| 1. |
- Andersson, Kerstin, et al.
(författare)
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Yersinia pseudotuberculosis-induced calcium signaling in neutrophils is blocked by the virulence effector YopH
- 1999
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 67:5, s. 2567-2574
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
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| 3. |
- Jerström Skarman, Petra, et al.
(författare)
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Subcellular distribution of annexins, gelsolin and filamentous actin in adherent human neutrophils during phagocytosis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Subcellular elevations of cytosolic free calcium concentration ([ea2+];), are critical for certain functional responses within the neutrophil, such asfilamentous actin (F-actin) reorganization and phagolysosome fusion (PLF). During this event, an accumulation of phospholipid- and calciumbinding proteins, annexins, can be seen in the periphagosomal area. A prerequisite for phagolysosome fusion is the elimination of F-actin around the phagosomes to facilitate the membrane contact between lysosomes and phagosomes. Gelsolin is a protein that severs F:actin by binding to the barbed ends, and thereby affect further polymerization. In this study, we used immunofluorescence staining and immunogold technique to analyse the distribution of annexin I, annexin III and gelsolin, in relation to the rearrangement ofF-actin during phagocytosos of complement-opsonized yeast particles by adherent human neutrophils. Iu unchallenged cells, both the aunexins and gelsolin were evenly distributed throughout the cells, whereas F-actin was found mostly in the protruding pseudopodia. Upon phagocytosis an accumulation of both. annexin I and annexin III, and gelsolin could be seen w1thm the vicimty of the phagocytic cups and phagosomes where they colocalized with Factin around the ingested particle.In calcium-depleted cells, the subcellular distributions of annexins and gelsolin were unaffected. On the other hand, there was a total increase inF-actin polymerization.Our data may indicate that gelsolin is important for the rearrangement of F-actin and that annexin I and annexin III, which are present in high concentrations in neutrophils, may participate in the following calciumdependent PLF in human neutrophils.
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| 4. |
- Kälvegren, Hanna, et al.
(författare)
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Chlamydia pneumoniae binds to platelets and triggers P-selectin expression and aggregation: A causal role in cardiovascular disease?
- 2003
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 23:9, s. 1677-1683
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Tidskriftsartikel (refereegranskat)abstract
- Objective - Evidence linking Chlamydia pneumoniae to atherosclerotic cardiovascular disease is expanding. Platelets are considered to play an essential role in cardiovascular diseases, however, so far platelets have not been associated with an infectious cause of atherosclerosis. This study aims to clarify the interaction between Cpneumoniae and platelets and possibly present a novel mechanism in the pathogenesis of atherosclerosis.Methods and Results - The effects of C pneumoniae on platelet aggregation and secretion were assessed with lumiaggregometry, and the ability of C pneumoniae to bind to platelets and stimulate expression of P-selectin was analyzed with flow cytometry. We found that Cpneumoniae, at a chlamydia:platelet ratio of 1:15, adheres to platelets and triggers P-selectin expression after 1 minute and causes an extensive aggregation and ATP secretion after 20 minutes of incubation. Inhibition of glycoprotein IIb/IIIa with Arg-Gly-Asp-Ser or abciximab markedly reduced C pneumoniae-induced platelet aggregation. Exposure of C pneumoniae to polymyxin B, but not elevated temperature, abolished the stimulatory effects on platelet activation, suggesting that chlamydial lipopolysaccharide has an active role. In contrast, other tested bacteria had no or only moderate effects on platelet functions.Conclusion - Our findings demonstrate a new concept of how C pneumoniae activates platelets and thereby may cause atherosclerosis and thrombotic vascular occlusion.
