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  • 1.
    Abrikossova, Natalia
    et al.
    Division of Molecular Surface Physics and Nanoscience, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden .
    Skoglund, Caroline
    Division of Molecular Surface Physics and Nanoscience, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden; Division of Clinical Medicine, Department of Biomedicine, Örebro University, Örebro, Sweden.
    Ahrén, Maria
    Division of Molecular Surface Physics and Nanoscience, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Division of Clinical Medicine, Department of Biomedicine, School of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    Uvdal, Kajsa
    Division of Molecular Surface Physics and Nanoscience, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
    Effects of gadolinium oxide nanoparticles on the oxidative burst from human neutrophil granulocytes2012Ingår i: Nanotechnology, ISSN 0957-4484, E-ISSN 1361-6528, Vol. 23, nr 27, artikel-id 275101Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have previously shown that gadolinium oxide (Gd2O3) nanoparticles are promising candidates to be used as contrast agents in magnetic resonance (MR) imaging applications. In this study, these nanoparticles were investigated in a cellular system, as possible probes for visualization and targeting intended for bioimaging applications. We evaluated the impact of the presence of Gd2O3 nanoparticles on the production of reactive oxygen species (ROS) from human neutrophils, by means of luminol-dependent chemiluminescence. Three sets of Gd2O3 nanoparticles were studied, i.e. as synthesized, dialyzed and both PEG-functionalized and dialyzed Gd2O3 nanoparticles. In addition, neutrophil morphology was evaluated by fluorescent staining of the actin cytoskeleton and fluorescence microscopy. We show that surface modification of these nanoparticles with polyethylene glycol (PEG) is essential in order to increase their biocompatibility. We observed that the as synthesized nanoparticles markedly decreased the ROS production from neutrophils challenged with prey (opsonized yeast particles) compared to controls without nanoparticles. After functionalization and dialysis, more moderate inhibitory effects were observed at a corresponding concentration of gadolinium. At lower gadolinium concentration the response was similar to that of the control cells. We suggest that the diethylene glycol (DEG) present in the as synthesized nanoparticle preparation is responsible for the inhibitory effects on the neutrophil oxidative burst. Indeed, in the present study we also show that even a low concentration of DEG, 0.3%, severely inhibits neutrophil function. In summary, the low cellular response upon PEG-functionalized Gd2O3 nanoparticle exposure indicates that these nanoparticles are promising candidates for MR-imaging purposes.

  • 2.
    Ahren, Maria
    et al.
    Linkoping Univ, Linkoping, Sweden.
    Selegard, Linnea
    Linkoping Univ, Linkoping, Sweden.
    Klasson, Anna
    Linkoping Univ, Linkoping, Sweden.
    Soderlind, Fredrik
    Linkoping Univ, Linkoping, Sweden.
    Abrikossova, Natalia
    Linkoping Univ, Linkoping, Sweden.
    Skoglund, Caroline
    Linkoping Univ, Linkoping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Hälsoakademin. Linkoping Univ, Linkoping, Sweden.
    Engstrom, Maria
    Linkoping Univ, Linkoping, Sweden.
    Kall, Per-Olov
    Linkoping Univ, Linkoping, Sweden.
    Uvdal, Kajsa
    Linkoping Univ, Linkoping, Sweden.
    Synthesis and Characterization of PEGylated Gd2O3 Nanoparticles for MRI Contrast Enhancement2010Ingår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 26, nr 8, s. 5753-5762Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recently, much attention has been given to the development of biofunctionalized nanoparticles with magnetic properties for novel biomedical imaging. Guided, smart, targeting nanoparticulate magnetic resonance imaging (MRI) contrast agents inducing high MRI signal will be valuable tools for future tissue specific imaging and investigation of molecular and cellular events. In this study. We report a new design of functionalized ultrasmall rare earth based nanoparticles to be used as a positive contrast agent in NI RI. The relaxivity is compared to commercially available Gd based chelates. The synthesis, PEGylation, and dialysis of small (3-5 nm) gadolinium oxide (DEG-Gd2O3) nanoparticles are presented. The chemical and physical properties of the nanomaterial were investigated with Fourier transform infrared spectroscopy. X-ray photoelectron spectroscopy, transmission electron microscopy, and dynamic light scattering. Neutrophil activation after exposure to this nanomaterial was studied by means of fluorescence microscopy. The proton relaxation times as a function of dialysis time and functionalization were measured at 1.5 T. A capping procedure introducing stabilizing properties was designed and verified, and the dialysis effects were evaluated. A higher proton relaxivity was obtained for as-synthesized diethylene glycol (DEG)-Gd2O3 nanoparticles compared to commercial Gd-DTPA. A slight decrease of the relaxivity for as-synthesized DEG-Gd2O3 nanoparticles as a function of dialysis time was observed. The results for functionalized nanoparticles showed a considerable relaxivity increase for particles dialyzed extensively with r(1) and r(2) values approximately 4 times the corresponding values for Gd-DTPA. The microscopy study showed that PEGylated nanoparticles do not activate neutrophils in contrast to uncapped Gd2O3. Finally, the nanoparticles are equipped with Rhodamine to show that our PEGylated nanoparticles are available for further coupling chemistry, and thus prepared for targeting purposes. The long term goal is to design a powerful, directed contrast agent for MRI examinations with specific targeting possibilities and with properties inducing local contrast, that is. an extremely high MR signal at the cellular and molecular level.

  • 3.
    Bengtsson, Torbjörn
    et al.
    Örebro universitet, Institutionen för läkarutbildning.
    Khalaf, Atika
    The PRO-CARE Group, School of Health and Society, Kristianstad University, Kristianstad, Sweden.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Secreted gingipains from Porphyromonas gingivalis colonies exert potent immunomodulatory effects on human gingival fibroblasts2015Ingår i: Microbiology Research, ISSN 0944-5013, E-ISSN 1618-0623, Vol. 178, s. 18-26Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Periodontal pathogens, including Polphyromonas gingivalis, can form biofilms in dental pockets and cause inflammation, which is one of the underlying mechanisms involved in the development of periodontal disease, ultimately leading to tooth loss. Although P. gingivalis is protected in the biofilm, it can still cause damage and modulate inflammatory responses from the host, through secretion of microvesicles containing proteinases. The aim of this study was to evaluate the role of cysteine proteinases in P. gingivalis colony growth and development, and subsequent immunomodulatory effects on human gingival fibroblast. By comparing the wild type W50 with its gingipain deficient strains we show that cysteine proteinases are required by P. gingivalis to form morphologically normal colonies. The lysine-specific proteinase (Kgp), but not arginine-specific proteinases (Rgps), was associated with immunomodulation. P. gingivalis with Kgp affected the viability of gingival fibroblasts and modulated host inflammatory responses, including induction of TGF-beta 1 and suppression of CXCL8 and IL-6 accumulation. These results suggest that secreted products from P. gingivalis, including proteinases, are able to cause damage and significantly modulate the levels of inflammatory mediators, independent of a physical host-bacterial interaction. This study provides new insight of the pathogenesis of P. gingivalis and suggests gingipains as targets for diagnosis and treatment of periodontitis.

  • 4.
    Bengtsson, Torbjörn
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Lönn, Johanna
    Department of Oral Biology, Institute of Odontology, Malmö University, Malmö, Sweden; PEAS Research Institute, Linköping, Sweden.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Palm, Eleonor
    Örebro universitet, Institutionen för medicinska vetenskaper.
    The lantibiotic gallidermin acts bactericidal against Staphylococcus epidermidis and Staphylococcus aureus and antagonizes the bacteria-induced proinflammatory responses in dermal fibroblasts2018Ingår i: MicrobiologyOpen, ISSN 2045-8827, E-ISSN 2045-8827, Vol. 7, nr 6, artikel-id e606Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antimicrobial resistance needs to be tackled from new angles, and antimicrobial peptides could be future candidates for combating bacterial infections. This study aims to investigate in vitro the bactericidal effects of the lantibiotic gallidermin on Staphylococcus epidermidis and Staphylococcus aureus, possible cytotoxic effects and its impact on host-microbe interactions. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of gallidermin were determined, and cytotoxicity and proinflammatory effects of gallidermin on fibroblasts, red blood cells (RBCs) and in whole blood were investigated. Both MIC and MBC for all four tested strains of S. epidermidis was 6.25 μg/ml. Both MIC and MBC for methicillin-sensitive S. aureus was 12.5 μg/ml and for methicillin-resistant S. aureus (MRSA) 1.56 μg/ml. Gallidermin displayed no cytotoxic effects on fibroblasts, only a high dose of gallidermin induced low levels of CXCL8 and interleukin-6. Gallidermin hemolyzed less than 1% of human RBCs, and did not induce reactive oxygen species production or cell aggregation in whole blood. In cell culture, gallidermin inhibited the cytotoxic effects of the bacteria and totally suppressed the bacteria-induced release of CXCL8 and interleukin-6 from fibroblasts. We demonstrate that gallidermin, expressing low cell cytotoxicity, is a promising candidate for treating bacterial infections caused by S. epidermidis and S. aureus, especially MRSA.

  • 5.
    Bengtsson, Torbjörn
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Selegård, Robert
    School of Medical Sciences, Örebro University, Örebro, Sweden; Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Musa, Amani
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Hultenby, Kjell
    Department of Laboratory Medicine, Division of Clinical Research Centre, Karolinska Institutet, Stockholm, Sweden.
    Utterström, Johanna
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Sivlér, Petter
    S2Medical AB, Linköping, Sweden.
    Skog, Mårten
    S2Medical AB, Linköping, Sweden.
    Nayeri, Fariba
    PEAS Research Institute, Department of Infection Control, Linköping, Sweden.
    Hellmark, Bengt
    Department of Clinical Microbiology, Örebro University Hospital, Örebro, Sweden.
    Söderquist, Bo
    Örebro universitet, Institutionen för medicinska vetenskaper. Department of Clinical Microbiology, Örebro University Hospital, Örebro, Sweden.
    Aili, Daniel
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Plantaricin NC8 αβ exerts potent antimicrobial activity against Staphylococcus spp. and enhances the effects of antibiotics2020Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 10, nr 1, artikel-id 3580Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The use of conventional antibiotics has substantial clinical efficacy, however these vital antimicrobial agents are becoming less effective due to the dramatic increase in antibiotic-resistant bacteria. Novel approaches to combat bacterial infections are urgently needed and bacteriocins represent a promising alternative. In this study, the activities of the two-peptide bacteriocin PLNC8 αβ were investigated against different Staphylococcus spp. The peptide sequences of PLNC8 α and β were modified, either through truncation or replacement of all L-amino acids with D-amino acids. Both L- and D-PLNC8 αβ caused rapid disruption of lipid membrane integrity and were effective against both susceptible and antibiotic resistant strains. The D-enantiomer was stable against proteolytic degradation by trypsin compared to the L-enantiomer. Of the truncated peptides, β1-22, β7-34 and β1-20 retained an inhibitory activity. The peptides diffused rapidly (2 min) through the bacterial cell wall and permeabilized the cell membrane, causing swelling with a disorganized peptidoglycan layer. Interestingly, sub-MIC concentrations of PLNC8 αβ substantially enhanced the effects of different antibiotics in an additive or synergistic manner. This study shows that PLNC8 αβ is active against Staphylococcus spp. and may be developed as adjuvant in combination therapy to potentiate the effects of antibiotics and reduce their overall use.

