liu.seSearch for publications in DiVA
Change search
Refine search result
12345 1 - 50 of 210
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1.
    Abdalla, Hana
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Diab, Asim
    Department of Neurology, University of Texas Southwestern Medical Center, Dallas, USA.
    Forslund, Tony
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Bakhiet, Moiz
    Department of Medicine, Divisions of lnfectious Diseases, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden.
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Expression of inducible nitric oxide synthases and nitrotyrosine during the course of Haemophilus influenzae type b meningitis in ratManuscript (preprint) (Other academic)
    Abstract [en]

    Bacterial meningitis continues to be a major health problem and despite great advances in antimicrobial therapy the fatality rate remains high. There is increasing evidence that leukocyte-endothelial interactions are involved in CNS damage during bacterial meningitis. Once leukocytes have entered the CSF they cause injury by releasing toxic molecules such as nitric oxide (NO) and reactive oxygen species (ROS). The induction of iNOS was examined by assessing intracerebral mRNA expression and protein production during the course of Haemophilus influenzae type b (Hib) meningitis in the rat. Induction of iNOS mRNA was detected 12h postinoculation (pi), followed by a gradual reduction. The increased number of intracerebral iN OS expressing cells was detected at 12h pi. followed by further elevation to peak expression at 72h pi. The iNOS positive tissue also bound antibodies specific for nitrotyrosine. The expression of iNOS and NO production, as shown by nitrotyrosine expression, correlated with disease severity, suggesting that activation of iNOS may play an important role in Haemophilus irifluenzae type b meningitis.

  • 2.
    Abrahams, M
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, MKC - Medicin och kirurgicentrum, Anestesi.
    Sjöberg, Folke
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Plastic Surgery, Hand Surgery and Burns. Östergötlands Läns Landsting, Reconstruction Centre, Department of Plastic Surgery, Hand surgery UHL.
    Oscarsson, Anna
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, MKC - Medicin och kirurgicentrum, Anestesi.
    Sundqvist, Tommy
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    The effects of human burn injury on urinary nitrate excretion. 1999In: Burns, ISSN 0305-4179, E-ISSN 1879-1409, Vol. 25, p. 29-33Article in journal (Refereed)
  • 3. Alvarado-Kristensson, M
    et al.
    Pörn-Ares, MI
    Grethe, S
    Smith, D
    Zheng, Limin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Andersson, T
    p38 Mitogen-activated protein kinase and phosphatidylinositol 3-kinase activities have opposite effects on human neutrophil apoptosis.2002In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 16, p. 129-131Article in journal (Refereed)
  • 4.
    Andersson, Kerstin
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Inhibition of phagocyte signaling by the Yersinia virulence protein YopH1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Yersinia pseudotuberculosis evades the immediate immune defense of a host organism by inhibiting bactericidal functions of phagocytes, including phagocytosis, cytokine release, and the oxidative burst. Consequently, this pathogen can survive and multiply in lymphatic tissues. An ensemble of proteins called Yops are involved in the virulence of Y. pseudotuberculosis. Through a polarized mechanism, certain Yops are translocated directly into the host cell, where they are assumed to exert their effects. The present studies were performed to gain further knowledge about the role of the tyrosine phosphatase YopH in Yersinia virulence, especially in regard to effects on the immediate functions and signals of phagocytes.

    Y. pseudotuberculosis was found to resist phagocytic uptake by a mechanism involving translocation of bacterially synthesized YopH into the target cell. The antiphagocytic mechanism had an impact on ingestion of both non-opsonized and IgO-opsonized bacteria. Phagocytosis of a YopH-negative strain was accompanied by induction of tyrosinephosphorylated cellular proteins (among them paxillin), and this involved binding of the bacterial surface protein invasin to ß1 integrins of the eukaryotic cell, which also initiated an immediate Ca2+ signal in the target cell. The phosphotyrosine proteins Cas and FYB were recruited to the focal complex area during phagocytosis of the YopH-negative strain. Furthermore, we found that a phosphatase-inactive YopH eo-localized with focal adhesion to the periphery of a host cell. In phagocytes infected with wild-type bacteria, phosphatase-active YopH dephosphorylated Cas and FYB, which caused disruption of focal complex structures, and inhibition of the Ca2+ signal. The phosphorylation events as well as the Ca2+ signal were rapid responses to bacterial attachment, suggesting that the action of YopH is instantaneous.

    Genetic studies revealed that the YopH protein contain an inherent sequence important for anchoring at focal complex structures. Specifically, deletion of the amino acids 223-226 disabled YopH to localize to the focal complexes and to inhibit phagocytosis and Ca2+-signaling. This indicates that Y opH must bind to a specific site in focal complexes to focus its activity on the appropriate substrates (i.e. Cas and FYB). Our results show that targeting such complexes is important for Y. pseudotuberculosis, not only as a means of avoiding ingestion by phagocytes, but also for its virulence in mice.

  • 5.
    Andersson, Kerstin
    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.
    Majeed, Meytham
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Fällman, Maria
    Department of Cell and Molecular Biology, University of Umeå, Umeå, Sweden.
    Yersinia pseudotuberculosis-induced calcium signaling in neutrophils is blocked by the virulence effector YopH1999In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 67, no 5, p. 2567-2574Article in journal (Refereed)
    Abstract [en]

    Pathogenic species of the genus Yersinia evade the bactericidal functions of phagocytes. This evasion is mediated through their virulence effectors, Yops, which act within target cells. In this study we investigated the effect of Yersinia pseudotuberculosis on Ca 2+ signaling in polymorphonuclear neutrophils. The intracellular free calcium concentration in single adherent human neutrophils was monitored during bacterial infection and, in parallel, the encounter between the bacteria and cells was observed. When a plasmid-cured strain was used for infection, adherence of a single bacterium to the cellular surface induced a β 1 integrin-dependent transient increase in the intracellular concentration of free calcium. This was, however, not seen with Yop-expressing wild-type bacteria, which adhered to the cell surface without generating any Ca 2+ signal. Importantly, the overall Ca 2+ homeostasis was not affected by the wild-type strain; the Ca 2+ signal mediated by the G-protein-coupled formyl-methionyl-leucyl- phenylalanine receptor was still functioning. Hence, the blocking effect was restricted to certain receptors and their signaling pathways. The use of different Yop mutant strains revealed that the protein tyrosine phosphatase YopH was responsible for the inhibition. This virulence determinant has previously been implicated in very rapid Yersinia-mediated effects on target cells as the key effector in the blockage of phagocytic uptake. The present finding, that Y. pseudotuberculosis, via YopH, specifically inhibits a self- induced immediate-early Ca 2+ signal in neutrophils, offers more-detailed information concerning the effectiveness of this virulence effector and implies an effect on Ca 2+-dependent, downstream signals.

  • 6.
    Asplund Persson, Anna
    et al.
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, Peter
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Larsson, Jenny
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    The nitrosoamine dephostatin interacts with nitric oxide/cGMP signalling and modulates cytosolic calcium responses in human plateletsManuscript (preprint) (Other academic)
    Abstract [en]

    1 The effects of the nitrosoamine dephostatin on cytosolic Ca2+ and nitric oxide (NO)/cGMP signallings in human platelets were investigated.

    2 Dephostatin has been characterised as a protein tyrosine phosphatase (PTP) inhibitor, and may on that account affect platelet responses. However, western blot analysis revealed that dephostatin (1-100 µM) did not increase tyrosine-specific protein phosphorylation.

    3 Dephostatin dose-dependently (0.003-3 µM) inhibited thrombin-, thrombin-receptor activating peptide-, and ADP-stimulated rises in cytosolic free Ca2+ concentration, [Ca2+]i. in fura-2-loaded platelets. Surprisingly, higher doses of dephostatin (10-30 µM) augmented thrombin-triggered Ca2+ response. This dual action of dephostatin mainly involved modulation of Ca2+ influx.

    4 The results revealed that dephostatin antagonised NO/cGMP- and prostaglandin E1/cAMP-mediated inhibition of [Ca2+]1. The action of the thrornbin-neutralising peptide hirudin was, however, unaffected.

    5 Dephostatin did not affect basal or NO-mediated rises in platelet cGMP content. On the other hand, dephostatin alone directly increased the phosphorylation of ser239 on vasodilator-stimulated phosphoprotein (VASP) and markedly enhanced NO/cGMP-dependent VASP phosphorylation.

    6 Cell functional studies revealed that dephostatin amplified NO-induced inhibition of platelet aggregation. Opposite to that, dephostatin diminished the inhibitory action of NO on phosphatidylserine exposure.

    7 In conclusion, the results revealed that dephostatin affects a wide range of mechanisms involved in Ca2+ homeostasis in platelets. These effects are apparently not due to inhibition of PTPs. However, enhancement of VASP phosphorylation represents another important molecular mechanism of dephostatin. Considering NO signalling, the results indicate that dephostatin may be an excellent tool when elucidating the relative importance of inhibition of Ca2+ versus VASP phosphorylation.

  • 7.
    Augustinsson (Nilsdotter-Augustinsson), Åsa
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Infectious Diseases. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Infectious Diseases in Östergötland.
    Frydén, Anders
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lindgren, Per-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Öhman, Lena
    Linköping University, Department of Molecular and Clinical Medicine, Infectious Diseases. Linköping University, Faculty of Health Sciences.
    Interaction of staphylococcus epidermidis from infected hip prostheses with neutrophil granulocytes2001In: Scandinavian Journal of Infectious Diseases, ISSN 0036-5548, E-ISSN 1651-1980, Vol. 33, no 6, p. 408-412Article in journal (Refereed)
    Abstract [en]

    This study focuses on the interaction of Staphylococcus epidermis isolated from granulation tissue covering infected hip prostheses and neutrophil granulocytes. Bacterial strains isolated from normal flora were used as controls. The bacteria were well characterized with routine methods and further characterized with random amplified polymorphic DNA analyses and slime tests. Phagocytosis and chemiluminescence (CL) assays were used in the neutrophil interaction studies. The prostheses strains were ingested to a lesser extent than strains from normal flora (p ≤ 0.001). There was no significant difference between the prostheses strains and the normal flora strains in terms of total CL response. However, the extracellular CL response from the neutrophils was lower in comparison with the normal flora when interacting with the prostheses strains. The results of this study support the notion that S. epidermidis strains isolated from infected hip prostheses have an enhanced capacity to resist phagocytosis and that most of these strains elicit a reduced inflammatory response, measured as the production of extracellular oxidative metabolites from the neutrophils, compared to normal flora.