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| 5. |
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| 6. |
- Majeed, Meytham, 1960-
(författare)
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Regulation of Attachment and Early Intracellular Development of Chlamydia trachomatis in Eucaryotic Cells
- 1994
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The obligate intracellular bacterium, Chlamydia trachomatis , a common human pathogen, causing different diseases in both males and females, as well as in infants born to infected mothers. Occasionally, these diseases involve serious complications, such as blindness and infertility. C. trachomatis has a unique biphasic life cycle: after initial inclosure in membrane~bound endosomes, the infectious elementary bodies (EBs) reorganize to the metabolically actiVe forms (RBs); the RBs divide by binary fission, and after multiple divisions, they again differentiate to form new EBs, which are subsequently released from the host cell to start a new infectious cycle. As an attempt to investigate the early events of chlamydial infection, my results show that EBs bind with high affinity to collagen type I and heparan sulfate, suggesting that this selective affinity may mediate the attachment of chlamydiae to the surface of a host cell. ER-containing endosomes avoid fusion with host celllysosomes. However, within 30 min of form ation, these endosomes form one local aggregate in the central orperinuclear region of individual cells. This aggregation is reversible and time and temperature dependent, and requires viable EBs. Clathrin and F-actin are mobilized and colocalized with EB aggregates, suggesting that the aggregation of EBs is an active process that may be biologically involved in the infectivity of chlamydiae. The aggregation and inclusion formation of EBs, and the redistribution of F-actin seem to be controlled by both extra- and intracellular Ca2+, whereas the attachment and ingestion of EBs occur independently of Ca2+ in the growth medium and at low intracellular free Ca2+ [Ca2+]i. Moreover, chlamydiae do not induce any changes in the level of [Ca2+]i, this indicates that the aggregation of EBs requires a normal homeostasis of intracellular Ca2+. By affecting F-act:in reorganization and, putatively, certain Ca2+ -binding proteins, [Ca2+]i plays a vital role in the process of chlamydiae infection. The ca2+_ and phospholipid-binding proteins, annex.ins, are selectivelytranslocated during ehlamydial infection, i.e. annexins III, IV, and V, but not annexins I and VI, translocate to the proximity of chlamydial aggregates and inclusions. Annexins differ in their ability to associate with chlamydia-containing vesicles and inclusions. This fact implies that different factors regulate the interaction of annexins I and Ill with the membrane and also suggests that there is a selective regulatorymechanism that guides endosome aggregation and that is responsible for endosome avoiding lysosome fusion during chlamydial infection. Chlamydiae also induce mobilization of intracellular Ca2+ stores. This suggests that in a chlamydia-infected cell localized [Ca2+]i changes may occur by mobilization of Ca2+ stores at the sites of ca2+ action. The physiological role of ca2+ stores redistribution during infection of the host cells with chlamydiae might be to generate subceJlular [Ca2+]i gradients needed for the intracellular itinerary of the membrane trafficking of BE-containing endosomes.
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| 7. |
- Majeed, Meytham, 1960-, et al.
(författare)
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Role of Src kinases and Syk in Fc? receptor-mediated phagocytosis and phagosome-lysosome fusion
- 2001
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Ingår i: Journal of Leukocyte Biology. - 0741-5400 .- 1938-3673. ; 70:5, s. 801-811
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Tidskriftsartikel (refereegranskat)abstract
- Phagocytosis is increased by Fc? receptors (Fc?Rs), and studies with syk-1- macrophages demonstrated that Syk kinase is required for Fc?TR phagocytosis. Similar studies with macrophages lacking the Src family kinases Hck, Fgr, and Lyn showed that these kinases are not required for phagocytosis but that they enhance the rate of particle engulfment. In this report we show that both wild-type and hck-1- fgr-1- macrophages expressed Fyn, Src, and Yes and that these kinases were activated on ingestion of immunoglobulin G (IgG)-coated particles and redistributed, together with Syk, to actin-rich phagocytic cups and the phagosomal membrane. At doses blocking IgG-dependent phagocytosis, the tyrosine kinase inhibitors PP1 and piceatannol inhibited both Src family kinase and Syk activities, as well as their redistribution to actin-rich phagocytic cups. Hck, Fgr, and Lyn were dispensable for lysosome-phagosome fusion (PLF) induced by IgG-coated particles. However, PP1 or piceatannol hampered unopsonized yeast-induced PLF despite the fact that they did not block yeast internalization.
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| 8. |
- Majeed, Meytham, et al.
(författare)
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Roles of Ca2+ and F-actin in intracellular aggregation of Chlamydia trachomatis in eucaryotic cells
- 1993
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 61:4, s. 1406-1414
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Tidskriftsartikel (refereegranskat)abstract
- The effect of intracellular free Ca2+ ([Ca2+]i) on the intracellular aggregation of Chlamydia trachomatis serovars L2 and E in McCoy and HeLa cells is investigated. Loading the cells with the Ca2+ chelator MAPT/AM (1,2-bis-5-methyl-amino-phenoxylethane-N,N-n'-tetra-acetoxymethyl acetate), thereby decreasing the [Ca2+]i from 67 to 19 nM, decreased the number of cells with a local aggregation of chlamydiae in a dose-dependent manner. Neither the attachment nor the uptake of elementary bodies (EBs) was, however, affected after depletion of Ca2+ from the cells. There was no significant difference in the level of measured [Ca2+]i between infected and uninfected cells. Reducing the [Ca2+]i also significantly inhibited chlamydial inclusion formation. Differences in the organization of the actin filament network were observed in response to [Ca2+]i depletion. In Ca(2+)-depleted cells, where few EB aggregates were formed, few local accumulations of F-actin were observed in the cytosol. These results suggest that the aggregation of EBs in eucaryotic cells requires a normal homeostasis of intracellular Ca2+. By affecting F-actin reorganization and putatively certain Ca(2+)-binding proteins, [Ca2+]i plays a vital role in the infectious process of chlamydiae.