  • 6.
    Bengtsson, Torbjörn
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Zhang, Boxi
    Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
    Selegård, Robert
    Örebro universitet, Institutionen för medicinska vetenskaper. Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Wiman, Emanuel
    School of Medical Sciences, Örebro University, Örebro, Sweden.
    Aili, Daniel
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Dual action of bacteriocin PLNC8 alpha beta through inhibition of Porphyromonas gingivalis infection and promotion of cell proliferation2017Ingår i: Pathogens and Disease, E-ISSN 2049-632X, Vol. 75, nr 5, artikel-id ftx064Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Periodontitis is a chronic inflammatory disease that is characterised by accumulation of pathogenic bacteria, including Porphyromonas gingivalis, in periodontal pockets. The lack of effective treatments has emphasised in an intense search for alternative methods to prevent bacterial colonisation and disease progression. Bacteriocins are bacterially produced antimicrobial peptides gaining increased consideration as alternatives to traditional antibiotics. We show rapid permeabilisation and aggregation of P. gingivalis by the two-peptide bacteriocin PLNC8 alpha beta. In a cell culture model, P. gingivalis was cytotoxic against gingival fibroblasts. The proteome profile of fibroblasts is severely affected by P. gingivalis, including induction of the ubiquitin-proteasome pathway. PLNC8 alpha beta enhanced the expression of growth factors and promoted cell proliferation, and suppressed proteins associated with apoptosis. PLNC8 alpha beta efficiently counteracted P. gingivalis-mediated cytotoxicity, increased expression of a large number of proteins and restored the levels of inflammatory mediators. In conclusion, we show that bacteriocin PLNC8 alpha beta displays dual effects by acting as a potent antimicrobial agent killing P. gingivalis and as a stimulatory factor promoting cell proliferation. We suggest preventive and therapeutical applications of PLNC8 alpha beta in periodontitis to supplement the host immune defence against P. gingivalis infection and support wound healing processes.

  • 7. Börgeson, Emma
    et al.
    Lönn, Johanna
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bergström, Ida
    Brodin, Veronika Patcha
    Ramström, Sofia
    Linköping University, Linköping, Sweden .
    Nayeri, Fariba
    Särndahl, Eva
    Örebro universitet, Hälsoakademin.
    Bengtsson, Torbjörn
    Örebro universitet, Hälsoakademin.
    Lipoxin A(4) inhibits porphyromonas gingivalis-induced aggregation and reactive oxygen species production by modulating neutrophil-platelet interaction and CD11b expression2011Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 79, nr 4, s. 1489-1497Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Porphyromonas gingivalis is an etiological agent that is strongly associated with periodontal disease, and it correlates with numerous inflammatory disorders, such as cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. Lipoxin A(4) (LXA(4)) is an endogenous anti-inflammatory and proresolving mediator that is protective of inflammatory disorders. The aim of this study was to investigate the effect of LXA(4) on the P. gingivalis-induced activation of neutrophils and platelets and the possible involvement of Rho GTPases and CD11b/CD18 integrins. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry and fluorescence microscopy. Integrin activity was studied by flow cytometry, detecting the surface expression of CD11b/CD18 as well as the exposure of the high-affinity integrin epitope, whereas the activation of Rac2/Cdc42 was examined using a glutathione S-transferase pulldown assay. The study shows that P. gingivalis activates Rac2 and Cdc42 and upregulates CD11b/CD18 and its high-affinity epitope on neutrophils, and that these effects are diminished by LXA(4). Furthermore, we found that LXA(4) significantly inhibits P. gingivalis-induced aggregation and ROS generation in whole blood. However, in platelet-depleted blood and in isolated neutrophils and platelets, LXA(4) was unable to inhibit either aggregation or ROS production, respectively. In conclusion, this study suggests that LXA(4) antagonizes P. gingivalis-induced cell activation in a manner that is dependent on leukocyte-platelet interaction, likely via the inhibition of Rho GTPase signaling and the downregulation of CD11b/CD18. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.

  • 8.
    Fälker, Knut
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Department of Biomedicine; Dept Clin & Expt Med, Linköping Univ, Linköping, Sweden.
    Klarström-Engström, Kristin
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Department of Biomedicine.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Department of Biomedicine.
    Lindahl, Tomas L.
    Dept Clin & Expt Med, Linköping Univ, Linköping, Sweden.
    Grenegård, Magnus
    Örebro universitet, Institutionen för läkarutbildning. Department of Biomedicine.
    The Toll-like receptor 2/1 (TLR2/1) complex initiates human platelet activation via the src/Syk/LAT/PLC gamma 2 signalling cascade2014Ingår i: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 26, nr 2, s. 279-286Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 9.
    Fürsatz, Marian
    et al.
    Department of Physics, Chemistry, and Biology, Linköping University, Linköping, Sweden.
    Skog, Mårten
    Department of Physics, Chemistry and Biology, Linkopings universitet, Linköping, Sweden.
    Sivlér, Petter
    Department of Physics, Chemistry and Biology, Linkopings universitet, Linköping, Sweden.
    Palm, Eleonor
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Aronsson, Christopher
    Department of Physics, Chemistry and Biology, Linkopings universitet, Linköping, Sweden.
    Skallberg, Andreas
    Department of Physics, Chemistry and Biology, Linkopings universitet, Linköping, Sweden.
    Greczynski, Grzegorz
    Department of Physics, Linköping University, Linkoping, Sweden.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Aili, Daniel
    Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
    Functionalization of bacterial cellulose wound dressings with the antimicrobial peptide ε-poly-L-Lysine2018Ingår i: Biomedical Materials, ISSN 1748-6041, E-ISSN 1748-605X, Vol. 13, artikel-id 025014Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Wound dressings based on bacterial cellulose (BC) can form a soft and conformable protective layer that can stimulate wound healing while preventing bacteria from entering the wound. Bacteria already present in the wound can, however, thrive in the moist environment created by the BC dressing which can aggravate the healing process. Possibilities to render the BC antimicrobial without affecting the beneficial structural and mechanical properties of the material would hence be highly attractive. Here we present methods for functionalization of BC with ε-Poly-L-Lysine (ε-PLL), a non-toxic biopolymer with broad-spectrum antimicrobial activity. Low molecular weight ε-PLL was cross-linked in pristine BC membranes and to carboxymethyl cellulose (CMC) functionalized BC using carbodiimide chemistry. The functionalization of BC with ε-PLL inhibited growth of S. epidermidis on the membranes but did not affect the cytocompatibility to cultured human fibroblasts as compared to native BC. The functionalization had no significant effects on the nanofibrous structure and mechanical properties of the BC. The possibility to functionalize BC with ε-PLL is a promising, green and versatile approach to improve the performance of BC in wound care and other biomedical applications.

  • 10.
    Jayaprakash, Kartheyaene
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Demirel, Isak
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Gunaltay, Sezin
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    PKC, ERK/p38 MAP kinases and NF-B targeted signalling play a role in the expression and release of IL-1β  and CXCL8 in Porphyromonas gingivalis-infected THP1 cells2017Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 125, nr 7, s. 623-633Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Porphyromonas gingivalis is a keystone pathogen in periodontitis and is gaining importance in cardiovascular pathogenesis. Protease-activated receptors (PARs), toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) on monocytes recognize the structural components on P. gingivalis, inducing inflammatory intermediates. Here, we elucidate the modulation of PARs, TLRs, NODs, and the role of MAPK and NF-B in IL-1 and CXCL8 release. THP1 cells were stimulated with P. gingivalis wild-type W50 and its isogenic gingipain mutants: Rgp mutant E8 and Kgp mutant K1A. We observed modulation of PARs, TLRs, NOD, IL-1 and CXCL8 expression by P. gingivalis. Gingipains hydrolyse IL-1 and CXCL8, which is more evident for IL-1 accumulation at 24 h. Inhibition of PKC (protein kinase C), p38 and ERK (extracellular signal-regulated kinases) partially reduced P. gingivalis-induced IL-1 at 6 h, whereas PKC and ERK reduced CXCL8 at both 6 and 24 h. Following NF-B inhibition, P. gingivalis-induced IL-1 and CXCL8 were completely suppressed to basal levels. Overall, TLRs, PARs and NOD possibly act in synergy with PKC, MAPK ERK/p38 and NF-B in P. gingivalis-induced IL-1 and CXCL8 release from THP1 cells. These pro-inflammatory cytokines could affect leucocytes in circulation and exacerbate other vascular inflammatory conditions such as atherosclerosis.