  • 8. Baniulis, Danas
    et al.
    Burritt, James B
    Taylor, Ross M
    Dinauer, Mary C
    Heyworth, Paul G
    Parkos, Charles A
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Jesaitis, Algirdas J
    Monoclonal antibody CL5 recognizes the amino terminal domain of human phagocyte flavocytochrome b558 large subunit, gp91phox2005In: European Journal of Haematology, ISSN 0902-4441, E-ISSN 1600-0609, Vol. 74, no 4, p. 337-347Article in journal (Refereed)
    Abstract [en]

    Human phagocyte flavocytochrome b558 (Cytb) is a heterodimeric integral membrane protein that serves as the electron transferase of the β-nicotinamide adenine dinucleotidephosphate, reduced (NADPH)-oxidase, an enzyme complex important in the host defense function of phagocytic cells. In this study, we report the characterization of monoclonal antibody (mAb) CL5 that is specific for the large subunit, gp91phox, of the oxidase protein. This antibody recognizes gp91phox by immunoblot analysis of membrane extracts and samples of the immunopurified gp91phox/p22 phox heterodimer, prepared on anti-p22phox affinity matrices. Phage display analysis confirmed this specificity, indicating that the CL5 epitope contains the region 135-DPYSVALSELGDR of gp91phox. The antibody was used to probe for the presence of gp91phox in membrane preparations from neutrophils of patients with nine genetically distinct forms of X-linked chronic granulomatous disease (CGD). The causative mutations included missense errors as well as nonsense errors that result in premature termination of gp91phox synthesis. Analysis of the CGD samples by immunoblotting indicated that CL5 recognizes only the full-length wild-type and two missense mutations, consistent with the absence of stable short gp91 phox peptide expression in CGD neutrophils. Interestingly, CL5 was also shown to be cross-reactive with cytosolic and membrane-bound gelsolin, identified by purification, mass spectrometry and immunoblot analysis. CL5 probably cross-reacts with the sequence 771-DPLDRAMAEL in the C-terminus of gelsolin. We conclude that mAb CL5 is a useful probe for detection of full length and possibly truncated N-terminal fragments of gp91phox from membranes of Cytb-producing cells. © Blackwell Munksgaard 2005.

  • 9. Baniulis, Danas
    et al.
    Nauseef, William M
    Burritt, James B
    Taylor, Ross M
    Heyworth, Paul G
    Dinauer, Mary C
    Bumelis, Vladas A
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Jesaitis, Algirdas J
    Unusual polyclonal anti-gp91phox peptide antibody interactions with X-linked chronic granulomatous disease-derived human neutrophils are not from compensatory expression of Nox proteins 1, 3, or 42005In: European Journal of Haematology, ISSN 0902-4441, E-ISSN 1600-0609, Vol. 74, no 3, p. 241-249Article in journal (Refereed)
    Abstract [en]

    To obtain topological information about human phagocyte flavocytochrome b558 (Cytb), rabbit anti-peptide antibodies were raised against synthetic peptides mimicking gp91phox regions: 1-9 (MGN), 30-44 (YRV), 150-159 (ESY), 156-166 (ARK), 247-257 (KIS-1, KIS-2). Following affinity purification on immobilized peptide matrices, all antibodies but not prebleed controls recognized purified detergent-solubilized Cytb by enzyme-linked immunosorbent assay (ELISA). Affinity-purified antibodies recognizing KIS, ARK and ESY but not YRV, MGN or prebleed IgG specifically detected gp91phox in immunoblot analysis. Antibodies recognizing MGN, ESY, ARK and KIS but not YRV or the prebleed IgG fraction labeled intact normal neutrophils. Surprisingly, all antibodies, with the exception of YRV and pre-immune IgG controls, bound both normal and Cytb-negative neutrophils from the obligate heterozygous mother of a patient with X-linked chronic granulomatous disease (X-CGD) and all neutrophils from another patient lacking the gp91phox gene. Further immunochemical examination of membrane fractions derived from nine genetically unrelated patients with X-CGD, using an antibody that recognizes other Nox protein family members, suggests that the unusual reactivity observed does not reflect the compensatory expression of gp91phox homologs Nox1, 3 or 4. These results suggest that an unusual surface reactivity exists on neutrophils derived from X-linked chronic granulomatous disease patients that most likely extends to normal neutrophils as well. The study highlights the need for caution in interpreting the binding of rabbit polyclonal antipeptide antibodies to human neutrophils in general and, in the specific case of antibodies directed against Cytb, the need for Cytb-negative controls.

  • 10. Bengtsson, T.
    et al.
    Jaconi, ME
    Gustafsson, Mikael
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Cardiology in Linköping.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Theler, JM
    Lew, DP
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium1993In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 62, no 1, p. 19-58Article in journal (Refereed)
    Abstract [en]

    The role of changes in cytosolic free calcium concentration ([Ca2+]i) in the assembly and disassembly of actin during adhesion and phagocytosis was evaluated. Rhodamine-phalloidin staining combined with quantitative fluorescence and confocal laser scanning microscopy was used to measure local F-actin changes in single adherent human neutrophils phagocytosing yeast particles on different surfaces and under different calcium conditions. Cells were suspended in a) calcium-containing medium (CCM) or b) calcium-free medium (CFM) or c) were first depleted of calcium (i.e., MAPT/AM-loaded in CFM) and then suspended in CFM (MAPT). In parallel, local [Ca2+]i changes were monitored using a fura-2 ratio imaging system. In CCM or CFM, attachment to the substrate and formation of pseudopods around a yeast particle generated, within a few seconds, rises in [Ca2+]i, both around the phagosome and in the cell body. During continued phagocytosis, [Ca2+]i was more elevated around the phagosome compared to the rest of the cell. No [Ca2+]i fluctuations were observed in MAPT cells. Adhesion and phagocytosis led to a several-fold increase in F-actin. The increase was transient in cells in CCM and CFM, but remained high in Ca-depleted neutrophils. A distinct ring of F-actin was formed around a phagosome with a yeast particle. Twenty min after ingestion the amount of this actin decreased more than 50% in CCM and CFM cells but increased by 40 to 100% in MAPT cells. The accumulation of F-actin in MAPT cells was reduced to resting levels by adding Ca2+ and ionomycin after ingestion. This treatment reestablished the periphagosomal [Ca2+]i rises, as observed in CCM cells. In conclusion, the present study shows that the actin polymerization, occurring in human neutrophils during adhesion and phagocytosis, is not influenced by changes in [Ca2+]i, whereas the subsequent depolymerization is. The accumulation of actin filaments around the phagosome in calcium-depleted cells could be involved in the inhibition of phagolysosome fusion seen in the absence of [Ca2+]i changes (Jaconi et al., J. Cell Biol. 110, 1555-1564 (1990)). This suggests that the actin network, controlled by [Ca2+]i, regulates the movement of granules during phagocytosis.

  • 11.
    Bengtsson, Torbjörn
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Frydén, A
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Zalavary, S
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Whiss, Per A
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Orselius, Kristina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Grenegård, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Platelets enhence neutrophil locomotion: evidence for a role of P-selectin1999In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 59, p. 439-450Article in journal (Refereed)
  • 12.
    Bengtsson, Torbjörn
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Grenegård, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Grenegård, M
    Leucocyte activation by collagen-stimulated platelets in whole blood2002In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 62, no 6, p. 451-462Article in journal (Refereed)
    Abstract [en]

    Interaction between vascular cells plays an important role in the initial phases of the inflammatory process, but the mechanisms responsible for cell-cell communication are not fully understood. In this study, activation of leucocytes and platelets in heparinized whole blood was assessed using lumi-aggregometry. This technique enables simultaneous measurement of aggregation and oxygen radical production by monitoring impedance and luminol-amplified chemiluminescence (CL), respectively. Collagen induced aggregation and CL, depending on dose, and markedly enhanced subsequent aggregation and CL-response triggered by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Collagen stimulation of whole blood down- and upregulated the expression of L-selectin and CD11b, respectively. Monoclonal antibodies against sialyl LewisX and P-selectin caused a pronounced inhibition of the oxidative burst, triggered by collagen itself or by a combination of collagen and fMet-Leu-Phe. Furthermore, the Arg-Gly-Asp-Ser(RGDS)-peptide effectively inhibited collagentriggered aggregation and CL, and the subsequent enhancement of the fMet-Leu-Phe-induced responses. This suggests that fibrinogen plays a part in linking platelet GpIIb/IIIa with CD11b on the leucocyte surface. However, neither anti-CD11b nor the PI-peptide (containing the ?-chain motif in fibrinogen that interacts with CD11b) counteracted the stimulatory effects of activated platelets on leucocyte functions. The selectin- and integrin-antagonizing substances were ineffective on the CL-responses induced by fMet-Leu-Phe itself. This study suggests that, through selectin- and integrin-dependent interaction, activated platelets potentiate leucocyte aggregation and oxygen radical production, which might be important for the outcome of inflammatory reactions.

  • 13.
    Bengtsson, Torbjörn
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Orselius, Kristina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Wetterö, Jonas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Rheumatology.
    Role of the actin cytoskeleton during respiratory burst in chemoattractant-stimulated neutrophils2006In: Cell Biology International, ISSN 1065-6995, E-ISSN 1095-8355, Vol. 30, no 2, p. 154-163Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to clarify the role of the actin cytoskeleton during chemotactic peptide fMet-Leu-Phe (fMLF)-stimulated respiratory burst in human neutrophil granulocytes. Reactive oxygen species (ROS) was measured as luminol-amplified chemiluminescence (CL) and F-actin content as bodipy phallacidin fluorescence in neutrophils treated with latrunculin B or jasplakinolide, an inhibitor and activator of actin polymerization, respectively. Latrunculin B markedly decreased, whereas jasplakinolide increased, the F-actin content in neutrophils, unstimulated or stimulated with fMLF. Latrunculin B enhanced the fMLF-triggered ROS-production more than tenfold. Jasplakinolide initially inhibited the fMLF-induced CL-response, however, caused a potent second sustained phase (>400% of control). Both actin drugs triggered a substantial CL-response when added 5-25 min after fMLF. This was also valid for chemotactic doses of fMLF, where latrunculin B and jasplakinolide amplified the ROS-production 5-10 times. By using specific signal transduction inhibitors, we found that the NADPH oxidase activation triggered by destabilization of the actin cytoskeleton occurs downstream of phospholipase C and protein kinase C but is mediated by Rho GTPases and tyrosine phosphorylation. In conclusion, rearrangements of the actin cytoskeleton are a prerequisite in connecting ligand/receptor activation, generation of second messengers and assembly of the NADPH oxidase in neutrophil granulocytes. © 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

  • 14.
    Berg, Cecilia
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Mechanisms of platelet-mediated fibroblast proliferation2003Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Wound healing is a multicomponent event that involves a network of molecular and cellular crosstalk between cells, including leukocytes, platelets and fibroblasts. Despite increased knowledge over the past decades regarding the regulation of cell and tissue growth, the inter- and intracellular systems that control wound healing are incompletely understood. The platelet is a rich source of growth factors essential to natural tissue repair. In the present thesis, the role of platelets and platelet-derived factors on fibroblast proliferation was evaluated, and related to the generation of reactive oxygen species (ROS) and eicosanoids.

