breeding boars, are confirmed as suitable biosensors for preclinical toxicology screening and ranking of lead compounds in the drug development processes.

  • 148.
    Vikström, Elena
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Bui, Lan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Role of calcium signalling and phosphorylations in disruption of the epithelial junctions by Pseudomonas aeruginosa quorum sensing molecule2010In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 89, no 8, p. 584-597Article in journal (Refereed)
    Abstract [en]

    In Pseudomonas aeruginosa. cell-cell communication based on acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms by the bacterium. Incidentally, these bacterial signals can also modulate mammalian cell behaviour. We demonstrate here that 3O-C-12-HSL can induce changes in calcium signalling through influx and release of calcium from thapsigargin-sensitive stores and delocalization of inositol 1,4,5-trisphosphate receptors (IP3R), but not of ryanodine receptors (RyR). In parallel, P. aeruginosa 3O-C-12-HSL disrupts junctions in human Caco-2 cells as evidenced by a reduction of the expression and distribution of ZO-3 and JAM-A. Using co-immunoprecipitation we also found an alteration in the binding of ZO-3 to JAM-A in protein complexes. Moreover, 3O-C-12-HSL-treatment resulted in tyrosine hyperphosphorylation of ZO-3 and JAM-A. On the contrary, serine and threonine residues of ZO-1 and JAM-A became less phosphorylated after exposition of 3O-C-12-HSL. The 3O-C-12-HSL-induced intracellular calcium signalling and alteration in the phosphorylation status of junction proteins furthermore correlated with changes in the association between JAM-A-ZO-3. The calcium inhibitors thapsigargin, xestospongin C. and dantrolene partly prevented the 3O-C-12-HSL-induced decreases in TER and increases in the paracellular flux of 10 kDa dextran. These findings clearly suggest that P. aeruginosa 3O-C-12-HSL can cause the loss of epithelial barrier function via calcium signalling and further alteration in the phosphorylation status of junction proteins; and that bacterial quorum sensing signals represent inter-kingdom signalling.

  • 149.
    Vikström, Elena
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Bui, Lan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    The junctional integrity of epithelial cells is modulated by Pseudomonas aeruginosa quorum sensing molecule through phosphorylation-dependent mechanisms2009In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 315, no 2, p. 313-326Article in journal (Refereed)
    Abstract [en]

    In Pseudomonas aeruginosa, cell-cell Communication based on acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms by the bacterium. Incidentally, these bacterial signals can also modulate mammalian cell behaviour. We report that 3O-C-12-HSL can disrupt adherens junctions in human epithelial Caco-2 cells as evidenced by a reduction of the expression and distribution of E-cadherin and beta-catenin. Using co-immunoprecipitation we also found that P. aeruginosa 3O-C-12-HSL-treatment resulted in tyrosine hyperphosphorylation of E-cadherin, beta-catenin, occludin and ZO-1. Similarly, serine and threonine residues of E-cadherin and ZO-1 became more phosphorylated after 3O-C-12-HSL treatment. On the contrary, occludin and beta-catenin underwent dephosphorylation on serine and threonine residues after exposition of 3O-C-12-HSL. These changes in the phosphorylation state were paralleled by alteration in the Structure of junction complexes and increased paracellular permeability. Moreover, pre-treatment of the Caco-2 cells with protein phosphatase and kinase inhibitors prevented 3O-C-12-HSL-induced changes in paracellular permeability and interactions between occludin-ZO-1 and the E-cadherin-beta-catenin. These findings clearly suggest that an alteration in the phosphorylation status of junction proteins are involved in the changes in cell junction associations and enhanced paracellular permeability, and that bacterial signals are indeed sensed by the host cells.

  • 150.
    Vikström, Olena
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Pivoriunas, Augustas
    Vilnius .
    The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L- homoserine lactone stimulates phagocytic activity in human macrophages through the p38 MAPK pathway2005In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 7, no 15, p. 1512-1518Article in journal (Refereed)
    Abstract [en]

    Quorum-sensing is an important mechanism for the regulation of bacteria-to-bacteria communication. Recent advances have demonstrated that the Pseudomonas aeruginosa signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) is also a potent modulator of eukaryotic cells and may thus play an important role in the host response during P. aeruginosa infections. Little is known, however, about specific effects of 3O-C 12-HSL molecules on human macrophages. To address this issue, we investigated the influence of 3O-C12-HSL on the phagocytic activity, production of reactive oxygen species, and activation of p38 and p42/44 MAPK signaling pathways in human macrophages. We show an effect of 3O-C 12-HSL on the phagocytic capacity in human macrophages, which depends on concentration and time of exposure. When cells were exposed to 100 μM 3O-C12-HSL for 30 min or 1 h, the phagocytic activity increased 1.8 and 1.6 times, respectively. The 3O-C12-HSL treatments had no significant effect on the level of reactive oxygen species production. Furthermore, the p38 MAPK, but not the p42/44 MAPK, signaling pathway was activated in response to 3O-C12-HSL. In addition, specific blocking of p38 MAPK activation with 10 μM SB 203580 prevented the 3O-C 12-HSL-induced increase in the phagocytic activity. These findings demonstrate that the bacterial quorum-sensing can play a significant role also in regulation of macrophage activity during infections caused by P. aeruginosa. © 2005 Elsevier SAS. All rights reserved.

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