  • 11.
    Jayaprakash, Kartheyaene
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Demirel, Isak
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Gunaltay, Sezin
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    PKC, ERK/p38 MAP kinases and NF-κB targeted signalling plays a crucial role in expression and release of IL-1β and CXCL8 in Porphyromonas gingivalis infected monocytesManuskript (preprint) (Övrigt vetenskapligt)
  • 12.
    Jayaprakash, Kartheyaene
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Demirel, Isak
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Porphyromonas gingivalis induced release of reactive oxygen species and interleukin-1 beta and the effects of low density lipoproteins in monocytes and whole bloodManuskript (preprint) (Övrigt vetenskapligt)
  • 13.
    Jayaprakash, Kartheyaene
    et al.
    Department of Medical Sciences, Örebro University, Örebro, Sweden.
    Demirel, Isak
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Porphyromonas gingivalis-induced inflammatory responses in THP1 cells are altered by native and modified low-density lipoproteins in a strain-dependent manner2018Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 126, nr 8, s. 667-677Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Strong epidemiological evidence supports an association between cardiovascular and periodontal disease and furthermore, the periodontopathogen Porphyromonas gingivalis has been identified in blood and from atheromatous plaques. Blood exposed to P.gingivalis shows an increased protein modification of low-density lipoprotein (LDL). In this study, we investigate the inflammatory responses of THP1 cells incubated with P.gingivalis and the effects of native or modified LDL on these responses. Reactive oxygen species (ROS) and IL-1 were observed in THP1 cells following infection with P.gingivalis ATCC33277 and W50. Caspase 1 activity was quantified in THP1 cells and correlated with IL-1 accumulation. Oxidized LDL (oxLDL) induced IL-1 release and CD36 expression on THP1 cells. Modified LDL co-stimulated with ATCC33277 exhibited regulatory effects on caspase 1 activity, IL-1 release and CD36 expression in THP1 cells, whereas W50 induced more modest responses in THP1 cells. In summary, we show that P.gingivalis is capable of inducing pro-inflammatory responses in THP1 cells, and native and modified LDL could alter these responses in a dose- and strain-dependent manner. Strain-dependent differences in THP1 cell responses could be due to the effect of P.gingivalis proteases, presence or absence of capsule and proteolytic transformation of native and modified LDL.

  • 14.
    Jayaprakash, Kartheyaene
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Demirel, Isak
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis2015Ingår i: Molecular Oral Microbiology, ISSN 2041-1006, E-ISSN 2041-1014, Vol. 30, nr 5, s. 361-375Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram-negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild-type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild-type strain ATCC33277, whereas the other wild-type strain W50 was less effective. Quantitative real-time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P.gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P.gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria.

  • 15.
    Jayaprakash, Kartheyaene
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Gingipains from Porphyromonas gingivalis play a significant role in induction and regulation of CXCL8 in THP-1 cells2014Ingår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 14, artikel-id 193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Porphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis. The bacterium expresses two kinds of cysteine proteases called gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). This study evaluated the interaction between P. gingivalis and THP-1 cells, a widely used monocytic cell line, in vitro with a focus on CXCL8 at the gene and protein levels and its fate thereafter in cell culture supernatants. THP-1 cells were stimulated with viable and heat-killed wild-type strains ATCC 33277 or W50 or viable isogenic gingipain mutants of W50, E8 (Rgp mutant) or K1A (Kgp mutant), for 24 hours.

    Results: ELISA and qPCR results show an elevated CXCL8 expression and secretion in THP-1 cells in response to P. gingivalis, where the heat-killed ATCC33277 and W50 induced higher levels of CXCL8 in comparison to their viable counterparts. Furthermore, the Kgp-deficient mutant K1A caused a higher CXCL8 response compared to the Rgp-deficient E8. Chromogenic quantification of lipopolysaccharide (LPS) in supernatant showed no significant differences between viable and heat killed bacteria except that W50 shed highest levels of LPS. The wild-type strains secreted relatively more Rgp during the co-culture with THP-1 cells. The CXCL8 degradation assay of filter-sterilized supernatant from heat-killed W50 treated cells showed that Rgp was most efficient at CXCL8 hydrolysis. Of all tested P. gingivalis strains, adhesion and internalization in THP-1 cells was least conspicuous by Rgp-deficient P. gingivalis (E8), as demonstrated by confocal imaging.

    Conclusions: W50 and its Kgp mutant K1A exhibit a higher immunogenic and proteolytic function in comparison to the Rgp mutant E8. Since K1A differs from E8 in the expression of Rgp, it is rational to conclude that Rgp contributes to immunomodulation in a more dynamic manner in comparison to Kgp. Also, W50 is a more virulent strain when compared to the laboratory strain ATCC33277.

  • 16.
    Khalaf, Hazem
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Division of Clinical Medicine,, Faculty of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning. Division of Clinical Medicine, Faculty of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    Altered T-cell responses by the periodontal pathogen Porphyromonas gingivalis2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 9, artikel-id e45192Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several studies support an association between the chronic inflammatory diseases periodontitis and atherosclerosis with a crucial role for the periodontal pathogen Porphyromonas gingivalis. However, the interplay between this pathogen and the adaptive immune system, including T-cells, is sparsely investigated. Here we used Jurkat T-cells to determine the effects of P. gingivalis on T-cell-mediated adaptive immune responses. We show that viable P. gingivalis targets IL-2 expression at the protein level. Initial cellular events, including ROS production and [Ca2+]i, were elevated in response to P. gingivalis, but AP-1 and NF-κB activity dropped below basal levels and T-cells were unable to sustain stable IL-2 accumulation. IL-2 was partially restored by Leupeptin, but not by Cathepsin B Inhibitor, indicating an involvement of Rgp proteinases in the suppression of IL-2 accumulation. This was further confirmed by purified Rgp that caused a dose-dependent decrease in IL-2 levels. These results provide new insights of how this periodontal pathogen evades the host adaptive immune system by inhibiting IL-2 accumulation and thus attenuating T-cell proliferation and cellular communication.

  • 17.
    Khalaf, Hazem
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Demirel, Isak
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Suppression of inflammatory gene expression in T cells by Porphyromonas gingivalis is mediated by targeting MAPK signaling2013Ingår i: Cellular & Molecular Immunology, ISSN 1672-7681, E-ISSN 2042-0226, Vol. 10, nr 5, s. 413-422Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is increasing awareness of the effects of Porphyromonas gingivalis on host immune responses. Degradation of cytokines and chemokines by cysteine proteinases has previously been reported. However, the precise mechanisms by which P. gingivalis is able to alter intracellular signaling, and thus proliferation and inflammation, have not been described. We have previously reported suppression of activator protein-1 (AP-1) and degradation of IL-2 by proteinases from P. gingivalis. In the present study, we have analyzed the effects of P. gingivalis on Jurkat T-cell signal transduction and subsequent IL-2 and CXCL8 expression. We found that CXCL8, but not IL-2, gene expression levels were significantly suppressed by viable P. gingivalis. Analysis of intracellular signaling revealed an inhibitory effect of P. gingivalis on c-Jun and c-Fos, but not NF kappa B (p50 and p65), NFAT or STAT5 expression. This inhibitory effect was not due to suppression of mitogen-activated protein kinase (MAPK) (p38, erk and JNK) gene expression, but was rather due to prevention of protein kinase C (PKC) and p38 phosphorylation, as demonstrated by western blot analysis. Furthermore, SOCS1 and SOCS3 expression levels decreased following treatment of Jurkat T cells with viable P. gingivalis. The results indicate that P. gingivalis is able to suppress inflammatory gene expression by targeting the activity of MAPK pathways in T cells, which was confirmed by using specific inhibitors of NF-kappa B, PKC, ERK, p38 and JNK.

  • 18.
    Khalaf, Hazem
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Lönn, Johanna
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Cytokines and chemokines are differentially expressed in patients with periodontitis: Possible role for TGF-beta 1 as a marker for disease progression2014Ingår i: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 67, nr 1, s. 29-35Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Periodontitis is a chronic inflammatory disease characterized by destruction of periodontal tissue ultimately leading to bone destruction and has been associated with other inflammatory diseases, such as atherosclerosis. Attachment loss of periodontal tissue is primarily caused by host cell-derived immune responses against subgingival biofilm. The aim of the present study was to determine the cytokine profile in serum, saliva and gingival crevicular fluid (GCF) patients with periodontitis and healthy controls. We show that periodontitis patients exhibit higher numbers of periodontal pathogens and their immune responses are significantly altered. The levels of IL-6 in saliva and GCF were significantly suppressed, and while CXCL8 was not altered in serum, its expression levels were significantly suppressed in saliva and elevated in GCF. The T-cell-derived cytokine IL-2 did not differ between patients and controls in serum and saliva, but there was a significant suppression in GCF of patients. Interestingly, TGF-beta(1) levels were significantly elevated in serum, saliva and GCF in patients compared to controls. Furthermore, by using cultured gingival fibroblasts stimulated with wild type and proteinase mutant strains of Porphyromonas gingivalis, we show that the suppression of CXCL8 and IL-6, and the induction of TGF-beta(1) is primarily mediated by the proteolytic activity of lysine-specific proteinases. These results indicate that P. gingivalis is a major contributor to the altered immune responses and the pathology of periodontitis. Furthermore, the ease of sampling and analyzing cytokine expression profiles, including TGF-beta(1), in saliva and GCF may serve to predict the progression of periodontitis and associated systemic inflammatory diseases.

  • 19.
    Khalaf, Hazem
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Nakka, Sravya Sowdamini
    Örebro universitet, Institutionen för hälsovetenskap och medicin. PEAS Institut AB, Söderleden 1, Linköping.
    Sandén, Camilla
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Svärd, Anna
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Hultenby, Kjell
    Division of Clinical Research Centre, Department of Laboratory Medicine, Karolinska.
    Scherbak, Nikolai
    Örebro universitet, Institutionen för naturvetenskap och teknik.
    Aili, Daniel
    PEAS Institut AB, Söderleden 1, Linköping.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Antibacterial effects of lactobacillus and bacteriocin NC8 αβ on the periodontal pathogen Porphyromonas gingivalisManuskript (preprint) (Övrigt vetenskapligt)
  • 20.
    Khalaf, Hazem
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Nakka, Sravya Sowdamini
    Örebro universitet, Institutionen för medicinska vetenskaper. PEAS Institut AB, Linköping, Sweden.
    Sandén, Camilla
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Svärd, Anna
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Hultenby, Kjell
    Division of Clinical Research Centre, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Scherbak, Nikolai
    Örebro universitet, Institutionen för naturvetenskap och teknik.
    Aili, Daniel
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Antibacterial effects of Lactobacillus and bacteriocin PLNC8 αβ on the periodontal pathogen Porphyromonas gingivalis2016Ingår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 16, nr 1, artikel-id 188Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The complications in healthcare systems associated with antibiotic-resistant microorganisms have resulted in an intense search for new effective antimicrobials. Attractive substances from which novel antibiotics may be developed are the bacteriocins. These naturally occurring peptides are generally considered to be safe and efficient at eliminating pathogenic bacteria. Among specific keystone pathogens in periodontitis, Porphyromonas gingivalis is considered to be the most important pathogen in the development and progression of chronic inflammatory disease. The aim of the present study was to investigate the antimicrobial effects of different Lactobacillus species and the two-peptide bacteriocin PLNC8 αβ on P. gingivalis.