    We found that whole platelets, platelet-derived growth factor (PDGF), transforming growth factor-ß (TGF-ß) and sphingosine-1-phosphate (S1P) induce fibroblast proliferation. Exposure of fibroblasts to these stimuli caused an extensive intracellular production of ROS, measured as increase in dichlorofluorescein fluorescence. Both fibroblast growth and the associated ROS production were inhibited by intracellular antioxidants (N-acetyl-L-cysteine (NAC) and pyrrolidinethiocarbamate (PDTC)) and NADPH-oxidase inhibitors (diphenyleneiodonium chloride (DPI) and apocynin). Moreover, platelet-mediated fibroblast proliferation was abrogated in the presence of the sphingosine kinase inhibitor DL-threo-dihydrosphingosine, but only slightly affected by antibodies directed against PDGF and TGF-ß.

    The production of the arachidonic acid metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) in fibroblasts, analysed by HPLC, was markedly elevated in the presence of platelets. Furthermore, inhibition of phospholipase A2, by 7,7-dimethyl-5,8-eicosadienoic acid (DMDA), or 5-lipoxygenase, by 5,8,11-eicosatriynoic acid (ETI) or 5,6-dehydro arachidonic acid (5,6-dAA), decreased the platelet-induced fibroblast proliferation and formation of 5-HETE. This indicate a role for transcellular metabolism of arachidonic acid during platelet-fibroblast interaction.

    We conclude that the production of ROS and 5-HETE is crucial in the plateletmediated stimulation of fibroblast growth. These findings may represent new targets in the future therapy for a successful wound healing.

    List of papers
    1. Platelets induce reactive oxygen species-dependent growth of human skin fibroblasts
    Open this publication in new window or tab >>Platelets induce reactive oxygen species-dependent growth of human skin fibroblasts
    2003 (English)In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 82, no 11, p. 565-571Article in journal (Refereed) Published
    Abstract [en]

    A growing amount of evidence suggests that reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, regulate intracellular signalling and have a role in cell proliferation. In the present study, we show that platelets increase the mitogenic rate in human fibroblasts and that this effect was inhibited by the intracellular antioxidant N-acetyl-L-cysteine (NAC) and the NADPH-oxidase inhibitor diphenyleneiodonium chloride (DPI). The mitogenic effects of platelets were mimicked by the platelet factors platelet-derived growth factor BB-isoform (PDGF-BB), transforming growth factor β1 (TGF-β1) and sphingosine-1-phosphate (S1P). The sphingosine kinase inhibitor DL-threo-dihydrosphingosine (DL-dihydro) abrogated the platelet-induced growth, while antibodies directed against PDGF or TGF-β had modest effects. Exposure of fibroblasts to platelets, PDGF-BB, TGF-β1 or S1P caused an extensive intracellular ROS production, measured as changes in dichlorofluorescein fluorescence. This ROS production was totally inhibited by NAC, pyrrolidinethiocarbamate (PDTC), DPI and apocynin. In conclusion, the results presented are indicative of a crucial role of ROS in the platelet-mediated regulation of fibroblast proliferation.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-26383 (URN)10.1078/0171-9335-00344 (DOI)000187637800004 ()10917 (Local ID)10917 (Archive number)10917 (OAI)
    Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13
    2. Platelet-induced growth of human fibroblasts is associated with an increased expression of 5-lipoxygenase
    Open this publication in new window or tab >>Platelet-induced growth of human fibroblasts is associated with an increased expression of 5-lipoxygenase
    Show others...
    2006 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 96, no 5, p. 652-659Article in journal (Refereed) Published
    Abstract [en]

    Proliferation of fibroblasts is vital for adequate wound healing but is probably also involved in different hyperproliferative disorders such as atherosclerosis and cancer. The regeneration of tissue usually starts with coagulation, involving release of mitogenic and inflammatory factors from activated platelets. This study focuses on the role of eicosanoids in the proliferative effects of platelets on human fibroblasts. We show that the phospholipase A2 inhibitor 7,7-dimethyl-5,8-eicosadienoic acid (DMDA), the combined cyclooxygenase (COX) and lipoxygenase (LOX) inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) and the LOX inhibitor 5,8,11-eicosatriynoic acid (ETI) block the platelet-induced proliferation of serum starved subconfluent human fibroblasts. Anti-proliferative effects were also obtained by specific inhibition of 5-LOX with 5,6-dehydro arachidonic acid (5,6-dAA), whereas the 12-LOX inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) did not affect the platelet-stimulated growth of fibroblasts. The expression of 5-LOX was analyzed by reverse-transcriptase-mediated PCR (RT-PCR), Western blotting and HPLC. 5-LOX message and protein was detected in fibroblasts but not in platelets. Incubation with platelets markedly increased, already after one hour, the expression of 5-LOX in the fibroblast culture. The increased 5-LOX activity was associated with an elevated level of the 5-LOX metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) reaching its maximum after 1-2 hours of co-incubation of fibroblasts and platelets. The 5-HETE production was reduced by the inhibitors DMDA, ETYA and ETI. In conclusion, this study suggests that platelet-stimulated proliferation of fibroblasts is mediated by an increased 5-LOX activity, which supports recent findings indicating a crucial role for this enzyme in proliferative disorders such as atherosclerosis. © 2006 Schattauer GmbH, Stuttgart.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-36111 (URN)10.1160/TH06-02-0069 (DOI)000242168400016 ()29986 (Local ID)29986 (Archive number)29986 (OAI)
    Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
  • 15.
    Berg, Cecilia
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Trofast, Catarina
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Platelets induce reactive oxygen species-dependent growth of human skin fibroblasts2003In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 82, no 11, p. 565-571Article in journal (Refereed)
    Abstract [en]

    A growing amount of evidence suggests that reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, regulate intracellular signalling and have a role in cell proliferation. In the present study, we show that platelets increase the mitogenic rate in human fibroblasts and that this effect was inhibited by the intracellular antioxidant N-acetyl-L-cysteine (NAC) and the NADPH-oxidase inhibitor diphenyleneiodonium chloride (DPI). The mitogenic effects of platelets were mimicked by the platelet factors platelet-derived growth factor BB-isoform (PDGF-BB), transforming growth factor β1 (TGF-β1) and sphingosine-1-phosphate (S1P). The sphingosine kinase inhibitor DL-threo-dihydrosphingosine (DL-dihydro) abrogated the platelet-induced growth, while antibodies directed against PDGF or TGF-β had modest effects. Exposure of fibroblasts to platelets, PDGF-BB, TGF-β1 or S1P caused an extensive intracellular ROS production, measured as changes in dichlorofluorescein fluorescence. This ROS production was totally inhibited by NAC, pyrrolidinethiocarbamate (PDTC), DPI and apocynin. In conclusion, the results presented are indicative of a crucial role of ROS in the platelet-mediated regulation of fibroblast proliferation.

  • 16.
    Berglund, Johnny
    et al.
    Umeå .
    Samuelsson, Kristina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Kull, Tomas
    Umeå .
    Müren, Umut
    Umeå .
    Andersson, Agneta
    Umeå .
    Relative strength of resource and predation limitation of heterotrophic nanoflagellates in a low-productive sea area2005In: Journal of Plankton Research, ISSN 0142-7873, E-ISSN 1464-3774, Vol. 27, no 9, p. 923-935Article in journal (Refereed)
    Abstract [en]

    The magnitude of resource and predation limitation of heterotrophic nanoflagellales (HMF) was studied in two short-term enclosure experiments performed in a low-productive sea area in the northern Baltic Sea in 2001. A cross-factorial design was used to simultaneously assess the relative importance of the two factors. Resource limitation was removed by adding bacteria, and predation limitation was eliminated by selective filtration. The first experiment was performed in June just after the spring bloom decline and the second in September at the end of the productive season. Resource limitation prevailed during both experiments, contributing to 60% of the net growth increase in June and 74% in September. Removal of predators had a significant effect only in June. Evidence for simultaneous resource and predation limitation was thus found only during the post-bloom situation. The results were applied to a model on resource and predation control of HNF abundances. To evaluate seasonal differences, field data on HNF and bacteria from a whole year study were applied to the model. Except for a few occasions during spring, the model indicated prevailing resource control of HNF at two locations with slightly different productivity. © The Author 2005. Published by Oxford University Press. All rights reserved.

  • 17.
    Bertilsson, PM
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Olsson, P
    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.
    Cytokines influence mRNA expression of cytochrome P450 3A4 and MDRI in intestinal cells.2001In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 90, p. 638-646Article in journal (Refereed)
  • 18.
    Bäckman, Jenny
    et al.
    Linköping University, Department of Physics, Chemistry and Biology.
    Kasimir Klemedtsson, Åsa
    Klemedtsson, Leif
    Lindgren, Per-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Clear-cutting affects the ammonia-oxidising community differently in limed and non-limed coniferous forest soils2004In: Biology and Fertility of Soils, ISSN 0178-2762, E-ISSN 1432-0789, Vol. 40, no 4, p. 260-267Article in journal (Refereed)
    Abstract [en]

    The effects of clear-cutting on the ammonia-oxidising bacterial community were studied in the soil of limed and non-limed spruce forest plots located in the central part of southern Sweden. The communities were studied using denaturing gradient gel electrophoresis (DGGE) profiling after polymerase chain reaction (PCR) amplification from total DNA with primers reported to be specific for β-subgroup ammonia-oxidising bacteria. The bands on the DGGE were sequenced and each unique sequence was interpreted as representing one ammonia-oxidising population. The relative abundance of each population was determined by measuring the fluorescence of the respective DGGE bands. In both limed and non-limed soil, the same two Nitrosospira populations were found, one belonging to cluster 2 (NScl2) and one to cluster 4 (NScl4). However, while NScl4 first appeared a year after the clear-cutting in the non-limed plot, it was present both before and after the cutting in the limed plot. Irrespective of previous liming, clear-cutting caused a shift in the ammonia-oxidiser community, from dominance by the NScl2 population to a community with approximately equal relative abundance of NScl2 and NScl4. In both plots the total size of the community increased after clear-cutting (based on increased DGGE band intensity), most likely due to increased NH4+ availability, but the growth response was faster in the limed plot. Hence, the prior liming increased the responsiveness of the ammonia-oxidisers to the changes caused by cutting. This is the first study to report the effects of clear-cutting on the ammonia-oxidising community, and the results show a clear correlation between increased potential nitrification and a shift in the ammonia-oxidiser community.