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| 9. |
- Majeed, Meytham, et al.
(författare)
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Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosisin human neutrophils
- 1998
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Ingår i: Microbial Pathogenesis. - : Elsevier BV. - 0882-4010 .- 1096-1208. ; 24:5, s. 309-320
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Tidskriftsartikel (refereegranskat)abstract
- The phagocytic function of neutrophils is a crucial element in the host defence against invading microorganisms. We investigated phagocytosis and intracellular killing of an attenuated strain of Mycobacterium tuberculosis(H37Ra) by human neutrophils focusing on the role of the cytosolic free calcium concentration [Ca2+]iand certain cytosolic calcium-dependent membrane-binding proteins annexins. Phagocytic uptake did not trigger a calcium rise and occurred independently of different calcium conditions, and in a serum-dependent manner. Changes in the viability of H37Ra were determined by agar plate colony count and a radiometric assay. Neutrophils showed a capacity to kill ingested mycobacteria and this occurred without a rise in [Ca2+]i. The ability to kill H37Ra decreased in the absence of extracellular calcium and when intra-extracellular calcium was reduced. Immunofluorescence staining revealed that during phagocytosis of H37Ra, annexins III, IV and VI translocated from cytoplasm to the proximity of the H37Ra-containing phagosomes, whereas the localization of annexin I and V remained unchanged. The translocation of annexin IV occurred even when Ca2+-depleted neutrophils ingested H37Ra in the absence of extracellular calcium. We concluded that neutrophil-mediated killing of mycobacteria is a Ca2+-dependent process. The fact that the association of certain annexins to the membrane vesicle containing H37Ra differ from other phagosomes suggests a selective regulatory mechanism during phagocytosis of mycobacteria by neutrophils.
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| 10. |
- Nimeri, G, et al.
(författare)
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Oxygen radical production in neutrophils interacting with platelets and surface-immobilized plasma proteins : role of tyrosine phosphorylation
- 2003
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Ingår i: Journal of Biomedical Materials Research. - : Wiley. - 0021-9304 .- 1097-4636 .- 1549-3296. ; 67A:2, s. 439-447
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between neutrophil granulocytes and platelets is considered to play an important role in the inflammatory process induced by an implanted foreign material. However, the cellular mechanisms involved remain incompletely understood. We used a luminol-dependent chemiluminescence (CL) technique to analyze the generation of reactive oxygen species (ROS) in human neutrophils interacting with different plasma protein-coated surfaces in the presence or absence of unstimulated or stimulated platelets. The role of tyrosine phosphorylation in the regulation of NADPH oxidase activity was evaluated with quantitative fluorescence microscopy and the specific tyrosine kinase inhibitor genistein. We found that the ROS-production is 2 to 3 times higher in neutrophils on immunoglobulin G (IgG)coated surfaces than in cells interacting with albumin- or fibrinogen-coated surfaces. Incubation with superoxide dismutase and catalase revealed that about 45% of the ROS was released extracellularly on IgG surfaces whereas corresponding values were 90% and 85% in neutrophils interacting with albumin and fibrinogen, respectively. The presence of platelets markedly increased the extracellular generation of ROS, mainly in neutrophils. interacting with IgG- or fibrinogen-coated surfaces whereas the intracellular production was only modestly affected. Quantitative fluorescence microscopy of neutrophils stained with FITC-conjugated anti-phosphotyrosine antibodies showed a correlation between tyrosine phosphorylation, cell spreading, and ROS production. Platelets markedly amplified the anti-phosphotyrosine staining on both fibrinogen- and IgG-coated surfaces whereas the low level of tyrosine phosphorylation in neutrophils on albumin-coated surfaces was not further elevated by platelets. Furthermore, the tyrosine kinase inhibitor genistein inhibited both extra- and intracellular ROS production in neutrophils regardless of the presence of platelets. We demonstrate that plasma protein coating and the presence of platelets are crucial for the inflammatory response of adhering neutrophils and that the oxidative response correlates with the extent of tyrosine phosphorylation of proteins in focal contacts. (C) 2003 Wiley Periodicals, Inc.
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