    Results: Growth inhibition of P. gingivalis was obtained by viable Lactobacillus and culture media from L. plantarum NC8 and 44048, but not L. brevis 30670. The two-peptide bacteriocin from L. plantarum NC8 (PLNC8 αβ) was found to be efficient against P. gingivalis through binding followed by permeabilization of the membranes, using Surface plasmon resonance analysis and DNA staining with Sytox Green. Liposomal systems were acquired to verify membrane permeabilization by PLNC8 αβ. The antimicrobial activity of PLNC8 αβ was found to be rapid (1 min) and visualized by TEM to cause cellular distortion through detachment of the outer membrane and bacterial lysis.

    Conclusion: Soluble or immobilized PLNC8 αβ bacteriocins may be used to prevent P. gingivalis colonization and subsequent pathogenicity, and thus supplement the host immune system against invading pathogens associated with periodontitis.

  • 21.
    Khalaf, Hazem
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Palm, Eleonor
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Cellular Response Mechanisms in Porphyromonas gingivalis Infection2017Ingår i: Periodontitis: A Useful Reference / [ed] Pachiappan Arjunan, InTech, 2017, s. 45-68Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    The pathogenicity of the periodontal biofilm is highly dependent on a few key species, of which Porphyromonas gingivalis is considered to be one of the most important pathogens. P. gingivalis expresses a broad range of virulence factors, of these cysteine proteases (gingipains) are of special importance both for the bacterial survival/proliferation and for the pathological outcome. Several cell types, for example, epithelial cells, endothelial cells, dendritic cells, osteoblasts, and fibroblasts, reside in the periodontium and are part of the innate host response, as well as platelets, neutrophils, lymphocytes, and monocytes/macrophages. These cells recognize and respond to P. gingivalis and its components through pattern recognition receptors (PRRs), for example, Toll-like receptors and protease-activated receptors. Ligation of PRRs induces downstream-signaling pathways modifying the activity of transcription factors that regulates the expression of genes linked to inflammation. This is followed by the release of inflammatory mediators, for example, cytokines and reactive oxygen species. Periodontal disease is today considered to play a significant role in various systemic conditions such as cardiovascular disease (CVD). The mechanisms by which P. gingivalis and its virulence factors interact with host immune cells and contribute to the pathogenesis of periodontitis and CVD are far from completely understood.

  • 22.
    Klarström-Engström, Kristin
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    The platelet response to various strains of Porphyromonas gingivalisManuskript (preprint) (Övrigt vetenskapligt)
  • 23.
    Klarström-Engström, Kristin
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Brommesson, Caroline
    Department of Physics, Chemistry and Biology, Division of Molecular Surface Physics and Nanoscience, Linköping University, Linköping, Sweden.
    Kälvegren, Hanna
    Department of Clinical Pathology and Clinical Genetics, Linköping University Hospital, Linköping, Sweden; Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden .
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Toll like receptor 2/1 mediated platelet adhesion and activation on bacterial mimetic surfaces is dependent on src/Syk-signaling and purinergic receptor P2X1 and P2Y12 activation2014Ingår i: Biointerphases, ISSN 1934-8630, E-ISSN 1559-4106, Vol. 9, nr 4, s. 041003-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Platelets are considered to have important functions in inflammatory processes as key players in innate immunity. Toll like receptors (TLRs), expressed on platelets, recognize pathogen associated molecular patterns and trigger immune responses. Pathogens are able to adhere to human tissues and form biofilms which cause a continuous activation of the immune system. The authors aimed to investigate how immobilized Pam(3)CSK(4) (a synthetic TLR2/1 agonist) and IgG, respectively, resembling a bacterial focus, affects adhesion and activation of platelets including release of two cytokines, regulated on activation normal T-cell expressed and secreted (RANTES) and macrophage migration inhibitory factor (MIF). The authors also aim to clarify the signaling downstream of TLR2/1 and Fc gamma RII (IgG receptor) and the role of adenine nucleotides in this process. Biolayers of Pam(3)CSK(4) and IgG, respectively, were confirmed by null-ellipsometry and contact angle measurements. Platelets were preincubated with signaling inhibitors for scr and Syk and antagonists for P2X1 or P2Y1 [adenosine triphosphate (ATP), adenosine diphosphate (ADP) receptors] prior to addition to the surfaces. The authors show that platelets adhere and spread on both Pam(3)CSK(4)- and IgG-coated surfaces and that this process is antagonized by scr and Syc inhibitors as well as P2X1 and P2Y antagonists. This suggests that Pam(3)CSK(4) activated platelets utilize the same pathway as Fc gamma RII. Moreover, the authors show that ATP-ligation of P2X1 is of importance for further platelet activation after TLR2/1-activation, and that P2Y12 is the prominent ADP-receptor involved in adhesion and spreading. RANTES and MIF were secreted over time from platelets adhering to the coated surfaces, but no MIF was released upon stimulation with soluble Pam(3)CSK(4). These results clarify the importance of TLR2/1 and Fc gamma RII in platelet adhesion and activation, and strengthen the role of platelets as an active player in sensing bacterial infections.

  • 24.
    Klarström-Engström, Kristin
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Kälvegren, H.
    Department of Clinical Pathology and Clinical Genetics, Linköping University Hospital, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    The role of Porphyromonas gingivalis gingipains in platelet activation and innate immune modulation2015Ingår i: Molecular Oral Microbiology, ISSN 2041-1006, E-ISSN 2041-1014, Vol. 30, nr 1, s. 62-73Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Platelets are considered to have important functions in inflammatory processes and as actors in the innate immunity. Several studies have shown associations between cardiovascular disease and periodontitis, where the oral anaerobic pathogen Porphyromonas gingivalis has a prominent role in modulating the immune response. Porphyromonas gingivalis has been found in atherosclerotic plaques, indicating spreading of the pathogen via the circulation, with an ability to interact with and activate platelets via e.g. Toll-like receptors (TLR) and protease-activated receptors. We aimed to evaluate how the cysteine proteases, gingipains, of P.gingivalis affect platelets in terms of activation and chemokine secretion, and to further investigate the mechanisms of platelet-bacteria interaction. This study shows that primary features of platelet activation, i.e. changes in intracellular free calcium and aggregation, are affected by P.gingivalis and that arg-gingipains are of great importance for the ability of the bacterium to activate platelets. The P.gingivalis induced a release of the chemokine RANTES, however, to a much lower extent compared with the TLR2/1-agonist Pam(3)CSK(4), which evoked a time-dependent release of the chemokine. Interestingly, the TLR2/1-evoked response was abolished by a following addition of viable P.gingivalis wild-types and gingipain mutants, showing that both Rgp and Kgp cleave the secreted chemokine. We also demonstrate that Pam(3)CSK(4)-stimulated platelets release migration inhibitory factor and plasminogen activator inhibitor-1, and that also these responses were antagonized by P.gingivalis. These results supports immune-modulatory activities of P.gingivalis and further clarify platelets as active players in innate immunity and in sensing bacterial infections, and as target cells in inflammatory reactions induced by P.gingivalis infection.

  • 25.
    Klarström-Engström, Kristin
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Magnusson, A.
    Demirel, I.
    Lönn, J.
    Starkhammar Johansson, C.
    Kälvegren, Hanna
    Department of Clinical Pathology and Clinical Genetics, Linköping University Hospital, Linköping, Sweden; Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden .
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Porphyromonas gingivalis-induced lipid peroxidation: the role of platelets, gingipains and periodontal status.Manuskript (preprint) (Övrigt vetenskapligt)
  • 26.
    Klarström-Engström, Kristin
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Skoglund, C.
    Linköping University, Linköping, Sweden.
    Kälvegren, Hanna
    Linköping University Hospital, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    The role of platelets in inflammation at sites of infection: toll like receptor 2/1 mediated platelet adhesion on bacterial peptide-mimetic surfaces2012Ingår i: Cardiovascular Research, ISSN 0008-6363, E-ISSN 1755-3245, Vol. 93, s. S8-S8Artikel i tidskrift (Övrigt vetenskapligt)
  • 27.
    Kälvegren, Hanna
    et al.
    Örebro universitet, Hälsoakademin. Fac Hlth Sci, Dept Med & Hlth Sci, Div Cardiovasc Med, Linköping Univ, Linköping, Sweden.
    Fridfeldt, Jonna
    Fac Hlth Sci, Dept Med & Hlth Sci, Div Drug Res, Linköping Univ, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Hälsoakademin. Fac Hlth Sci, Dept Med & Hlth Sci, Div Drug Res, Linköping Univ, Linköping, Sweden.
    The role of plasma adenosine deaminase in chemoattractant-stimulated oxygen radical production in neutrophils2010Ingår i: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 89, nr 6, s. 462-467Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Objectives: Adenosine deaminase (ADA) has a role in many immunity mediated disorders, such as asthma, tuberculosis and coronary artery disease. This study aims to investigate the ability of plasma ADA to modulate reactive oxygen species (ROS) production in neutrophils, and examine the involvement of adenosine and the cyclic AMP signaling pathway in this process. Methods: Neutrophils were stimulated, in the absence or presence of plasma, with the chemotactic peptide fMLP (formyl-methionyl-leucyl-phenylalanine), and the ROS production was determined with luminol-enhanced chemiluminescence. Activity of ADA was measured spectrophotometrically. Results: Plasma dose-dependently amplified the ROS generation in fMLP-stimulated neutrophils. In parallel, incubation of neutrophils in plasma elevated the total ADA-activity approximately 10 times from 1.3 U/ml to 12 U/ml. Inhibition of ADA, or type IV phosphodiesterases, significantly lowered the plasma-mediated ROS production. Furthermore, the high-affinity adenosine A(1) receptor antagonists DPCPX and 8-phenyltheophylline markedly inhibited the plasma-induced respiratory burst in neutrophils, suggesting an AI receptor-mediated mechanism. Conclusions: This study suggests that plasma ADA amplifies the release of toxic oxygen radicals from neutrophils through a downregulation of the inhibitory adenosine/cAMP-system and an enhanced activation of the stimulatory adenosine A(1)-receptor. This mechanism has probably a crucial role in regulating neutrophil function and in the defence against microbial infections. However, a sustained neutrophil activation could also contribute to inflammatory disorders such as atherosclerosis. (C) 2010 Elsevier GmbH. All rights reserved.