  • 19. Connolly-Andersen, AM
    et al.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Mirazimi, A
    Basolateral entry and release of Crimean-Congo hemorrhagic fever virus in polarized MDCK-1 cells2007In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 81, no 5, p. 2158-2164Article in journal (Refereed)
    Abstract [en]

    Crimean-Congo hemorrhagic fever virus (CCHFV) is an etiological agent of a disease with mortality rates in patients averaging 30%. The disease is characterized by fever, myalgia, and hemorrhage. Mechanisms underlying the hemorrhage have to our knowledge not been elucidated for CCHFV. Possibly, a direct or indirect viral effect on tight junctions (TJ) could cause the hemorrhage observed in patients, as TJ play a crucial role in vascular homeostasis and can cause leakage upon deregulation. Moreover, there is no knowledge regarding the site of entry and release of CCHFV in polarized epithelial cells. Such cells represent a barrier to virus dissemination within the host, and as a site of viral entry and release, they could play a key role in further spread. For the first time, we have shown preferential basolateral entry of CCHFV in Madin-Darby canine kidney 1 (MDCK-1) epithelial cells. Furthermore, we demonstrated basolateral release of CCHFV in polarized epithelial cells. Interestingly, by measuring transepithelial electrical resistance, we found no effect of CCHFV replication on the function of TJ in this study. Neither did we observe any difference in the localization of the TJ proteins ZO-1 and occludin in CCHFV-infected cells compared to mock-infected cells. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

  • 20. Constantin, G
    et al.
    Majeed, Meytham
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Giagulli, C
    Piccio, L
    Kim, JY
    Butcher, EC
    Laudanna, C
    Chemokines trigger immediate beta2 integrin affinity and mobility changes: Differential regulation and roles in lymphocytes arrest under flow.2000In: Immunity, ISSN 1074-7613, E-ISSN 1097-4180, Vol. 13, p. 759-769Article in journal (Refereed)
  • 21.
    Dahlfors, Gunilla
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Arnqvist, Hans J.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Effect of nitric oxide on vascular smooth muscle cell proliferation and insulin-like growth factor binding protein expressionManuscript (preprint) (Other academic)
    Abstract [en]

    A possible interaction between nitric oxide (NO) and the insulin-like growth factor (IGF)-system was studied in cultured rat aortic smooth muscle cells. The NO-donor SNAP markedly inhibited basal and sernm-induced DNA synthesis while addition of L-NAME, an inhibitor of endogenous NO production, had no effect. L-NAME did also not significantly affect IGF-I, angiotensin II or TGF-ß1- induced effects on DNA synthesis. IGF-I was shown to stimulate the expression of IGFBP-4 mRNA, as measured by an RNase-protection assay, and angiotensin II inhibited expression of IGFBP-2 mRNA. Addition of L-NAME did not significantly change the effect of IGF-I or angiotensin II on IGFBP mRNA expression, neither did L-NAME or SNAP affect basal expression of IGFBP-2, -4 or -6 mRNA. In conclusion, we found no evidence for interaction of NO with the IGF-system in smooth muscle cells. Nitric oxide did not regulate the expression of IGFBPs and IGF-I-induced smooth muscle cell proliferation was not affected by inhibition of endogenous NO production.

  • 22.
    Favre, Cécile J.
    et al.
    Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
    Jerström, Petra
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Foti, Michelangelo
    Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Huggler, Elzbieta
    Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
    Lew, Daniel P.
    Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
    Krause, Karl-Heinz
    Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
    Organization of Ca2+ stores in myeloid cells: association of SERCA2b and the type-1 inositol-1,4,5-trisphosphate receptor1996In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 316, no 1, p. 137-142Article in journal (Refereed)
    Abstract [en]

    In this study, we have analysed the relationship between Ca2+ pumps and Ins(1,4,5)P3-sensitive Ca2+ channels in myeloid cells. To study whether sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-type Ca2+-ATPases are responsible for Ca2+ uptake into Ins(1,4,5)P3-sensitive Ca2+ stores, we used the three structurally unrelated inhibitors thapsigargin, 2,5-di-t-butylhydroquinone and cyclopiazonic acid. In HL-60 cells, all three compounds precluded formation of the phosphorylated intermediate of SERCA-type Ca2+-ATPases. They also decreased, in parallel, ATP-dependent Ca2+ accumulation and the amount of Ins(1,4,5)P3-releasable Ca2+. Immunoblotting with subtype-directed antibodies demonstrated that HL-60 cells contain the Ca2+ pump SERCA2 (subtype b), and the Ca2+-release-channel type-1 Ins(1,4,5)P3 receptor. In subcellular fractionation studies, SERCA2 and type-1 Ins(1,4,5)P3 receptor co-purified. Immunofluorescence studies demonstrated that both type-1 Ins(1,4,5)P3 receptor and SERCA2 were evenly distributed throughout the cell in moving neutrophils. During phagocytosis both proteins translocated to the periphagosomal space. Taken together, our results suggest that in myeloid cells (i) SERCA-type Ca2+-ATPases function as Ca2+ pumps of Ins(1,4,5)P3-sensitive Ca2+ stores, and (ii) SERCA2 and type-1 Ins(1,4,5)P3 receptor reside either in the same or two tightly associated subcellular compartments.

  • 23.
    Filbey, Derek
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Alloimmunization during pregnancy with special emphasis on anti-D: Laboratory and clinical management1995Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The laboratory and clinical management of alloimmunized in pregnancy has been investigated according to a protocol currently in use in Örebro region. A 12 year epidemiological study showed the prevalence of alloimmunization to be 0.57% in this Swedish populationwith a 0.24% incidence of clinically significant antibodies that can induce haemolytic disease of the newborn (HDN). Rh antibodies, predominantly anti-D, are still the causes of most cases of severe HDN in which 45/47 babies required exchange transfusion. During the studyperiod, 14 mothers were successfully treated with plasma exchange during pregnancy owing to high anti-D antibody concentrations. Only two other blood group syswms, Kell and Duffy, besides Rh affected newborns to alloimmunized mothers to such a grade that exchange transfusion of the newborns was necessary. All generally accepted for the fetus clinically nonsignificant antibodies were also followed and shown not to cause HDN. In 3 instances, anti-D was detected in partial RhO-positive mothers who were carrying normal RhO-positive fetuses,a study to identify these partial RhD individuals and to group them into D-categories was performed. The ability of the indirect antiglobulin titre (IAT), AutoAnalyzer (AA) quantitation and chemiluminescence l£st (CLT) performed on maternal anti-D serum during pregnancy to discriminal£ babies affected or unaffected by HDN has been studied. It was found that all methods had their weaknesses, but AA-quantitation and CLT improved speeifieity when compared to the IAT-titration method. However, a great improvement was achieved when the results of IAT-titration and AA-quantitation, as determined by the cut-offlimits applied to discriminate unaffected from affected newborns, were combined; specificity was then found to increase from 60-78% to 93% and was further increased with the addition of the CLT test to 95%. Finally, the detection of HLA class II monocyte-reactive antibodies and their potential protective role in ameliorating HDN has been viewed.

  • 24.
    Filippini, Daniel
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Tejle, Katarina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    ELISA test for anti-neutrophil cytoplasm antibodies detection evaluated by a computer screen photo-assisted technique2005In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 21, no 2, p. 266-272Article in journal (Refereed)
    Abstract [en]

    The computer screen photo-assisted technique (CSPT), a method for substance classification based on spectral fingerprinting, which involves just a computer screen and a web camera as measuring platform is used here for the evaluation of a prospective enzyme-linked immunosorbent assay (ELISA). A anti-neutrophil cytoplasm antibodies (ANCA-ELISA) test, typically used for diagnosing patients suffering from chronic inflammatory disorders in the skin, joints, blood vessels and other tissues is comparatively tested with a standard microplate reader and CSPT, yielding equivalent results at a fraction of the instrumental costs. The CSPT approach is discussed as a distributed measuring platform allowing decentralized measurements in routine applications, whereas keeping centralized information management due to its natural network embedded operation. © 2004 Elsevier B.V. All rights reserved.

  • 25.
    Follin, Per
    Linköping University, Department of Molecular and Clinical Medicine, Infectious Diseases. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    The primed neutrophil: a friend or a foe in inflammation1991Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Human neutrophils are the most abundant of the white blood cells in circulation and represent the first line of defense against invading microorganisms. With a membrane-bound enzyme system (the NADPH oxidase), these cells can generate reactive oxygen metabolites that serve efficiently in antimicrobial defense. Neutrophils are normally dormant in the circulation but may become primed; in that state they can produce an enhanced respiratory burst response upon activation and thereby strengthen the immune response.

    During bacterial infections, endogenous inflammatory mediators orbacterial products induce metabolic priming of neutrophils, which thenexpose an increased number of receptors to the peptide f-Meth-Leu-Phe(fMLP). There is, however, no correlation between the increased level ofrespiratory burst response and the level of receptor upregulation, indicating that post-receptor events in the activation sequence are also involved. Neutrophils isolated from an inflammatory focus were found tobe metabolically deactivated as far as the agonists NAP-1/IL 8 and C5awere concerned but primed in relation to tMLP. Further characterizationof exudated cells revealed that the mechanism of priming involves protein kinase C but not a rise in intracellular Ca2+ or a decreased inactivation rate of the oxidase. In primed cells most of the increased production of reactive oxygen species induced by fMLP is located intracellularly, whereas, an increased extracellular release of reactive oxygen species occurs during phagocytosis. The fact that primed cells can both produce and, under certain conditions, release increased amounts of hydrogen peroxide raises the question of whether the primed cell is a friend or a foe in the inflammatory reaction.

  • 26.
    Forsberg, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Blomgran, Robert
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lem, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Särndahl, Eva
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Sebti, Said M.
    Drug Discovery Program, H. Lee Moffitt Cancer Center & Research Institute, Department of Oncology, University of South Florida, Tampa.
    Hamilton, Andrew
    Department of Chemistry, Yale University, New Haven, Connecticut .
    Stendahl, Olle
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Zheng, Limin
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages: potential role of Rho–GTPases and Akt2003In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 74, no 4, p. 620-629Article in journal (Refereed)
    Abstract [en]

    In addition to direct activation of caspase-1 and induction of apoptosis by SipB, invasive Salmonella stimulates multiple signaling pathways that are key regulators of host cell survival. Nevertheless, little is known about the relative contributions of these pathways to Salmonella-mediated death of macrophages. We studied human monocytic U937 cells and found that apoptosis was induced by invading wild-type Salmonella typhimurium but not by phagocytosed, serum-opsonized, noninvasive Salmonella mutants. Pretreating U937 cells with inhibitors of tyrosine kinases or phosphatidylinositol-3 kinase (PI-3K) completely blocked phagocytosis of opsonized Salmonella mutants but did not affect invasion by wild-type Salmonella or the apoptosis caused by invasion. However, pretreatment with GGTI-298, a geranylgeranyltransferase-1 inhibitor that prevents prenylation of Cdc42 and Rac1, suppressed Salmonella-induced apoptosis by ∼70%. Transduction of Tat fusion constructs containing dominant-negative Cdc42 or Rac1 significantly inhibited Salmonella-induced cell death, indicating that the cytotoxicity of Salmonella requires activation of Cdc42 and Rac. In contrast to phagocytosis of opsonized bacteria, invasion by S. typhimurium stimulated Cdc42 and Rac1, regardless of the activities of tyrosine- or PI-3K. Moreover, Salmonella infection activated Akt protein in a tyrosine-kinase or PI-3K-dependent manner, and a reduced expression of Akt by antisense transfection rendered the cells more sensitive to apoptosis induced by opsonized Salmonella. These results indicate that direct activation of Cdc42 and Rac1 by invasive Salmonella is a prerequisite of Salmonella-mediated death of U937 cells, whereas the simultaneous activation of Akt by tyrosine kinase and PI-3K during receptor-mediated phagocytosis protects cells from apoptosis.

  • 27.
    Forsberg, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Druid, Pia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Zheng, Limin
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Särndahl, Eva
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Activation of Rac2 and Cdc42 on Fc and complement receptor ligation in human neutrophils2003In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 74, no 4, p. 611-619Article in journal (Refereed)
    Abstract [en]

    Phagocytosis is a complex process engaging a concerted action of signal-transduction cascades that leads to ingestion, subsequent phagolysosome fusion, and oxidative activation. We have previously shown that in human neutrophils, C3bi-mediated phagocytosis elicits a significant oxidative response, suggesting that activation of the small GTPase Rac is involved in this process. This is contradictory to macrophages, where only Fc receptor for immunoglobulin G (FcγR)-mediated activation is Rac-dependent. The present study shows that engagement of the complement receptor 3 (CR3) and FcγR and CR3- and FcγR-mediated phagocytosis activates Rac, as well as Cdc42. Furthermore, following receptor-engagement of the CR3 or FcγRs, a downstream target of these small GTPases, p21-activated kinase, becomes phosphorylated, and Rac2 is translocated to the membrane fraction. Using the methyltransferase inhibitors N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine, we found that the phagocytic uptake of bacteria was not Rac2- or Cdc42-dependent, whereas the oxidative activation was decreased. In conclusion, our results indicate that in neutrophils, Rac2 and Cdc42 are involved in FcR- and CR3-induced activation and for properly functioning signal transduction involved in the generation of oxygen radicals.

  • 28.
    Forsberg, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Löfgren, Ragnhild
    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.
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Tumour necrosis factor-α potentiates CR3-induced respiratory burst by activating p38 MAP kinase in human neutrophils2001In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 103, no 4, p. 465-472Article in journal (Refereed)
    Abstract [en]

    CR3 and FcγRs are the main receptors involved in the phagocytic process leading to engulfment and killing of microbes by production of reactive oxygen intermediates (ROI) and degranulation. Various inflammatory mediators, such as tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS), are known to prime neutrophils leading to increased bactericidal responses, but the underlying mechanism of priming has only been partially elucidated. The purpose of this study was to investigate how TNF-α primes neutrophils for subsequent stimuli via either CR3 or FcγR. The receptors were specifically activated with pansorbins (protein-A-positive Staphylococcus aureus) coated with anti-CR3, anti-FcγRIIa, or anti-FcγRIIIb monoclonal antibody. Activation of neutrophils with these particles resulted in ROI production as measured by chemiluminescence. Anti-CR3 pansorbins induced the most prominent ROI production in neutrophils. TNF-α potentiated the CR3-mediated respiratory burst but had little effect on that mediated by FcγRs. The priming effect of TNF-α on CR3-mediated ROI production is associated with an increased activation of p38 MAPK as well as tyrosine phosphorylation of p72syk. Pretreatment of neutrophils with the inhibitors for p38 MAPK and p72syk markedly suppressed the respiratory burst induced by CR3. Furthermore, TNF-α induced about a three-fold increase in the expression of CR3 in neutrophils, an effect which is blocked by the p38 MAPK inhibitor. Taken together, these results showed that TNF-α potentiates the CR3-mediated respiratory burst in neutrophils not only by triggering a p38 MAPK-dependent up-regulation of CD11b/CD18 but also by modulating the signalling pathways.

  • 29.
    Forslund, Tony
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Nitric oxide modulates neutrophil function1997Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    An important role of the neutrophils is to attack and destroy microbial intruders. In order to do so, these leukocytes must adhere to the blood vessel wall, pass through the endothelial cell layer, move through the tissues to the point of infection, ingest the intruder and destroy it.

    The aim of this thesis was to study the effects of nitric oxide (NO) on the different steps in the inflammatory response of the neutrophil.

    Adhesion to a collagen surface was decreased by pretreatment with a NO-releasing substance, and this was mimicked by pretreatment with a functional cGMP-analogue, indicating a role of the NO/cGMP pathway. Homotypic adhesion, aggregation, was not affected by external NO, but when the precursor of endogenous NO-production CLarginine) was added, the aggregation increased. Measurements of total F-actin content in cells showed that a NO-releasing substance decreased the total amount of F-actin, while cGMP increased it. Treatment with L-arginine had no effect.

    Phagocytosis was neither affected by endogenous NO, nor by a NO-releasing substance. However, if the prey itself released NO, both the adhesion to and phagocytosis of the target was decreased. This NO-particle also inhibited the production of oxygen metabolites, as measured using Luminal-dependent chemiluminescence. The inhibition was almost exclusively affecting the intracellular production of oxygen metabolites, as could be seen when neutrophils were stimulated with FMLP or PMA after an incubation with the NO-releasing particles.

    Treatment of neutrophils with the NO-releasing substance nitroprusside resulted in a decrease of the respiratory burst. It was primarily the extracellular release that was diminished. This effect can be explained by an inhibition of the enzyme producing oxygen metabolites. Endogenous NO-production increased the chemiluminescence whereas an inhibitor of the NO-synthase decreased it. A possible explanation for these effects is an NO-inhibition of the protection against hydrogen peroxide, i.e. catalase activity, resulting in an increased amount of oxygen metabolites.

    In conclusion, these results shows that NO has different effects depending on where it is produced and in what quantities. It modulates the different steps of the inflammatory response of neutrophils. Control of NO-formation could therefore be a way to control inflammation.

  • 30.
    Forslund, Tony
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Nilsson, Harriet
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Nitric Oxide Regulates the Aggregation of Stimulated Human Neutrophils2000In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 274, no 2, p. 482-487Article in journal (Refereed)
    Abstract [en]

    Neutrophil aggregation is mediated by both CD18 integrin and L-selectin. Nitric oxide attenuates the integrin-mediated adhesion of neutrophils to collagen and to endothelium and may therefore affect aggregation as well. FMLP-stimulated neutrophils exposed to -arginine showed increased and prolonged aggregation, whereas cells pretreated with L-NAME did not differ from FMLP-stimulated controls. Nitric oxide is known to induce ADP ribosylation of G-actin, which inhibits polymerization. We detected equivalent levels of total F-actin in cells pretreated with -arginine or L-NAME and non-pretreated controls. However, neutrophils pretreated with -arginine and stimulated by CD18 integrin cross-linking exhibited a more limited increase in total F-actin, compared to control and L-NAME-pretreated cells. Thus at least two signaling pathways may be involved FMLP-stimulated aggregation, mediated by CD18 integrins. More specifically, it is plausible that FMLP-receptor signaling upregulates CD18 integrins and endogenous NO subsequently modulates CD18-mediated signaling to prolong aggregation, possibly through ADP-ribosylation of actin.

  • 31.
    Gustafsson, Mikael
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Motility of human polymorphonuclear leukocytes: An image processing approach1993Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cell motility is essential for polymorphonuclear leukocyte (PMN) function in the defense against invading microorganisms and in inflammatory processes. Using digital video microscopy and image processing, individual cells were studied during random locomotion, chemotactic locomotion and phagocytosis. When PMN moved in a specific direction, the lipid membrane was found to flow in the same direction, contradicting the rearward lipid flow model of cell locomotion. This was assessed with video photobleaching of a fluorescent lipid probe, Dii. The motility of PMN could not be blocked by ca2+ depletionand /or inhibition of myosin light chain kinase, suggesting that ea2+ and myosin are not necessary for locomotion. Temporal and spatial characteristics of intracellular free ea2+ and intracellular pH were assessed by loading the cells with esters of Fura-2 and the fluorcscein derivative BCECF, respectively. There was a distinct correlation between the direction of PMN locomotion and the slope of the Ca2+ gradient of the cells. PMN moving randomly on a surface often exhibited a rearward gradient with higher ca2+ concentration in the rear of the cell. Experimental reversal of the gradient, however, did not change the direction of motility. During phagocytosis of yeast particles, Saccharomyces cerevisiae, intracellular free calcium rose within seconds after contact with the prey. Intracellular pH varied between 7.1 and 7.3 and was uniform across the cells. After phagocytosis, phagosomal pH first decreased and then returned to neutraL A role for pH in phagosome-lysosome formation is proposed. PMN loaded with self-quenching concentrations of BCECF exhibited a strict correlation between pseudopod protrusions and increase in fluorescence, indicating water influx and dilution of the probe. This finding may be essential for the understanding of actin cytoskeleton dynamics during cell locomotion.