  • 28.
    Kälvegren, Hanna
    et al.
    Örebro universitet, Hälsoakademin. Fac Hlth Sci, Linkoping Univ, Linkoping, Sweden.
    Skoglund, Caroline
    Fac Hlth Sci, Linkoping Univ, Linkoping, Sweden.
    Helldahl, Christian
    Fac Hlth Sci, Linkoping Univ, Linkoping, Sweden.
    Lerm, Maria
    Fac Hlth Sci, Linkoping Univ, Linkoping, Sweden.
    Grenegård, Magnus
    Fac Hlth Sci, Linkoping Univ, Linkoping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Hälsoakademin. Fac Hlth Sci, Linkoping Univ, Linkoping, Sweden.
    Toll-like receptor 2 stimulation of platelets is mediated by purinergic P2X(1)-dependent Ca2+ mobilisation, cyclooxygenase and purinergic P2Y(1) and P2Y(12) receptor activation2010Ingår i: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 103, nr 2, s. 398-407Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Toll-like receptor 2 (TLR2), which recognise and respond to conserved microbial pathogen-associated molecular patterns, is expressed on the platelet surface. Furthermore, it has recently been shown that the TLR2/1 agonist Pam(3)CSK(4) stimulates platelet activation. The aim of the present study was to clarify important signalling events in Pam(3)CSK(4)-induced platelet aggregation and secretion. Platelet interaction with Pam(3)CSK(4) and the TLR2/6 agonist MALP-2 was studied by analysing aggregation, ATP-secretion, [Ca2+](i) mobilisation and thromboxane B2 (TxB(2)) production. The results show that Pam(3)CSK(4) but not MALP-2 induces [Ca2+](i) increase, TxB(2) production, dense granule secretion and platelet aggregation. Preincubation of platelets with MALP-2 inhibited the Pam(3)CSK(4)-induced responses. The ATP-secretion and aggregation in Pam(3)CSK(4)-stimulated platelets was impeded by the purinergic P2X(1) inhibitor MRS 2159, the purinergic P2Y(1) and P2Y(12) antagonists MRS 2179 and cangrelor, the phospholipase C inhibitor U73122, the calcium chelator BAPT-AM and aspirin. The calcium mobilisation was lowered by MRS 2159, aspirin and U73122 whereas the TxB(2) production was antagonised by MRS 2159, aspirin and BAPT-AM. When investigating the involvement of the myeloid differentiation factor-88 (MyD88) -dependent pathway, we found that platelets express MyD88 and interleukin 1 receptor-associated kinase (IRAK-1), which are proteins important in TLR signalling. However, Pam(3)CSK(4) did not stimulate a rapid (within 10 minutes) phosphorylation of IRAK-1 in platelets. In conclusion, the results show that Pam(3)CSK(4)-induced platelet aggregation and secretion depends on a P2X(1)-mediated Ca2+ mobilisation, production of TxA(2) and ADP receptor activation. The findings in this study further support a role for platelets in sensing bacterial components.

  • 29.
    Ljunggren, Stefan
    et al.
    Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Karlsson, Helen
    Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Johansson Starkhammar, Carin
    Centre for Oral Rehabilitation, Public Dental Health Care County Council of Östergötland, Linköping, Sweden.
    Palm, Eleonor
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Nayeri, Fariba
    PEAS Institute, Linköping, Sweden.
    Ghafouri, Bijar
    Pain and Rehabilitation Centre, and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden.
    Davies, Julia
    Department of Oral Biology, Malmö University, Malmö, Sweden.
    Svensäter, Gunnel
    Department of Oral Biology, Malmö University, Malmö, Sweden.
    Lönn, Johanna
    Örebro universitet, Institutionen för medicinska vetenskaper. PEAS Research Institute, Department of Infection Control, Linköping, Sweden; Department of Oral Biology, Faculty of Odontology, Malmö University, Malmö, Sweden .
    Modified lipoproteins in periodontitis: a link to cardiovascular disease?2019Ingår i: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 39, nr 3, artikel-id BSR20181665Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is a strong association between periodontal disease and atherosclerotic cardiovascular disorders. A key event in the development of atherosclerosis is accumulation of modified lipoproteins within the arterial wall. We hypothesize that patients with periodontitis have an altered lipoprotein profile towards an atherogenic form. Therefore, this study aims at identifying modifications of plasma lipoproteins in periodontitis. Lipoproteins from ten female patients with periodontitis and gender- and age-matched healthy controls were isolated by density-gradient-ultracentrifugation. Proteins were separated by two-dimensional gel-electrophoresis and identified by map-matching or by nano-liquid chromatography followed by mass spectrometry. ApoA-I methionine oxidation, Oxyblot, total antioxidant capacity and a multiplex of 71 inflammation-related plasma proteins were assessed.Reduced levels of apoJ, phospholipid transfer protein, apoF, complement C3, paraoxonase 3 and increased levels of alpha-1-antichymotrypsin, apoA-II, apoC-III were found in HDL from the patients. In LDL/VLDL, the levels of apoL-1 and platelet-activating factor acetylhydrolase as well as apo-B fragments were increased. Methionine oxidation of apoA-I was increased in HDL and showed a relationship with periodontal parameters. Alpha-1 antitrypsin and alpha-2-HS glycoprotein were oxidised in LDL/VLDL and antioxidant capacity was increased in the patient group. 17 inflammation-related proteins were important for group separation with the highest discriminating proteins identified as IL-21, Fractalkine, IL-17F, IL-7, IL-1RA and IL-2.Patients with periodontitis have an altered plasma lipoprotein profile, defined by altered protein levels as well as posttranslational and other structural modifications towards an atherogenic form, which supports a role of modified plasma lipoproteins as central in the link between periodontal and cardiovascular disease (CVD).

  • 30.
    Lönn, J.
    et al.
    Department of Oral Biology, Institute of Odontology, Malmö University, Malmö, Sweden; PEAS Institute AB, Linköping, Sweden.
    Ljunggren, S.
    Department of Clinical and Experimental Medicine, Occupational and Environmental Medicine Center, Linköping University, Linköping, Sweden.
    Klarström-Engström, Kristin
    Department of Medical Sciences, Örebro University, Örebro, Sweden.
    Demirel, Isak
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Karlsson, H.
    Department of Clinical and Experimental Medicine, Occupational and Environmental Medicine Center, Linköping University, Linköping, Sweden.
    Lipoprotein modifications by gingipains of Porphyromonas gingivalis2018Ingår i: Journal of Periodontal Research, ISSN 0022-3484, E-ISSN 1600-0765, Vol. 53, nr 3, s. 403-413Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND AND OBJECTIVE: Several studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD, and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis is the major pathogen in the disease. Several studies support a role of modified low-density lipoprotein (LDL) in atherogenesis; however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims to identify alterations in plasma lipoproteins induced by the periodontopathic species of bacterium, P. gingivalis, in vitro.

    MATERIAL AND METHODS: Plasma lipoproteins were isolated from whole blood treated with wild-type and gingipain-mutant (lacking either the Rgp- or Kgp gingipains) P. gingivalis by density/gradient-ultracentrifugation and were studied using 2-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry. Porphyromonas gingivalis-induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid-reactive substances and antioxidant assay kits, respectively, and lumiaggregometry was used for measurement of reactive oxygen species (ROS) and aggregation.

    RESULTS: Porphyromonas gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing 2 apoE fragments, as well as 2 apoB-100 fragments, in LDL, and the Kgp gingipain produced an unidentified fragment in high-density lipoproteins. Porphyromonas gingivalis and its different gingipain variants induced ROS and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation.

    CONCLUSIONS: Porphyromonas gingivalis has the potential to change the expression of lipoproteins in blood, which may represent a crucial link between periodontitis and CVD.

  • 31.
    Lönn, Johanna
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Department of Clinical Medicine, Faculty of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    Hallström, J.
    Department of Clinical Medicine, Faculty of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Department of Clinical Medicine, Faculty of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    P. gingivalis-induced aggregation and ros production in whole blood is dependent on gingipains2012Ingår i: Cardiovascular Research, ISSN 0008-6363, E-ISSN 1755-3245, Vol. 93, s. S35-S35Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    A large body of data accumulated over the past several years suggests that the periodontal pathogen Porphyromonas gingivalis is associated with cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. We have previously demonstrated that P. gingivalis induces aggregation and ROS production in whole blood, and that the anti-inflammatory mediator lipoxin A4 (LXA4) inhibits these responses by modulating plateletneutrophil interaction through a down-regulation of the bacterium-induced surface expression of CD11b/CD18 on neutrophils, likely by inhibiting Rac2 and Cdc42 signaling pathways. Furthermore, P. gingivalis, unlike other periodontopathic bacteria, has been shown to trigger platelet aggregation, mainly through the interaction between bacterial gingipains and protease-activating receptors (PARs) on the platelets. Since platelet aggregation precedes thromboembolic events, this is an important pathogenic feature of the bacterium. The aim of this study was to investigate the effect of gingipains on P. gingivalis-induced cell activation in whole blood. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry. This study shows that leupeptin, a protease inhibitor of gingipains, inhibits P. gingivalis-induced aggregation and ROS production in whole blood. Supernatants of bacteria suspensions induced no ROS-production, but an aggregatory response that was also inhibited by leupeptin. In conclusion, P. gingivalis-induced aggregation and ROS production in whole blood is mainly dependent on gingipains. However, since bacterial supernatants (containing soluble gingipains) stimulate only aggregation, this suggests that a gingipain/PAR-mediated mechanism in combination with phagocytosis of whole bacterium is a prerequisite for inducing a respiratory burst and an inflammatory response. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.

  • 32.
    Lönn, Johanna
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin. PEAS Institute, Linköping, Sweden; Clinical Research Center, Örebro University, Örebro, Sweden.
    Johansson, Carin Starkhammar
    Division of Cardiovascular Medicine, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden; Centre for Oral Rehabilitation, Public Dental Health Care, County Council of Östergötland, Linköping, Sweden.
    Nakka, Sravya
    The Institution for Protein Environment Affinity Surveys (PEAS Institute), Linköping, Sweden.
    Palm, Eleonor
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Nayeri, Fariba
    The Institution for Protein Environment Affinity Surveys (PEAS Institute), Linköping, Sweden ; Div Infect Dis, Linköping Univ Hosp, Linköping, Sweden.
    Ravald, Nils
    Division of Cardiovascular Medicine, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden; Centre for Oral Rehabilitation, Public Dental Health Care, County Council of Östergötland, Linköping, Sweden.
    High Concentration but Low Activity of Hepatocyte Growth Factor in Periodontitis2014Ingår i: Journal of Periodontology, ISSN 0022-3492, E-ISSN 1943-3670, Vol. 85, nr 1, s. 113-122Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: High levels of hepatocyte growth factor (HGF), a healing factor with regenerative and cytoprotective effects, are associated with inflammatory diseases, including periodontitis. HGF biological activity requires binding to its receptors, the proto-oncogene c-Met (c-Met) and heparan sulphate proteoglycan (HSPG). Here we investigated HGF expression and its relationship to subgingival microbiota in medically healthy individuals with and without periodontitis.

    Methods: Saliva, gingival crevicular fluid (GCF), and blood samples from 30 patients with severe periodontitis and 30 healthy controls were analyzed for HGF concentration using enzyme-linked immunosorbent assay (ELISA), and binding affinity for HSPG and c-Met using surface plasmon resonance (SPR). The regenerative effects of saliva from three patients and controls were analyzed in an in vitro model of cell injury. Subgingival plaques were analyzed for the presence of 18 bacterial species.

    Results: Patients with periodontitis showed higher HGF concentrations in saliva, GCF, and serum (P < 0.001); however, the binding affinities for HSPG and c-Met were reduced in GCF and saliva (P < 0.002). In contrast to the controls, saliva from patients showed no significant regenerative effect over time on gingival epithelial cells. Compared to controls, patients had a higher prevalence of periodontal-related bacteria.

    Conclusion: Higher circulatory HGF levels indicate a systemic effect of periodontitis. However, the HGF biological activity at local inflammation sites was reduced, and this effect was associated with the amount of periodontal bacteria. Loss of function of healing factors may be an important mechanism in degenerative processes in periodontally susceptible individuals.

  • 33.
    Lönn, Johanna
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin. PEAS Institute, Linköping, Sweden.
    Shahzad, Faisal
    PEAS Institute, Linköping, Sweden.
    Uhlin, Fredrik
    Department of Nephrology UHL, County Council of Östergötland, Linköping, Sweden; Department of Medicine and Health Science, Faculty of Health Science, Linköping University, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning. Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Almroth, Gabriel
    Department of Nephrology UHL, County Council of Östergötland, Department of Medicine and Health Science, Faculty of Health Science, Linköping University, Linköping, Sweden.
    Nayeri, Fariba
    PEAS Institute, Linköping, Sweden; Department of Molecular and Clinical Medicine, Division of Infectious Diseases, University Hospital, Linköping, Sweden.
    High concentration but low biological activity of hepatocyte growth factor in patients with chronic renal failure2012Ingår i: Advances in Bioscience and Biotechnology, ISSN 2156-8456, E-ISSN 2156-8502, Vol. 3, nr 4, s. 516-523Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hepatocyte growth factor (HGF) is a renotropic, antifibrotic and regenerative factor with cytoprotective effects that is produced by mesenchymal cells and shows high affinity to components of extra cellular matrix, such as heparan sulphate proteoglycan (HS-PG), in healthy. Patients with chronic renal failure (CRF) suffer from a chronic inflammatory disorder. In order to assess the underlying mechanisms for development of CRF we aimed to assess the amounts and affinity of HGF in this patient group. Elisa, western blot and surface plasmon resonance (SPR) were used to study HGF in blood samples, as well as in isolated neutrophils, in CRF patients compared to healthy controls. Patients with CRF showed higher HGF levels in serum (P < 0.0001), but decreased affinity to HSPG (P < 0.0001), compared to healthy controls. Addition of protease inhibitors decreased the difference between patients with CRF compared to healthy individuals. HGF with potent regenerative function during injury lacks affinity to HSPG in patients with CRF that may depend on production of proteases from activated immune cells. This information might be used to highlight underlying mechanisms for chronicity and leading to new strategies for treatment of chronic injuries.

  • 34.
    Lönn, Johanna
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin. PEAS Institute,Linköping, Sweden.
    Starkhammar Johansson, C.
    Division of Cardiovascular Medicine, Department of Medical and Health Sciences, Linköping University, Linköping, sweden; Centre for Oral Rehabilitation, Public Dental Health Care, County Council of Östergötland, Linköping, Sweden.
    Kälvegren, H.
    Division of Clinical Pathology and Clinical Genetics, Linköping University, Faculty of Health Sciences, Linköping, Sweden.
    Brudin, L.
    Department of Medical and Health Sciences, University Hospital, Linköping, Sweden.
    Skoglund, C.
    Department of Medical and Health and Sciences, Division of Drug Research, Faculty of Health Sciences, Linköping University, Linköping, Sweden; Department of Physics, Chemistry and Biology, Division of Molecular Physics and Nanoscience, Linköping University, Linköping, Sweden.
    Garvin, P.
    Division of Community Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
    Särndahl, Eva
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Department ofCardiology, Örebro UniversityHospital, Örebro, Sweden.
    Ravald, N.
    Division of Cardiovascular Medicine, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden; Centre for Oral Rehabilitation, Public Dental Health Care, County Council of Östergötland, Linköping, Sweden.
    Richter, A.
    Department of Cardiology, Heart Center, Linköping University Hospital, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Nayeri, F.
    PEAS Institute, Linköping, Sweden; Department of Molecular and Clinical Medicine, Division of Infectious Diseases, University Hospital, Linköping, Sweden.
    Hepatocyte growth factor in patients with coronary artery disease and its relation to periodontal condition2012Ingår i: Results in Immunology, ISSN 0105-1121, E-ISSN 2211-2839, Vol. 2, s. 7-12Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hepatocyte growth factor (HGF) is an angiogenic, cardioprotective factor important for tissue and vascular repair. High levels of HGF are associated with chronic inflammatory diseases, such as coronary artery disease (CAD) and periodontitis, and are suggested as a marker of the ongoing atherosclerotic event in patients with CAD. Periodontal disease is more prevalent among patients with CAD than among healthy people. Recent studies indicate a reduced biological activity of HGF in different chronic inflammatory conditions. Biologically active HGF has high affinity to heparan sulfate proteoglycan (HSPG) on cell-membrane and extracellular matrix. The aim of the study was to investigate the serum concentration and the biological activity of HGF with ELISA and surface plasmon resonance (SPR), respectively, before and at various time points after percutaneous coronary intervention (PCI) in patients with CAD, and to examine the relationship with periodontal condition. The periodontal status of the CAD patients was examined, and the presence of P. gingivalis in periodontal pockets was analyzed with PCR. The HGF concentration was significantly higher, at all time-points, in patients with CAD compared to the age-matched controls (P< 0.001), but was independent of periodontal status. The HGF concentration and the affinity to HSPG adversely fluctuated over time, and the biological activity increased one month after intervention in patients without periodontitis. We conclude that elevated concentration of HGF but with reduced biological activity might indicate a chronic inflammatory profile in patients with CAD and periodontitis.

  • 35.
    Nakka, Sravya Sowdamini
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin. The Institution for Protein Environmental Affinity Surveys, PEAS Institut AB, Linköping, Sweden.
    Lönn, Johanna
    Örebro universitet, Institutionen för hälsovetenskap och medicin. The Institution for Protein Environmental Affinity Surveys, PEAS Institut AB, Linköping, Sweden.
    Starkhammar Johansson, Carin
    Centre for Oral Rehabilitation, Public Dental Health Care, County Council of Östergötland, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Nayeri, Fariba
    The Institution for Protein Environmental Affinity Surveys, PEAS Institut AB, Linköping, Sweden; Division of Infectious Diseases, University Hospital, Linköping, Sweden.
    Antibodies produced in vitro in the detection of periodontal bacteria by using surface plasmon resonance analysis2015Ingår i: Clinical and Experimental Dental Research, ISSN 2057-4347, Vol. 1, nr 1, s. 32-44Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Porphyromonas gingivalis (P. gingivalis) is a major etiological agent associated with periodontitis. This study aims to develop antibodies to P. gingivalis in vitro for real-time detection of bacteria in clinical samples. Lymphocytes were isolated from whole blood of patient treated for periodontitis and were stimulated with P. gingivalis ATCC 33277. B-cell maturation to long-living antibody secreting-plasma cells was studied using flow cytometry and immunofluorescence staining. The antibodies developed in vitro were immobilized onto a CM-5 sensor chip of a biosensor to detect the presence of P. gingivalis in the gingival crevicular fluid of patients with periodontitis compared to periodontally healthy controls (n = 30). Surface plasmon resonance (SPR) analysis was performed to evaluate specific interactions of bacteria in samples with the immobilized antibodies. The results of SPR analysis were compared to the detection of P. gingivalis in the samples using DNA–DNA checkerboard hybridization technique. A clear and distinct change in lymphocyte morphology upon stimulation with P. gingivalis was observed. Anti-P. gingivalis antibodies secreted by CD38+ plasma cells showed the presence of all the four IgG subclasses. The results of DNA–DNA checkerboard analysis were in agreement with that of SPR analysis for the detection of P. gingivalis in patient samples. Furthermore, incubation with anti-P. gingivalis attenuated the bacterial response in SPR. The in vitro method for antibody production developed during this study could be used for an efficient real-time detection of periodontitis, and the attenuating effects of in vitro antibodies suggest their role in passive immunization to prevent periodontitis and their associated risk factors.

  • 36.
    Nakka, Sravya Sowdamini
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper. The Institution for Protein Environmental Affinity Surveys, PEAS Institut AB, Linköping, Sweden.
    Palm, Eleonor
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Bacteriocin plantaricin NC8 αβ antagonizes Porphyromonas gingivalis infection and induces proliferation of gingival epithelial cellsManuskript (preprint) (Övrigt vetenskapligt)
  • 37.
    Nakka, Sravya Sowdamini
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper. The Institution for Protein Environmental Affinity Surveys, PEAS Institut AB, Linköping, Sweden.
    Palm, Eleonor
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Nayeri, Fariba
    The Institution for Protein Environmental Affinity Surveys, PEAS Institut AB, Linköping, Sweden; Division of Infectious Diseases, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Effects of plantaricin NC8 αβ and antibodies on gingival epithelial cells infected by Porphyromonas gingivalisManuskript (preprint) (Övrigt vetenskapligt)
  • 38.
    Nixon Tangi, Tebeng
    et al.
    Department of Clinical Medicine, School of Health and Medical Science, Örebro University, Örebro, Sweden.
    Elmabsout, Ali Ateia
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Sirsjö, Allan
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Fransén, Karin
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Role of NLRP3 and CARD8 in the regulation of TNF-α induced IL-1β release in vascular smooth muscle cells2012Ingår i: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 30, nr 3, s. 697-702Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Interleukin (IL)-1β is known to be activated by the inflammasome. Inflammasome activities depend on a plethora of moieties including NLRP3 and CARD8, which have been reported to be associated with several inflammatory diseases. Aortic smooth muscle cells (AOSMCs) were transfected with siRNA targeting the NLRP3 and CARD8 genes, followed by tumor necrosis factor-α (TNF-α) treatment. We found that TNF-α induces IL-1β, IL-1Ra and NLRP3 genes but not CARD8. Silencing of the NLRP3 gene significantly decreased IL-1β expression and release, the IL-1Ra expression showed a borderline non-significant increment, while CARD8 knockdown did not affect the IL-1β and IL-1Ra mRNA expression or IL-1β protein release. Our results suggest that mainly NLRP3 plays a role in the regulation of IL-1β expression and release in AOSMC and could be a potential future target for the treatment of atherosclerosis and other inflammatory diseases.

  • 39.
    Palm, Eleonor
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Demirel, Isak
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    The role of toll-like and protease-activated receptors and associated intracellular signalling in Porphyromonas gingivalis-infected gingival fibroblastsManuskript (preprint) (Övrigt vetenskapligt)
  • 40.
    Palm, Eleonor
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Demirel, Isak
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    The role of toll-like and protease-activated receptors in the expression of cytokines by gingival fibroblasts stimulated with the periodontal pathogen Porphyromonas gingivalisManuskript (preprint) (Övrigt vetenskapligt)
  • 41.
    Palm, Eleonor
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Department of Biomedicine, Örebro University Hospital, Örebro, Sweden.
    Demirel, Isak
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Department of Biomedicine, Örebro University Hospital, Örebro, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning. Department of Biomedicine, Örebro University Hospital, Örebro, Sweden.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Department of Biomedicine, Örebro University Hospital, Örebro, Sweden.
    The role of toll-like and protease-activated receptors in the expression of cytokines by gingival fibroblasts stimulated with the periodontal pathogen Porphyromonas gingivalis2015Ingår i: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 76, nr 2, s. 424-432Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Porphyromonas gingivalis is a periodontitis-associated pathogen and interactions between the bacterium and gingival fibroblasts play an important role in development and progression of periodontitis, an inflammatory disease leading to degeneration of tooth-supporting structures. Gingival fibroblasts, which expresses protease activated receptors (PARs) as well as toll-like receptors (TLRs), produces inflammatory mediators upon bacterial challenges. In this study, we elucidated the importance of PAR1, PAR2, TLR2 and TLR4 for the expression and secretion of CXCL8, interleukin-6 (IL-6), transforming growth factor-beta 1 (TGF-beta 1) and secretory leukocyte inhibitor (SLPI). Human gingival fibroblasts were transfected with small-interfering RNA against the target genes, and then stimulated with P. gingivalis wild-type W50 and W50-derived double rgp mutant E8 and kgp mutant K1A. TLR2-silencing reduced P. gingivalis-induced CXCL8 and IL-6. IL-6 was also reduced after PAR1-silencing. No effects were observed for TGF-beta 1. SLPI was suppressed by P. gingivalis and silencing of PAR1 as well as TLR2, gave additional suppression at the mRNA level. TLR4 was not involved in the regulation of the investigated mediators. CXCL8 and IL-6 are important for progression and development of periodontitis, leading to a chronic inflammation that may contribute to the tissue destruction that follows an exacerbated host response. Therefore, regulating the expression of TLR2 and subsequent release of CXCL8 and IL-6 in periodontitis could attenuate the tissue destruction seen in periodontitis.

  • 42.
    Palm, Eleonor
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Porphyromonas gingivalis downregulates the immune response of fibroblasts2013Ingår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, s. 155-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Porphyromonas gingivalis is a key pathogen in periodontitis, an inflammatory disease leading to destruction of bone and tooth-supporting tissue. P. gingivalis possesses a number of pathogenic properties to enhance growth and survival, including proteolytic gingipains. Accumulating data shows that gingipains are involved in the regulation of host inflammatory responses. The aim of this study was to determine if P. gingivalis infection modulates the inflammatory response of fibroblasts, including the release of chemokines and cytokines. Human gingival fibroblasts or primary dermal fibroblasts were pre-stimulated with tumor-necrosis factor-alpha (TNF-alpha) and cocultured with P. gingivalis. Gingipain inhibitors were used to explore the effect of gingipains. CXCL8 levels were determined with ELISA and the relative levels of various inflammatory mediators were determined by a cytokine assay.

    Results: TNF-alpha-triggered CXCL8 levels were completely abolished by viable P. gingivalis, whereas heat-killed P. gingivalis did not suppress CXCL8. Accumulation of CXCL8 was partially restored by an arginine-gingipain inhibitor. Furthermore, fibroblasts produced several inflammatory mediators, notably chemokines, all of which were suppressed by viable P. gingivalis.

    Conclusion: These findings provide evidence that fibroblast-derived inflammatory signals are modulated by heat-instable gingipains, whereby the bacteria can escape killing by the host immune system and promote its own growth and establishment. In addition, we show that fibroblasts are important mediators of inflammation in response to infection and thereby play a crucial role in determining the nature and magnitude of the invasion of immune cells.

  • 43.
    Palm, Eleonor
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Suppression of inflammatory responses of human gingival fibroblasts by gingipains from Porphyromonas gingivalis2015Ingår i: Molecular Oral Microbiology, ISSN 2041-1006, E-ISSN 2041-1014, Vol. 30, nr 1, s. 74-85Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The interaction between human gingival fibroblasts (HGFs) and Porphyromonas gingivalis plays an important role in the development and progression of periodontitis. Porphyromonas gingivalis possesses several virulence factors, including cysteine proteases, the arginine-specific (Rgp) and lysine-specific (Kgp) gingipains. Studying the mechanisms that P.gingivalis, and its derived virulence, use to propagate and interact with host cells will increase the understanding of the development and progression of periodontitis. In this study, we aimed to elucidate how P.gingivalis influences the inflammatory events in HGFs regarding transforming growth factor-(1) (TGF-(1)), CXCL8, secretory leucocyte protease inhibitor (SLPI), c-Jun and indoleamine 2,3-dioxygenase (IDO). HGFs were inoculated for 6 and 24h with the wild-type strains ATCC 33277 and W50, two gingipain-mutants of W50 and heat-killed ATCC 33277. The P.gingivalis regulated CXCL8 and TGF-(1) in HGFs, and the kgp mutant gave significantly higher immune response with increased CXCL8 (P<0.001) and low levels of TGF-(1). We show that HGFs express and secrete SLPI, which was significantly suppressed by P.gingivalis (P<0.05). This suggests that by antagonizing SLPI, P.gingivalis contributes to the tissue destruction associated with periodontitis. Furthermore, we found that P.gingivalis inhibits the expression of the antimicrobial IDO, as well as upregulating c-Jun (P<0.05). In conclusion, P.gingivalis both triggers and suppresses the immune response in HGFs. Consequently, we suggest that the pathogenic effects of P.gingivalis, and especially the activity of the gingipains on the inflammatory and immune response of HGFs, are crucial in periodontitis.

  • 44.
    Paramel, Geena
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Fransén, Karin
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Hurtig-Wennlöf, Anita
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Jansson, Jan-Håkan
    Department of Internal Medicine, Skellefteå Hospital and Umeå University Hospital.
    Sirsjö, Allan
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Q705K variant in NLRP3 gene confers protection against myocardial infarction in female individuals2013Ingår i: Biomedical Reports, ISSN 2049-9434, Vol. 1, nr 6, s. 879-882Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Inflammation is a multifaceted process that underlies the pathophysiology of acute myocardial infarction (MI). Variations in the inflammasome‑related NLRP3 gene have been associated with risk for a number of different inflammatory diseases. Therefore, Q705K polymorphism in NLRP3 gene likely confers susceptibility to risk for MI. A First‑ever myocardial Infarction study in Ac‑county (FIA) cohort comprising 555 MI patients and 1,016 controls was used to genotype rs35829419 in the NLRP3 gene by TaqMan single‑nucleotide polymorphism assay. C‑reactive protein (CRP) was measured in the study participants by ELISA. The results showed no significant association between the variant rs35829419 and MI. However, the minor A allele of the rs35829419 polymorphism conferred a protective effect against the risk of developing MI in females. The minor A allele of rs35829419 polymorphism was also associated with increased CRP levels in males. Results of the study suggested a gender‑specific deregulation of NLRP3 gene mediated by rs35829419 polymorphism that confers protection against MI in females but has no effect on MI susceptibility in males. However, the rs35829419 polymorphism was associated with increased CRP levels among the male subjects, thereby demonstrating the possible effect of the Q705K polymorphism in elevating the basal active state of innate immune response.

  • 45.
    Paramel Varghese, Geena
    et al.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Zhang, Boxi
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Khalaf, Hazem
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Ljungberg, Liza U.
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Sirsjö, Allan
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för läkarutbildning.
    Fransén, Karin
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Poryphyromonas gingivalis induces IL-1β in aortic smooth muscle cells: possible role of gingipains?Manuskript (preprint) (Övrigt vetenskapligt)
  • 46.
    Selegård, Robert
    et al.
    Faculty of Medicine & Health, School of Medical Sciences, Örebro University, Örebro, Sweden.
    Musa, Amani
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Nyström, Pontus
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Aili, Daniel
    Division of Molecular Physics, Department of Physics, Chemistry & Biology (IFM), Linköping University, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Khalaf, Hazem
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Plantaricins markedly enhance the effects of traditional antibiotics against Staphylococcus epidermidis2019Ingår i: Future microbiology, ISSN 1746-0913, Vol. 14, nr 3, s. 195-206Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    AIM: Bacteriocins are considered as promising alternatives to antibiotics against infections. In this study, the plantaricins (Pln) A, E, F, J and K were investigated for their antimicrobial activity against Staphylococcus epidermidis.

    MATERIALS & METHODS: The effects on membrane integrity were studied using liposomes and viable bacteria, respectively.

    RESULTS: We show that PlnEF and PlnJK caused rapid and significant lysis of S. epidermidis, and induced lysis of liposomes. The PlnEF and PlnJK displayed similar mechanisms by targeting and disrupting the bacterial cell membrane. Interestingly, Pln enhanced the effects of different antibiotics by 30- to 500-fold.

    CONCLUSION: This study shows that Pln in combination with low concentrations of antibiotics is efficient against S. epidermidis and may be developed as potential treatment of infections.

  • 47.
    Skoglund, Caroline
    et al.
    Department of Biomedicine, School of Health and Medical Sciences, Örebro University, Örebro, Sweden; Division of Molecular Surface Physics and Nanoscience, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
    Wettero, Jonas
    Rheumatology/Autoimmunity and Immune Regulation Unit (AIR), Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Institutionen för hälsovetenskap och medicin. Department of Biomedicine, Faculty of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    C1q regulates collagen-dependent production of reactive oxygen species, aggregation and levels of soluble P-selectin in whole blood2012Ingår i: Immunology Letters, ISSN 0165-2478, E-ISSN 1879-0542, Vol. 142, nr 1-2, s. 28-33Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Blood platelets express several receptors involved in immunity (e.g. complement-, toll-like- and Fc gamma-receptors) and release inflammatory mediators. Furthermore, formation of platelet-leukocyte aggregates has an important role during inflammatory conditions such as coronary artery disease. Thus, apart from their well-known role in haemostasis, platelets are today also recognized as cells with immunomodulatory properties. We have previously reported regulatory effects of complement protein 1q (C1q) on platelet activation in experimental setups using isolated cells. In the present study we have proceeded by investigating effects of C1q on collagen-induced aggregation, production of reactive oxygen species (ROS), formation of platelet-leukocyte aggregates and levels of soluble P-selectin in whole blood. Impedance measurements showed that C1q inhibited collagen-induced aggregation whereas it potentiated the collagen-provoked production of ROS in a luminol-dependent chemiluminescence assay. The effects of C1q on aggregation and ROS-production were dependent upon platelets, as they were no longer observed in presence of the platelet (GpIIb/IIIa) inhibitor Reopro. Furthermore, the levels of soluble P-selectin were found to be lowered upon treatment with C1q prior to addition of collagen. There was also a trend towards a decreased formation of large platelet-leukocyte aggregates in collagen-stimulated whole blood following C1q treatment. In conclusion, our data indicate that C1q could have a role in regulating platelet activation and associated leukocyte recruitment during vessel wall injury. This has implications for inflammatory disorders such as coronary artery disease.

  • 48.
    Skoglund, Caroline
    et al.
    Div Drug Res, Dept Med & Hlth Sci, Linkoping Univ, Linkoping, Sweden.
    Wetterö, Jonas
    Rheumatol Autoimmun & Immune Regulat Unit, Dept Clin & Expt Med, Linkoping Univ, Linkoping, Sweden.
    Tengvall, Pentti
    Inst Clin Sci, Dept Biomat, Sahlgrenska Acad, Univ Gothenburg, Gothenburg, Sweden.
    Bengtsson, Torbjörn
    Örebro universitet, Hälsoakademin. Div Drug Res, Dept Med & Hlth Sci, Linkoping Univ, Linkoping, Sweden.
    C1q induces a rapid up-regulation of P-selectin and modulates collagen- and collagen-related peptide-triggered activation in human platelets2010Ingår i: Immunobiology, ISSN 0171-2985, E-ISSN 1878-3279, Vol. 215, nr 12, s. 987-995Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Blood platelets are emerging as important immunomodulatory cells, but complement interaction with platelets is not well understood. Several platelet structures have been described as complement protein 1q (C1q) binding receptors, such as C1qRp/CD93 and gC1qR. However, there are conflicting results whether these receptors are C1q binding structures, or even at all expressed on the cell surface. Recently, the collagen-binding integrin alpha II beta I was reported to bind C1q on mast cells, and this receptor is also present on platelets. The aim of this study was to further characterize the effects of C1q on platelets, by quantifying the platelet surface expression of P-selectin (CD62P) and monitoring the formation of platelet-neutrophil aggregates. Using flow cytometry, we found that C1q dose-dependently triggered a rapid but moderate and transient up-regulation of P-selectin already within 5s of C1q exposure. Pre-incubation with an antibody directed against gC1qR significantly inhibited (with 57% compared to control) the up-regulation, whereas an antibody towards the alpha II beta I-integrin showed no effect. Stimulation with C1q did not change the cytosolic calcium-levels, as measured with the fluorescent ratiometric probe Fura-2, however, a protein kinase C inhibitor (GF109203x) blocked the C1q-induced P-selectin expression. Furthermore, pre-incubation of platelets with C1q diminished both the collagen as well as the collagen-related peptide-induced up-regulation of P-selectin, most evident after 90 s of stimulation. This indicates that C1q may regulate platelet activation via the GPVI receptor, which is a novel finding. Moreover, C1q antagonized the collagen-induced formation of platelet-neutrophil aggregates, indicating a reduced interaction between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1(PSGL-1/CD162). In summary, C1q induces a moderate rapid platelet P-selectin expression, modulates subsequent collagen and collagen-related peptide stimulation of platelets, and inhibits the formation of platelet-neutrophil aggregates. These immuno-regulatory effects of C1q may have a crucial role in innate immunity and inflammation. (C) 2009 Elsevier GmbH. All rights reserved.

  • 49. Svensson Holm, A.-C.
    et al.
    Bengtsson, Torbjörn
    Örebro universitet, Hälsoakademin.
    Grenegård, M.
    Lindström, E. G.
    Platelets stimulate airway smooth muscle cell proliferation through mechanisms involving 5-lipoxygenase and reactive oxygen species2008Ingår i: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 19, nr 7, s. 528-536Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Continuous recruitment and inappropriate activity of platelets in the airways may contribute to airway remodeling, a characteristic feature of inflammatory airway diseases that includes increased proliferation of the smooth muscle. The aim of the present investigation was to examine the effect of platelets on proliferation of airway smooth muscle cells (ASMC) in culture and to determine the possible role of 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) in this context. ASMC obtained from guinea pigs were cultured and co-incubated with washed platelets for 24 hours. Thereafter, the proliferation was measured with the MTS-assay; the results were also verified by using thymidine incorporation, DNA measurements and manual counting. The interaction between platelets and ASMC was visualized with fluorescence microscopy. We found that platelets bind to the ASMC and the presence of platelets caused a significant dose-dependent increase in ASMC proliferation. Co-incubation of ASMC with platelets also increased ROS-production, detected by the fluorescent probe DCFDA. Furthermore, the platelet-induced proliferation was reduced in the presence of the NADPH-oxidase inhibitors DPI and apocynin. A possible role of 5-LOX in platelet-induced proliferation and ROS-generation was evaluated by using the 5-LOX inhibitor AA-861 and the PLA2-inhibitor ATK. The results showed that inhibition of these enzymes significantly reduced the platelet-induced proliferation. Moreover, Western blot analysis revealed that the ASMC but not the platelets express 5-LOX. In addition, our experiments revealed that the presence of AA-861 and ATK significantly inhibited the ROS-production generated upon co-incubation of platelets and ASMC. In conclusion, we show that platelets have a marked capacity to induce ASMC proliferation. Furthermore, our study indicates that the interaction between platelets and ASMC leads to activation of 5-LOX in the ASMC followed by an increased ROS-production, events resulting in enhanced ASMC proliferation. The new findings are of importance in understanding possible mechanisms contributing to airway remodeling. © 2008 Informa UK Ltd.

  • 50. Svensson Holm, Ann-Charlotte B.
    et al.
    Bengtsson, Torbjörn
    Örebro universitet, Hälsoakademin.
    Grenegard, Magnus
    Lindström, Eva G.
    Platelet membranes induce airway smooth muscle cell proliferation2011Ingår i: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 22, nr 1, s. 45-55Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The role of platelets in airway disease is poorly understood although they have been suggested to influence on proliferation of airway smooth muscle cells (ASMC). Platelets have been found localized in the airways in autopsy material from asthmatic patients and have been implicated in airway remodeling. The aim of the present study was to investigate the effects of various platelet fractions on proliferation of ASMC obtained from guinea pigs (GP-ASMC) and humans (H-ASMC). Proliferation of ASMC was measured by the MTS assay and the results confirmed by measurements of the DNA content. A key observation was that the platelet membrane preparations induced a significant increase in the proliferation of both GP-ASMC (129.9 +/- 3.0 %) and H-ASMC (144.8 +/- 12.2). However, neither supernatants from lysed or filtrated thrombin stimulated platelets induced ASMC proliferation to the same extent as the membrane preparation. We have previously shown that platelet-induced proliferation is dependent on 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) pathways. In the present work we established that platelet membrane-induced ASMC proliferation was reduced in the presence of the NADPH oxidase inhibitor DPI and the 5-LOX inhibitor AA-861. In conclusion, our results showed that platelet membranes significantly induced ASMC proliferation, demonstrating that the mitogenic effect of platelets and platelet membranes on ASMC is mainly due to membrane-associated factors. The effects of platelet membranes were evident on both GP-ASMC and H-ASMC and involved 5-LOX and ROS. These new findings are of importance in understanding the mechanisms contributing to airway remodeling and may contribute to the development of new pharmacological tools in the treatment of inflammatory airway diseases.

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