  • 32.
    Gustafsson, Mikael
    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.
    A distributed image-processing system for measurements of intracellular calcium in living cells1991In: Computer Methods and Programs in Biomedicine, ISSN 0169-2607, E-ISSN 1872-7565, Vol. 36, no 4, p. 199-221Article in journal (Refereed)
    Abstract [en]

    During the last decade, image-processing techniques have been introduced as a valuable tool in biologically oriented research. In combination with novel fluorescent probes, these techniques permit assessment of subcellular distributions of several intracellularly important cations, such as free calcium ions and protons. Typically, systems used for image processing are located centrally around the experimental setup. This configuration has drawbacks, mainly because the laborious extraction and processing of data that generally follow an experimental session limits the access to the system for other investigators. We describe here the principles of a distributed image processing system, based on IBM-compatible personal computers (PCs), that without extra hardware can cope with all the necessary image processing involved in imaging of intracellular cations. The potential of the PC as an image processor, however, reaches beyond this specific application and many image processing tasks can be carried out successfully on a standard PC. Thus, the centrally located dedicated image processor is used only for image acquisition in the experimental situation. This in turn optimizes the utilization of expensive resources and increases efficiency. The mouse-operated software is described in detail, so that interested investigators can extract useful parts for integration into their own applications and experimental environment.

  • 33.
    Gustafsson, Mikael
    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.
    A novel principle for quantitation of fast intracellular calcium changes using Fura-2 and a modified image processing system: applications in studies of neutrophil motility and phagocytosis1992In: Cell Calcium, ISSN 0143-4160, E-ISSN 1532-1991, Vol. 13, no 8, p. 473-486Article in journal (Refereed)
    Abstract [en]

    A new principle is described for imaging intracellular free calcium [Ca2+]i changes in single, living cells utilizing the fluorescent probe Fura-2. It is based upon video color mixing in real time and allows high-speed visualization, at maximum image resolution, of [Ca2+]i changes without digital image ratioing. The epifluorescence images produced by 340 and 380 nm excitations are stored in two memory buffers of a personal computer-based image processing system. Two video signals are generated independently from each buffer and connected to the red and green inputs of a video display. An image is this way created, in which [Ca2+]i shows up as a specific hue, whereas changes in dye concentration, light intensity, cell thickness show up as variations in brightness of the imaged cells. The method has advantages over conventional ratio imaging, notably simplicity and speed, since no calculations are made. Yet it can be combined with traditional digital image processing. The imaging technique allows monitoring of [Ca2+]i changes in rapidly moving cells, like neutrophils. It is demonstrated that during random locomotion on serum-coated glass surfaces, [Ca2+]i levels appeared to oscillate and that the frequency of the oscillations are related to locomotive activity. Furthermore, in Ca2+ free medium, the cells continue to move and phagocytose in the presence of Ca2+ ionophore (ionomycin) and 2 mM EGTA. In the presence of 1 mM extracellular Ca2+, ionomycin-treated cells were not able to move or phagocytose.

  • 34.
    Gustafsson, Mikael
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Cardiology in Linköping.
    Sundqvist, Tommy
    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.
    Lateral diffusion of the secretory component (SC) in the basolateral membrane of the human colon carcinoma cell line HT29 assessed with fluorescence recovery after photobleaching1988In: Journal of Cellular Physiology, ISSN 0021-9541, E-ISSN 1097-4652, Vol. 137, no 3, p. 608-611Article in journal (Refereed)
    Abstract [en]

    The lateral diffusion of the secretory component (SC), acting as a receptor for dimeric IgA in the basolateral side of intestinal epithelial cells, was studied in the human colonic carcinoma cell line HT29. The HT29 cells were grown in Dulbecco's modified Eagle's medium in which galactose had been substituted for glucose to promote development of small intestine-like cells, with a distinct separation of the basolateral side from the apical surface. The SC was stained with rhodamine-labeled polyclonal anti-human SC rabbit antibodies (Ig) or Fab fragments, and the lateral mobility was assessed with the fluorescence recovery after photobleaching technique. The average lateral diffusion was consistent with a diffusion constant of 7.7 ± 2.0 (mean value ± SD; n = 29) and 7.1 ± 2.3 (n = 30) × 10−10 cm2s−1 for Ig-and Fab-labeled receptors, respectively, which is slower than lipid diffusion but is similar to that found for other membrane receptors. The corresponding values for the fraction of mobile receptors were 66 ± 13% and 71 ± 12%, respectively. Cells were labeled from the top of the culture plate, and cells adjacent to a mechanically made rift or a natural opening in the cell monolayer were labeled more strongly, confirming the microscope-based impression that the basolateral surface primarily harboured the SC receptor.

  • 35.
    Gustavsson, Johanna
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Parpal, Santiago
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Karlsson, Margareta
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Ramsing, Cecilia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Thorn, Hans
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Borg, Marie
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Lindroth, Margaretha
    Linköping University, Department of Biomedicine and Surgery, Cell biology. 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.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Localization of the insulin receptor in caveolae of adipocyte plasma membrane1999In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 13, no 14, p. 1961-1971Article in journal (Refereed)
    Abstract [en]

    The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with β-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. β-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.

  • 36. Hallin, S
    et al.
    Lindgren, Per-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    PCR detection of genes encoding nitrite reductase in denitryifying bacteria.1999In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 65, p. 1652-1657Article in journal (Refereed)
  • 37. Hallin, S
    et al.
    Lydmark, P
    Kokalj, S
    Hermansson, M
    Sorensson, F
    Jarvis, A
    Lindgren, Per-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Community survey of ammonia-oxidizing bacteria in full-scale activated sludge processes with different solids retention time2005In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 99, no 3, p. 629-640Article in journal (Refereed)
    Abstract [en]

    Aims: To study the effects of different solids retention time (SRT) on the nitrification activity and community composition of ammonia-oxidizing bacteria (AOB) in two full-scale activated sludge processes during a 5-month period. Methods and Results: The AOB community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT reduced the nitrification activity, but neither the number per mixed liquor suspended solids nor community composition of AOB were affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. Conclusions: The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition. Significance and Impact of the Study: Physiological alterations of a stable AOB community are possible and may stabilize activated sludge processes. The commonly used FISH probes designed to target all beta-proteobacterial AOB does not detect certain Nitrosomonas oligotropha populations, leading to an underestimation of AOB if a wider set of probes is not used. © 2005 The Society for Applied Microbiology.

  • 38. Hamid, N
    et al.
    Gustavsson, A
    Andersson, K
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    McGee, K
    Persson, C
    Rudd, CE
    Fällman, M
    YopH dephosphorylates Cas and Fyn-binding protein in macrophages. 1999In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 27, p. 231-242Article in journal (Refereed)
  • 39.
    Hammarström, Sven
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Trinks, Cecilia
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Wigren, Jane
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Surapureddi, S
    Söderström, Mats
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Glass, CK
    Novel eicosanoid activators of PPAR? formed by raw 264.7 macrophage cultures2002In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 507, p. 343-349Article in journal (Refereed)
    Abstract [en]

    [No abstract available]

  • 40.
    Hansson, Kenny
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Tosatti, Samuele
    Isaksson, Joakim
    Linköping University, The Institute of Technology. Linköping University, Department of Science and Technology.
    Wetterö, Jonas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Textor, Marcus
    Lindahl, Tomas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of clinical chemistry. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
    Tengvall, Pentti
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics.
    Whole blood coagulation on protein adsorption-resistant PEG and peptide functionalised PEG-coated titanium surfaces2005In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 26, no 8, p. 861-872Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to investigate whole blood coagulation on low blood plasma protein adsorbing surfaces. For this purpose, the polycationic graft copolymer poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), PLL-g-PEG grafted with a cell adhesive peptide containing the amino acid sequence -Arg-Gly-Asp- (RGD), and PLL-g-PEG with a control peptide -Arg-Asp-Gly- (RDG) were adsorbed onto titanium (oxide), forming stable monomolecular adlayers through electrostatic attraction. Free oscillation rheometry and complementary techniques were used to measure the coagulation time (CT) and other interactions of the surfaces with native whole blood, recalcified platelet-rich plasma (PRP), and recalcified citrated platelet-free plasma (PFP). The results show that the uncoated titanium surfaces (reference) activated platelets and quickly triggered the coagulation cascade via the intrinsic pathway, whereas the PLL-g-PEG surfaces displayed a prolonged CT, approximately 2-3 times longer compared to uncoated titanium. We hypothesise that blood coagulates outside the vascular system independent of low protein adsorption to or activation by surfaces, due to the absence of an active down-regulation of procoagulative processes by the vascular endothelium.

  • 41. Herbertsson, H
    et al.
    Bengtsson, Torbjörn
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Role of platelets and the arachidonic acid pathway in the regulation of neutrophil oxidase activity2001In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 61, no 8, p. 641-649Article in journal (Refereed)
    Abstract [en]

    The intercellular mechanisms involved in platelet-mediated regulation of neutrophil function remain incompletely understood. This study investigated the role of the arachidonic acid pathway in the modulation of chemoattractant-induced production of oxygen metabolites, measured as luminol-amplified chemiluminescence (CL). We demonstrate that platelets dose-dependently inhibit the CL response in neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Incubation with eicosatetrayonic acid (ETYA), a combined cyclooxygenase and lipooxygenase inhibitor, dramatically decreased the fMLP-induced CL response in neutrophils, an effect that was further enhanced in the presence of platelets. The separate effects of eicosatriyonic acid (ETI) and indomethacin, specific inhibitors of lipoxygenase and cyclooxygenase, respectively, were significantly lower compared to the action of ETYA. On the contrary, impediment of arachidonic acid release with the phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone (ATK) markedly increased the production of oxygen radicals triggered by fMLP. The addition of exogenous arachidonic acid clearly decreased the fMLP-induced CL response in neutrophils, which further strengthens a downregulating effect of arachidonic acid on oxidase activity. This inhibitory action of arachidonic acid, however, was reversed upon co-incubation with platelets. In conclusion, this study suggests that an accumulation of arachidonic acid, following chemotactic peptide stimulation, turns off neutrophil oxidase activity. Furthermore, platelets may support the synthesis of reactive arachidonic acid metabolites, which modulate oxygen radical production in neutrophils.

  • 42. Hermansson, A
    et al.
    Hermansson, A
    Lindgren, Per-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Quantification of ammonia-oxidizing bacteria in arable soil by real-time PCR2001In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 67, no 2, p. 972-976Article in journal (Refereed)
    Abstract [en]

    Real-time PCR was used to quantify populations of ammonia-oxidizing bacteria representing the ▀ subdivision of the class Proteobacteria in samples of arable soil, both nitrogen fertilized and unfertilized, from Mellby, Sweden. Primers and probes targeting a 16S ribosomal DNA region of the ammonia-oxidizing bacteria were designed and used. In the fertilized soil there were ~6.2 x 107 ammonia-oxidizing bacteria per g of soil, three times more than the number of bacteria in the unfertilized soil. The lyric efficiency of bead beating in these soils was investigated by using populations of free or loosely attached bacteria, bacteria tightly bound to particles, and bacteria in nonfractionated samples. The shapes of the curves generated in these tests showed that the concentration of template DNA released at various times remained constant after 10 to 100 s of bead beating.

  • 43. Hermansson, Anna
    et al.
    Bäckman, Jenny
    Linköping University, Department of Physics, Chemistry and Biology.
    Svensson, Bo
    Linköping University, The Tema Institute.
    Lindgren, Per-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Quantification of ammonia-oxidising bacteria in limed and non-limed acidic coniferous forest soil using real-time PCR2004In: Soil Biology and Biochemistry, ISSN 0038-0717, E-ISSN 1879-3428, Vol. 36, no 12, p. 1935-1941Article in journal (Refereed)
    Abstract [en]

    Ammonia-oxidising bacteria (AOB) in limed and non-limed acidic coniferous forest soil were investigated using real-time PCR. Two sites in southern Sweden were studied, 244 Åled and Oxafällan. The primers and probe used earlier appeared to be specific to the 16S rRNA gene of AOB belonging to the β-subgroup of the Proteobacteria [Appl. Environ. Microbiol. 67 (2001) 972]. Plots treated with two different doses of lime, 3 or 6 t ha-1, were compared with non-limed control plots on two occasions during a single growing season. Three different soil depths were analysed to elucidate possible differences in the density of their AOB communities. The only clear effect of liming on the AOB was recorded in the beginning of the growing season at 244 Åled. In samples taken in April from this site, the numbers of AOB were higher in the limed plots than in the control plots. At the end of the growing season the AOB communities were all of a similar size in the different plots at both sites, irrespective of liming. The number of AOB, determined using real-time PCR, ranged between 6×106 and 1×109 cells g-1 soil (dw) at the two sites, and generally decreased with increasing soil depth. The results showed no correlation between community density and potential nitrification. This may indicate a partly inactive AOB community. Furthermore, more than 107 cells g-1 soil (dw) were recorded using real-time PCR in the control plot at 244 Åled, although Bäckman et al. [Soil Biol. Biochem. 35 (2003) 1337] detected no AOB like sequences in the same plots using PCR followed by DGGE. Taken together our results strongly suggest that the primers and probe set used are not well suited for quantifying AOB in acidic forest soils, which is probably due to an insufficient specificity. This shows that it is extremely important to re-evaluate any primers and probe set when used in a new environment. Consideration should be given to the specificity and sensitivity, both empirically and using bioinformatic tools.

  • 44.
    Hernández-Pando, R.
    et al.
    Experimental Pathology Laboratory. Department of Pathology, Instituto Nacional De Ciencias Médicas y Nutrición “Salvador Zubirán”, Mexico city, Mexico.
    Schön, Thomas
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Orozco, E. H.
    Experimental Pathology Laboratory. Department of Pathology, Instituto Nacional De Ciencias Médicas y Nutrición “Salvador Zubirán”, Mexico city, Mexico.
    Serafin, J.
    Department of Immunology, Escuela Nacional de Ciencias Biológicas. Instituto Politecnico Nacional, México city, Mexico.
    Estradea-Garcia, I.
    Department of Immunology, Escuela Nacional de Ciencias Biológicas. Instituto Politecnico Nacional, México city, Mexico.
    Expression of inducible nitric oxide synthase and nitrotyrosineduring the evolution of experimental pulmonary tuberculosis2001In: Experimental and Toxicological Pathology, ISSN 0940-2993, E-ISSN 1618-1433, Vol. 53, no 4, p. 257-265Article in journal (Refereed)
    Abstract [en]

    Nitric oxide (NO) is a relevant antimycobacterial factor in mouse macrophages. NO is a product of inducible nitric oxide synthase (iNOS). NO toxicity is greatly enhanced by reacting with superoxide to form peroxynitrite that reacts with many biological molecules. Tyrosine is one of the molecules with which NO reacts and the product is nitrotyrosine (NT). The production of peroxynitrite and the nitrosylation of proteins might play a role in bacterial killing and also in mediating host injury. In this study, we used a well-characterized mouse model of pulmonary tuberculosis to examine the local kinetics of expression and cellular distribution of iNOS and NT at the cellular and subcellular level. The histopathological study showed two phases of the disease: early and late. The early phase was characterized by mononuclear inflammation and granuloma formation. During this phase, high percentages of activated macrophages were observed that were immunostained for iNOS and NT. Immuno-electronmicroscopy showed NT immunoreactivity in lysosomes and mycobacterial wall and cytoplasm. The concentration of iNOS mRNA and NO metabolites were also elevated. The late phase was characterized by progressive pneumonia with focal necrosis and a decrease of iNOS mRNA and NO metabolites. The strongest NT immunostained areas were the necrotic tissue. Macrophages became foamy cells with scarce iNOS immunostaining but strong NT immunoreactivity. At the ultrastructural level, these cells showed NT immunolabeling in cytoskeleton, mitochondria, lysosomes and cell membrane. NT was also located in bronchial epithelial cell mitochondria, in cell membranes and cytoplasm of endothelial cells and in actin bundles within smooth muscle cells. These results suggest an important role of NO in mycobacterial killing, particularly during the early phase of the infection. They also suggest an important participation by NO in tissue damage during the late phase of the disease.

  • 45.
    Holm, Åsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Leishmania donovani lipophosphoglycan: effects on actin and phagosomal maturation2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Leishmania donovani promastigotes survive intracellularly in macrophages by inhibiting phagosomal maturation. This ability is conferred by surface lipophosphoglycan (LPG), which is transferred to the host-cell plasma and phagosomal membranes during phagocytosis. LPG modulates the biophysical properties of membranes and has several effects on the host cell, including inhibition of protein kinase C alpha (PKCα)-mediated signaling. Our studies were focused on molecular mechanisms operating in the establishment of L. donovani infection, especially effects on host-cell F-actin.

    We found that L. donovani promastigotes induced accumulation of periphagosomal F-actin, an effect directly dependent on LPG. The F-actin accumulation correlated to inhibition of phagosomal maturation. Cortical F-actin was increased as well. Macrophages overexpressing dominant-negative (DN) PKCα also displayed elevated cortical F-actin, decreased phagocytic capacity, elevated periphagosomal F-actin, and defective phagosomal maturation, effects similar to those seen when exposing the cells to LPG. LPG colocalized with lipid rafts in the host-cell membrane, and lipid rafts were necessary both for translocation of activated PKCα to the membrane, and for the effects of LPG on host cell actin dynamics and phagosomal maturation. Introduction of constitutively active Rac1 and Cdc42 into the host macrophage mimicked the effects of LPG on actin dynamics and phagosomal maturation while DN Rac1 and Cdc42 abrogated LPG's effects on actin.

    Taken together, our results show that LPG partitions into lipid rafts in macrophages and induces an accumulation of periphagosomal F-actin, which is correlated to inhibition of phagosomal maturation. The effect of LPG on actin involves inhibition of PKCα and depends on active Rac1 and Cdc42.

    List of papers
    1. Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation
    Open this publication in new window or tab >>Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation
    Show others...
    2001 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 3, no 7, p. 439-447Article in journal (Refereed) Published
    Abstract [en]

    Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide–phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. In the present study, we have characterized the dynamics of F-actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F-actin accumulated progressively around phagosomes containing wild-type L. donovani promastigotes during the first hour of phagocytosis. Using LPG-defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG-dependent accumulation of periphagosomal F-actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCα to the phagosome. Accumulation of periphagosomal F-actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-13848 (URN)10.1046/j.1462-5822.2001.00127.x (DOI)
    Available from: 2006-05-23 Created: 2006-05-23 Last updated: 2017-12-13Bibliographically approved
    2. Role of protein kinase C α for uptake of unopsonized prey and phagosomal maturation in macrophages
    Open this publication in new window or tab >>Role of protein kinase C α for uptake of unopsonized prey and phagosomal maturation in macrophages
    Show others...
    2003 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 302, no 4, p. 653-658Article in journal (Refereed) Published
    Abstract [en]

    Protein kinase C α (PKCα) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Leishmania donovani promastigotes, which inhibit phagosomal maturation, cause accumulation of periphagosomal F-actin instead of the dissassembly observed around other prey [Cell. Microbiol. 7 (2001) 439]. This accumulation is induced by promastigote lipophosphoglycan (LPG), which has several effects on macrophages including inhibition of PKCα. To investigate a possible connection between PKCα and LPG’s effects on actin dynamics, we utilized RAW264.7 macrophages overexpressing dominant-negative PKCα (DN PKCα). We found increased cortical F-actin and decreased phagocytic capacity, as well as defective periphagosomal F-actin breakdown and inhibited phagosomal maturation in the DN PKCα-overexpressing cells, effects similar to those seen in controls subjected to LPG-coated prey. The results indicate that PKCα is involved in F-actin turnover in macrophages and that PKCα-dependent breakdown of periphagosomal F-actin is required for phagosomal maturation, and endorse the hypothesis that intracellular survival of L. donovani involves inhibition of PKCα by LPG.

    Keywords
    PKCα, Actin, Phagocytosis, Macrophage, Lipophosphoglycan
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-13850 (URN)10.1016/S0006-291X(03)00231-6 (DOI)
    Available from: 2006-05-23 Created: 2006-05-23 Last updated: 2017-12-13Bibliographically approved
    3. Lipid rafts are required for the effects of Leishmania donovani lipophosphoglycan on periphagosomal F-actin and phagosomal maturation
    Open this publication in new window or tab >>Lipid rafts are required for the effects of Leishmania donovani lipophosphoglycan on periphagosomal F-actin and phagosomal maturation
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Lipophosphoglycan (LPG) is the major surface glycoconjugate on Leishmania donovani promastigotes, and is cmcial for pro mastigote survival following phagocytosis by macrophages. LPG consists of a chain of repeating phosphodisaccharides anchored to the parasite membrane by a lysophosphatidylinositol lipid anchor with an unusually long saturated fatty acid residue. During phagocytosis, LPG transfers from the parasite surface to the plasma membrane of the host macrophage. The presence of LPG alters the biophysical properties of the host cell membrane and the signaling capacity of the macrophage. LPG induces accumulation ofF-actin around the phagosome, and inhibits phagosome maturation. The effects of LPG on the host ce!l include inhibition of PKCα, a PKC isoenzyme involved in F-actin tumover.

    The biophysical properties of the LPG lipid anchor suggest that it partitions into caveolae or lipid rafts, which are cholesterol-rich plasma membrane microdomains central for signal transduction. Since PKCa is enriched in caveolae/lipid rafts in other cell types, we investigated if lipid rafts constitute a platform for the interaction of LPG and PKCα. We found that the plasma membrane of human monocyte-derived macrophages were rich in lipid rafts, but did not contain caveolae. LPG colocalized with lipid raft markers after interaction with WT L. donovani promastigotes. The presence of LPG inhibited the translocation of PKCα to the plasma membrane. Destruction of lipid rafts by cholesterol depletion lead to a complete eradication of LPG's effects on periphagosomal F-actin and phagosomal maturation. We also found that cholesterol depletion reduced uptake of WT L. donovani promastigotes, while uptake of an LPG-defective mutant was not affected.

    We conclude that LPG partitions to lipid rafts in the plasma membrane of human macrophages and inhibits the translocation of PKCα to the membrane. The presence of lipid rafts is a prerequisite for LPG to exert its effects on host cell actin and phagosomal maturation.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-84524 (URN)
    Available from: 2012-10-11 Created: 2012-10-11 Last updated: 2012-10-11Bibliographically approved
    4. Rac1 and Cdc42 are involved in the periphagosomal F-actin accumulation and inhibition of phagosomal maturation caused by Leishmania donovani lipophosphoglycan
    Open this publication in new window or tab >>Rac1 and Cdc42 are involved in the periphagosomal F-actin accumulation and inhibition of phagosomal maturation caused by Leishmania donovani lipophosphoglycan
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    The intracellular parasite Leishmania donovani survives inside macrophage phagosomes by inhibiting phagosornal maturation. Its main surface glycoconjugate, lipophosphoglycan (LPG), is crucial for survival and essential for the build-up of a coat of F-actin surrounding the phagosome. Previous studies have shown that inhibition of PKCα by LPG is partly responsible for the elevated levels of F-actin around the phagosome (1, 2). This study shows that simultaneous inhibition of Cdc42 and Rac1, members of the Rho family of small GTPases, prevented the accumulation of F-actin around L. donovani containing phagosomes in murine macrophages. Moreover, an LPG-defective L. donovani mutant normally not capable of accumulating F-actin around it's phagosome, displayed elevated amounts of periphagosomal F-actin in cells pre-treated with permanently active forms of Cdc42 and Rac. The lysosomal marker LAMP1 did not translocate normally to phagosomes in these cells, indicating defective phagosomal maturation. We conclude that Cdc42 and Rac are activated by L. donovani in an LPG-dependent manner, and that this activation contributes to the accumulation of periphagosomal F-actin around L. donovani phagosomes. Our results also indicate a direct link between the build-up of periphagosomal F-actinand inhibition of phagosomal mahuation.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-84526 (URN)
    Available from: 2012-10-11 Created: 2012-10-11 Last updated: 2012-10-11Bibliographically approved
  • 46.
    Holm, Åsa
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Öberg, Åke
    Linköping University, Department of Biomedical Engineering. 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.
    Mechanical manipulation of polymorphonuclear leukocyte plasma membranes with optical tweezers causes influx of extracellular calcium through membrane channels1999In: Medical and Biological Engineering and Computing, ISSN 0140-0118, E-ISSN 1741-0444, Vol. 37, no 3, p. 410-412Article in journal (Refereed)
    Abstract [en]

    Optical tweezers are used mechanically to manipulate the plasma membrane of polymophonuclear leukocytes attached to the bottom of a glass manipulation chamber. The laser trapping beam is dragged across the membrane of cells in calcium-containing and calcium-depleted extracellular medium. This treatment causes a significant rise in the intracellular calcium concentration compared with controls, in cells in calcium-containing medium (239.8±49.0% against 75.4±16.4%, respectively), but not in cells in calcium-depeleted medium (69.1±9.6% against 83.4±18.5%, respectively), indicating that the calcium rise is caused by an influx of calcium from the environment. The rise in calcium concentration is blocked (23.5±7.1% against 17.1±4.1%, respectively) by the addition of lansoprazole, indicating that the influx is not due to unspecific membrane damage caused by the mechanical manipulation of the cell. It can therefore be concluded that mechanical manipulation of the neutrophil membrane, in the piconewton force range exerted by the optical tweezer, does not damage the plasma membrane but stimulates a mechanically inducible, membrane channel-mediated influx of extracellular calcium.

  • 47.
    Holm, Åsa
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Tejle, Katarina
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, T.
    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.
    Descoteaux, A.
    INRS - Institut Armand-Frappier, Université du Québec.
    Rasmusson, Birgitta
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Protein C α regulates phagocytosis actin dynamics and phagosomal maturation in macrophagesManuscript (preprint) (Other academic)
    Abstract [en]

    Protein kinase C α (PKCα) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Promastigotes of the protozoan parasite Leishmania donovani cause an accumulation of periphagosomal F-actin instead of the normal decrease seen with other prey [1]. This accumulation is dependent on promastigote lipophosphoglycan (LPG), which has several detrimental effects on the cell including inhibition of PKCα activity.

    To directly address the role of PKCα and LPG for actin remodeling in macrophages, we investigated F-actin dynamics in RAW 264.7 macrophages overexpressing a dominant-negative mutant of PKCα (DN PKCα). We found that DN PKCα-overexpressing cells displayed increased levels of cortical F-actin and decreased phagocytic capacity, which was augmented when the cells were subjected to LPG-coated prey. The DN PKCα-overexpressing cells also showed defective breakdown of periphagosomal F-actin and inhibition of phagosomal maturation. The level of periphagosomal F-actin was similar to that of controls subjected to LPG-coated prey. Our results show that PKCα regulates phagocytosis and F-actin turnover in macrophages, and that PKCα-dependent breakdown of periphagosomal F-actin is required for normal phagosomal maturation.

  • 48.
    Holm, Åsa
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Tejle, Katarina
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, T.
    Linköping University, Department of Molecular and Clinical 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.
    Descoteaux, A.
    INRS—Institut Armand-Frappier, Université du Québec, Laval, Qué., Canada.
    Rasmusson, Birgitta
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Role of protein kinase C α for uptake of unopsonized prey and phagosomal maturation in macrophages2003In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 302, no 4, p. 653-658Article in journal (Refereed)
    Abstract [en]

    Protein kinase C α (PKCα) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Leishmania donovani promastigotes, which inhibit phagosomal maturation, cause accumulation of periphagosomal F-actin instead of the dissassembly observed around other prey [Cell. Microbiol. 7 (2001) 439]. This accumulation is induced by promastigote lipophosphoglycan (LPG), which has several effects on macrophages including inhibition of PKCα. To investigate a possible connection between PKCα and LPG’s effects on actin dynamics, we utilized RAW264.7 macrophages overexpressing dominant-negative PKCα (DN PKCα). We found increased cortical F-actin and decreased phagocytic capacity, as well as defective periphagosomal F-actin breakdown and inhibited phagosomal maturation in the DN PKCα-overexpressing cells, effects similar to those seen in controls subjected to LPG-coated prey. The results indicate that PKCα is involved in F-actin turnover in macrophages and that PKCα-dependent breakdown of periphagosomal F-actin is required for phagosomal maturation, and endorse the hypothesis that intracellular survival of L. donovani involves inhibition of PKCα by LPG.

  • 49.
    Holm, Åsa
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Tejle, Katarina
    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.
    Descoteaux, A.
    INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
    Rasmusson, Birgitta
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation2001In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 3, no 7, p. 439-447Article in journal (Refereed)
    Abstract [en]

    Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide–phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. In the present study, we have characterized the dynamics of F-actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F-actin accumulated progressively around phagosomes containing wild-type L. donovani promastigotes during the first hour of phagocytosis. Using LPG-defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG-dependent accumulation of periphagosomal F-actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCα to the phagosome. Accumulation of periphagosomal F-actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.

  • 50.
    Holm, Åsa
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Winberg, M. E.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Särndahl, E.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. 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.
    Descoteaux, A.
    INRS- lnstitut Annand-Frappier, Université du Quebec, Laval, Québéc, Canada.
    Rasmusson, Birgitta
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lipid rafts are required for the effects of Leishmania donovani lipophosphoglycan on periphagosomal F-actin and phagosomal maturationManuscript (preprint) (Other academic)
    Abstract [en]

    Lipophosphoglycan (LPG) is the major surface glycoconjugate on Leishmania donovani promastigotes, and is cmcial for pro mastigote survival following phagocytosis by macrophages. LPG consists of a chain of repeating phosphodisaccharides anchored to the parasite membrane by a lysophosphatidylinositol lipid anchor with an unusually long saturated fatty acid residue. During phagocytosis, LPG transfers from the parasite surface to the plasma membrane of the host macrophage. The presence of LPG alters the biophysical properties of the host cell membrane and the signaling capacity of the macrophage. LPG induces accumulation ofF-actin around the phagosome, and inhibits phagosome maturation. The effects of LPG on the host ce!l include inhibition of PKCα, a PKC isoenzyme involved in F-actin tumover.

    The biophysical properties of the LPG lipid anchor suggest that it partitions into caveolae or lipid rafts, which are cholesterol-rich plasma membrane microdomains central for signal transduction. Since PKCa is enriched in caveolae/lipid rafts in other cell types, we investigated if lipid rafts constitute a platform for the interaction of LPG and PKCα. We found that the plasma membrane of human monocyte-derived macrophages were rich in lipid rafts, but did not contain caveolae. LPG colocalized with lipid raft markers after interaction with WT L. donovani promastigotes. The presence of LPG inhibited the translocation of PKCα to the plasma membrane. Destruction of lipid rafts by cholesterol depletion lead to a complete eradication of LPG's effects on periphagosomal F-actin and phagosomal maturation. We also found that cholesterol depletion reduced uptake of WT L. donovani promastigotes, while uptake of an LPG-defective mutant was not affected.

    We conclude that LPG partitions to lipid rafts in the plasma membrane of human macrophages and inhibits the translocation of PKCα to the membrane. The presence of lipid rafts is a prerequisite for LPG to exert its effects on host cell actin and phagosomal maturation.

12345 1 - 50 of 210
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf