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  • 1.
    Bengtsson, Torbjörn
    et al.
    Örebro University, School of Medicine, Örebro University, Sweden.
    Khalaf, Atika
    The PRO-CARE Group, School of Health and Society, Kristianstad University, Kristianstad, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Secreted gingipains from Porphyromonas gingivalis colonies exert potent immunomodulatory effects on human gingival fibroblasts2015In: Microbiology Research, ISSN 0944-5013, E-ISSN 1618-0623, Vol. 178, p. 18-26Article in journal (Refereed)
    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.

  • 2.
    Bengtsson, Torbjörn
    et al.
    Örebro University, School of Medical Sciences.
    Lönn, Johanna
    Department of Oral Biology, Institute of Odontology, Malmö University, Malmö, Sweden; PEAS Research Institute, Linköping, Sweden.
    Khalaf, Hazem
    Örebro University, School of Medical Sciences.
    Palm, Eleonor
    Örebro University, School of Medical Sciences.
    The lantibiotic gallidermin acts bactericidal against Staphylococcus epidermidis and Staphylococcus aureus and antagonizes the bacteria-induced proinflammatory responses in dermal fibroblasts2018In: MicrobiologyOpen, ISSN 2045-8827, E-ISSN 2045-8827, Vol. 7, no 6, article id e606Article in journal (Refereed)
    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.

  • 3.
    Bengtsson, Torbjörn
    et al.
    Örebro University, School of Medical Sciences.
    Zhang, Boxi
    Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
    Selegård, Robert
    Örebro University, School of Medical Sciences. 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 University, School of Medical Sciences.
    Dual action of bacteriocin PLNC8 alpha beta through inhibition of Porphyromonas gingivalis infection and promotion of cell proliferation2017In: Pathogens and Disease, E-ISSN 2049-632X, Vol. 75, no 5, article id ftx064Article in journal (Refereed)
    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.

  • 4.
    El Marghani, Ahmed M.
    et al.
    Örebro University, School of Science and Technology.
    Khalaf, Hazem
    Örebro University, School of Science and Technology.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Immune cell activation by sewage treatment plant effluents and inland waters in SwedenManuscript (preprint) (Other academic)
  • 5.
    Elmarghani, Ahmed
    et al.
    Örebro University, School of Science and Technology.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Seyoum, Asmerom
    Örebro University, School of Science and Technology.
    Khalaf, Hazem
    Örebro University, School of Science and Technology.
    Ros, Torbjön
    Pelagia Miljokonsult AB, Umeå, Sweden.
    Forsberg, Lars-Håkan
    Mälarenergi AB, Vasterås, Sweden.
    Nermark, Tomas
    Karlskoga Energi MO AB, Karlskoga, Sweden.
    Osterman, Lisa
    Skebäcks Reningsverk, Örebro, Sweden.
    Wiklund, Ulf
    Tyrens AB, Umeå, Sweden.
    Ivarsson, Per
    ALS Scandinavia AB, Täby, Sweden.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Contribution of pharmaceuticals, fecal bacteria and endotoxin to the inflammatory responses to inland waters2014In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 488-489, p. 228-235Article in journal (Refereed)
    Abstract [en]

    The increasing contamination of freshwater with pharmaceuticals, surfactants, pesticides and other organic compounds are of major concern. As these contaminants are detected at trace levels in the environment it is important to determine if they elicit biological responses at the observed levels. In addition to chemical pollutants, there is also a concern for increasing levels of bacteria and other microorganisms in freshwater systems. In an earlier study, we observed the activation of inflammatory systems downstream of a wastewater treatment plant (WWTP) in southern Sweden. We also observed that the water contained unidentified components that were pro-inflammatory and potentiated the immune response in human urinary bladder epithelial cells. In order to determine if these effects were unique for the studied site or represent a common response in Swedish water, we have now performed a study on three WWTPs and their recipient waters in central Sweden. Analysis of immune responses in urinary bladder epithelial cells, monocyte-like cells and blood mononuclear cells confirm that these waters activate the immune system as well as induce pro-inflammatory responses. The results indicate that the cytokine profiles correlate to the endotoxin load of the waters rather than to the levels of pharmaceuticals or culturable bacteria load, suggesting that measurements of endotoxin levels and immune responses would be a valuable addition to the analysis of inland waters.

  • 6.
    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 University, School of Medical Sciences.
    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 University, School of Medical Sciences.
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences.
    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-Lysine2018In: Biomedical Materials, ISSN 1748-6041, E-ISSN 1748-605X, Vol. 13, article id 025014Article in journal (Refereed)
    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.

  • 7.
    Jayaprakash, Kartheyaene
    et al.
    Örebro University, School of Medical Sciences.
    Demirel, Isak
    Örebro University, School of Medical Sciences.
    Gunaltay, Sezin
    Örebro University, School of Medical Sciences.
    Khalaf, Hazem
    Örebro University, School of Medical Sciences.
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences.
    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 cells2017In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 125, no 7, p. 623-633Article in journal (Refereed)
    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.

  • 8.
    Jayaprakash, Kartheyaene
    et al.
    Örebro University, School of Medical Sciences.
    Demirel, Isak
    Örebro University, School of Medical Sciences.
    Gunaltay, Sezin
    Örebro University, School of Medical Sciences.
    Khalaf, Hazem
    Örebro University, School of Medical Sciences.
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences.
    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 monocytesManuscript (preprint) (Other academic)
  • 9.
    Jayaprakash, Kartheyaene
    et al.
    Örebro University, School of Medical Sciences.
    Demirel, Isak
    Örebro University, School of Medical Sciences.
    Khalaf, Hazem
    Örebro University, School of Medical Sciences.
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences.
    Porphyromonas gingivalis induced release of reactive oxygen species and interleukin-1 beta and the effects of low density lipoproteins in monocytes and whole bloodManuscript (preprint) (Other academic)
  • 10.
    Jayaprakash, Kartheyaene
    et al.
    Department of Medical Sciences, Örebro University, Örebro, Sweden.
    Demirel, Isak
    Örebro University, School of Medical Sciences.
    Khalaf, Hazem
    Örebro University, School of Medical Sciences.
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences.
    Porphyromonas gingivalis-induced inflammatory responses in THP1 cells are altered by native and modified low-density lipoproteins in a strain-dependent manner2018In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 126, no 8, p. 667-677Article in journal (Refereed)
    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.

  • 11.
    Jayaprakash, Kartheyaene
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Demirel, Isak
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis2015In: Molecular Oral Microbiology, ISSN 2041-1006, E-ISSN 2041-1014, Vol. 30, no 5, p. 361-375Article in journal (Refereed)
    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.

  • 12.
    Jayaprakash, Kartheyaene
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Gingipains from Porphyromonas gingivalis play a significant role in induction and regulation of CXCL8 in THP-1 cells2014In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 14, article id 193Article in journal (Refereed)
    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.

  • 13.
    Karlsson, Mattias
    et al.
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Substances released from probiotic Lactobacillus rhamnosus GR-1 potentiate NF-κB activity in Escherichia coli-stimulated urinary bladder cells2012In: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 66, no 2, p. 147-156Article in journal (Refereed)
    Abstract [en]

    Lactobacillus rhamnosus GR-1 is a probiotic bacterium used to maintain urogenital health. The putative mechanism for its probiotic effect is by modulating the host immunity. Urinary tract infections (UTI) are often caused by uropathogenic Escherichia coli that frequently evade or suppress immune responses in the bladder and can target pathways, including nuclear factor-kappaB (NF-κB). We evaluated the role of L. rhamnosus GR-1 on NF-κB activation in E. coli-stimulated bladder cells. Viable L. rhamnosus GR-1 was found to potentiate NF-κB activity in E. coli-stimulated T24 bladder cells, whereas heat-killed lactobacilli demonstrated a marginal increase in NF-κB activity. Surface components released by trypsin- or LiCl treatment, or the resultant heat-killed shaved lactobacilli, had no effect on NF-κB activity. Isolation of released products from L. rhamnosus GR-1 demonstrated that the induction of NF-κB activity was owing to released product(s) with a relatively large native size. Several putative immunomodulatory proteins were identified, namely GroEL, elongation factor Tu and NLP/P60. GroEL and elongation factor Tu have previously been shown to elicit immune responses from human cells. Isolating and using immune-augmenting substances produced by lactobacilli is a novel strategy for the prevention or treatment of UTI caused by immune-evading E. coli.

  • 14.
    Khalaf, Hazem
    Örebro University, School of Science and Technology.
    Characterization and environmental influences on inflammatory and physiological responses2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Pharmaceuticals are regularly released into the environment, in particular nonsteroidalanti-inflammatory drugs (NSAIDs) and antibiotics. The measuredconcentrations are relatively low and have therefore been considered to be harmless.However, several pharmaceuticals, including naproxen and atenolol, are stable for upto 1 year in the environment, which increases the risk for accumulation. Evaluation ofthe effects of pharmaceuticals on induced inflammatory responses is thereforenecessary for the assessment of potential risks. Since NF-κB and MAPK are the mainpathways known to be critical regulators of inflammatory responses, intracellularsignalling and effects on these systems were examined in vitro using human cell-lines.NSAIDs were shown to significantly reduce NF-κB activity at environmentallyrelevant concentrations. Suppression of immune responses may lead to progressiveinfections since inflammatory responses are controlled by a cooperative activity ofAP-1 and NF-κB. Alterations in the activity of transcription factors and proinflammatorycytokine and chemokine levels such as TNF, IL-6 and CXCL8 areassociated with several human diseases including cystic fibrosis and AIDS. PMAexposure resulted in a rapid NF-κB activation, while extended treatment suppressedNF-κB and activated AP-1. Suppression of NF-κB activity may be due to PKCdependentBcl10 degradation, which decreased in response to PMA and correlatedwith the NF-κB activity. Regulation of cytokine expression revealed that NF-κB wasessential for IL-6 but not CXCL8 expression following specific inhibition of NF-κB,without affecting AP-1 activity. Furthermore, several reports have indicated theimportance of a functional NF-κB complex in zebrafish embryogenesis, whereblockage of NF-κB activation resulted in a deformation of the tail. Our results indicatea suppression of apoptotic pathways following activation of inflammatory mediatorsin response to HK E. coli treatment. These signals acted to direct zebrafish sexdifferentiation towards feminization. NF-κB was shown to regulate zp2 geneexpression, an indicator of oocyte development. Zebrafish sex determination was alsoshown to start early, prior to 16 days post fertilization. The results support thetransition through a juvenile ovary stage and suggests that steriodogenesis is aconsequence of sex differentiation rather than a regulatory mechanism.Control of prescription, use and disposal of pharmaceuticals is therefore importantto preserve human health, biotic processes and to avoid developmental alterations inaquatic organisms. The complexity of regulatory systems involved in inflammationsuggest that there is a need to further evaluate the signalling pathways involved inorder to provide a better understanding of cellular responses to manmade substances,but also to offer an insight into possible development of alternative treatments forhuman diseases with elevated cytokine/chemokine levels.

  • 15.
    Khalaf, Hazem
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Division of Clinical Medicine,, Faculty of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden. Division of Clinical Medicine, Faculty of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    Altered T-cell responses by the periodontal pathogen Porphyromonas gingivalis2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 9, article id e45192Article in journal (Refereed)
    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.

  • 16.
    Khalaf, Hazem
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Demirel, Isak
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Suppression of inflammatory gene expression in T cells by Porphyromonas gingivalis is mediated by targeting MAPK signaling2013In: Cellular & Molecular Immunology, ISSN 1672-7681, E-ISSN 2042-0226, Vol. 10, no 5, p. 413-422Article in journal (Refereed)
    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.

  • 17.
    Khalaf, Hazem
    et al.
    Örebro University, School of Health and Medical Sciences.
    Jass, Jana
    Örebro University, School of Science and Technology. Lawson Hlth Res Inst, Univ Western Ontario, London ON, Canada; Dept Microbiol & Immunol, Univ Western Ontario, London ON, Canada.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Differential cytokine regulation by NF-κB and AP-1 in Jurkat T-cells2010In: BMC Immunology, ISSN 1471-2172, E-ISSN 1471-2172, Vol. 11, article id 26Article in journal (Refereed)
    Abstract [en]

    Background: Activator protein (AP)-1 and nuclear factor (NF)-κB largely control T-cell activation, following binding offoreign antigens to the T-cell receptor leading to cytokine secretion. Elevated levels of pro-inflammatory cytokines andchemokines such as TNF, IL-6 and CXCL8 are associated with several human diseases including cystic fibrosis, pulmonary fibrosis and AIDS. The aim of this study was to investigate the role of the transcription factors, AP-1 and NF-κB, in IL-6 and CXCL8 regulation in Jurkat T-cells.

    Results: Phorbol myristate acetate (PMA) exposure resulted in an up-regulation of AP-1 and down-regulation of NF-κBactivity, however, exposure to heat killed (HK) Escherichia. coli MG1655 resulted in a dose-dependent increase in NF-κBactivity without affecting AP-1. The cytokine profile revealed an up-regulation of the chemokine CXCL8 and the pro-inflammatory cytokines TNF, IL-2 and IL-6 following treatment with both PMA and HK E. coli, while the levels of the anti-inflammatory cytokine IL-10 were not affected by PMA but were significantly down-regulated by HK E. coli. AP-1activation was significantly increased 2 h after PMA exposure and continued to increase thereafter. In contrast, NF-κBresponded to PMA exposure by a rapid up-regulation followed by a subsequent down-regulation. Increased intracellular Ca2+ concentrations countered the down-regulation of NF-κB by PMA, while similar treatment with calcium ionophore resulted in a reduced NF-κB activity following induction with HK E. coli. In order to further study NF-κB activation, we considered two up-stream signalling proteins, PKC and Bcl10. Phosphorylated-PKC levels increased inresponse to PMA and HK E. coli, while Bcl10 levels significantly decreased following PMA treatment. Using an NF-κBactivation inhibitor, we observed complete inhibition of IL-6 expression while CXCL8 levels only decreased by 40% atthe highest concentration. Treatment of Jurkat T-cells with PMA in the presence of JNK-inhibitor suppressed both CXCL8 and IL-6 while PKC-inhibitor primarily decreased CXCL8 expression.

    Conclusion: The present study shows that NF-κB regulated IL-6 but not CXCL8. This complex regulation of CXCL8suggests that there is a need to further evaluate the signalling pathways in order to develop new treatment fordiseases with elevated CXCL8 levels, such as AIDS and autoimmune diseases.

  • 18.
    Khalaf, Hazem
    et al.
    Örebro University, School of Health and Medical Sciences.
    Jass, Jana
    Örebro University, School of Health and Medical Sciences.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Influence of growth conditions on in vitro regulation of NF-κB activity in Jurkat T-cells2010Manuscript (preprint) (Other academic)
  • 19.
    Khalaf, Hazem
    et al.
    Örebro University, School of Science and Technology.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    The role of calcium, NF-κB and NFAT in the regulation of CXCL8 and IL-6 expression in Jurkat T-cells2013In: International Journal of Biochemistry and Molecular Biology, ISSN 2152-4114, Vol. 4, no 3, p. 150-156Article in journal (Refereed)
    Abstract [en]

    T-cells play an important role in host immunity against invading pathogens. Determining the underlying regulatory mechanisms will provide a better understanding of T-cell-derived immune responses. In this study, we have shown the differential regulation of IL-6 and CXCL8 by NF-κB and NFAT in Jurkat T-cells, in response to PMA, heat killed Escherichia coli and calcium. CXCL8 was closely associated with the activation pattern of NFAT, while IL-6 expression was associated with NF-κB. Furthermore, increasing the intracellular Ca(2+) concentration by calcium ionophore treatment of the cells resulted in NFAT induction without affecting the NF-κB activity. Interestingly, NF-κB activation by heat killed E. coli, as well as CXCL8 and IL-6 expression was significantly suppressed following addition of the calcium ionophore. This indicates that calcium plays an important role in regulating protein trafficking and T-cell signalling, and the subsequent inflammatory gene expression infers an involvement of NFAT in CXCL8 regulation.Understanding these regulatory patterns provide clarification of conditions that involve altered intracellular signalling leading to T-cell-derived cytokine expression.

  • 20.
    Khalaf, Hazem
    et al.
    Örebro University, School of Science and Technology.
    Larsson, Anders
    Örebro University, School of Science and Technology.
    Berg, Håkan
    Örebro University, School of Science and Technology.
    McCrindle, Robert
    Wellington Laboratories Inc., Research Division, Guelph, Ontario, Canada.
    Arsenault, Gilles
    Wellington Laboratories Inc., Research Division, Guelph, Ontario, Canada.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Diastereomers of the Brominated Flame Retardant 1,2-dibromo-4-(1,2 dibromoethyl)cyclohexane Induce Androgen Receptor Activation in the HepG2 Hepatocellular Carcinoma Cell Line and the LNCaP Prostate Cancer Cell Line2009In: Journal of Environmental Health Perspectives, ISSN 0091-6765, E-ISSN 1552-9924, Vol. 117, no 12, p. 1853-1859Article in journal (Refereed)
    Abstract [en]

    Background: Reported incidences of prostate cancer and masculinization of animals indicate a release of compounds with androgenic properties into the environment. Large numbers of environmental pollutants have been screened to identify such compounds; however, not until recently was 1,2-dibromo-4-(1,2-dibromoethyl)cyclohex​ane (TBECH) identified as the first potent activator of the human androgen receptor (hAR). TBECH has been found in beluga whales and bird eggs and has also been found to be maternally transferred in zebrafish.

    Objectives: In the present study we investigated interaction energies between TBECH diastereomers (α, β, γ, and δ) and the hAR, and their ability to activate the receptor and induce prostate-specific antigen (PSA) expression in vitro.

    Methods: We performed computational modeling to determine interaction energies between the ligand and the AR ligand-binding site, and measured in vitro competitive binding assays for AR by polarization fluorometry analysis. We used enzyme-linked immunosorbent assays to determine PSA activity in LNCaP and HepG2 cells.

    Results: We found the γ and δ diastereomers to be more potent activators of hAR than the α and β diastereomers, which was confirmed in receptor binding studies. All TBECH diastereomers induced PSA expression in LNCaP cells even though the AR present in these cells is mutated (T877A). Modeling studies of LNCaP AR revealed that TBECH diastereomers bound to the receptor with a closer distance to the key amino acids in the ligand-binding domain, indicating stronger binding to the mutated receptor.

    Conclusions: The present study demonstrates the ability of TBECH to activate the hAR, indicating that it is a potential endocrine disruptor.

  • 21.
    Khalaf, Hazem
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Lönn, Johanna
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Cytokines and chemokines are differentially expressed in patients with periodontitis: Possible role for TGF-beta 1 as a marker for disease progression2014In: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 67, no 1, p. 29-35Article in journal (Refereed)
    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.

  • 22.
    Khalaf, Hazem
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Nakka, Sravya Sowdamini
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. 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 University, School of Science and Technology.
    Aili, Daniel
    PEAS Institut AB, Söderleden 1, Linköping.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Antibacterial effects of lactobacillus and bacteriocin NC8 αβ on the periodontal pathogen Porphyromonas gingivalisManuscript (preprint) (Other academic)
  • 23.
    Khalaf, Hazem
    et al.
    Örebro University, School of Medical Sciences.
    Nakka, Sravya Sowdamini
    Örebro University, School of Medical Sciences. 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 University, School of Science and Technology.
    Aili, Daniel
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences.
    Antibacterial effects of Lactobacillus and bacteriocin PLNC8 αβ on the periodontal pathogen Porphyromonas gingivalis2016In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 16, no 1, article id 188Article in journal (Refereed)
    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.

  • 24.
    Khalaf, Hazem
    et al.
    Örebro University, School of Science and Technology.
    Ochsner, Scott A.
    Koskinen, Jarno
    Karlsson, Marie
    Karlsson, Jesper
    Sreenivasan, Rajini
    McKenna, Neil J.
    Orban, Laszlo
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Zebrafish feminization in response to heat killed bacterial exposure suggests a function for anti-apoptotic genes in oocyte maintenanceManuscript (preprint) (Other academic)
  • 25.
    Khalaf, Hazem
    et al.
    Örebro University, School of Medical Sciences.
    Palm, Eleonor
    Örebro University, School of Medical Sciences.
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences.
    Cellular Response Mechanisms in Porphyromonas gingivalis Infection2017In: Periodontitis: A Useful Reference / [ed] Pachiappan Arjunan, InTech, 2017, p. 45-68Chapter in book (Refereed)
    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.

  • 26.
    Khalaf, Hazem
    et al.
    Örebro University, School of Health and Medical Sciences.
    Salste, Lotta
    Karlsson, Patrik
    Ivarsson, Per
    Jass, Jana
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    In vitro analysis of inflammatory responses following environmental exposure to pharmaceuticals and inland waters2009In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 407, no 4, p. 1452-1460Article in journal (Refereed)
    Abstract [en]

    Pharmaceuticals are regularly released into the environment; in particular non-steroidalanti-inflammatory drugs (NSAIDs) and antibiotics. Erythromycin, naproxen, furosemideand atenolol are reported to be stable for up to 1 year in the environment, which increasesthe risk for accumulation. In the present study we have measured the occurrence andconcentration of pharmaceuticals in river Viskan (Jössabron) downstream of a sewagetreatment plant in Borås, Sweden. Pharmaceuticals and water samples were tested forpotential human risk by evaluating inflammatory responses (NF-κB and AP-1) using humanT24 bladder epithelial cells and Jurkat T-cells. NF-κB activity in T24 cells was significantlyreduced by all NSAIDs analysed (diclofenac, ketoprofen, naproxen, ibuprophen anddextropropoxyphene), but also by trimethoprim, using environmentally relevantconcentrations. NF-κB and AP-1 activation was further analysed in response to watersamples collected from different locations in Sweden. Dose-dependent down-regulation ofAP-1 activity in Jurkat cells was observed at all locations. At two locations (Jössabron andAlmenäs) down-regulation of NF-κB was observed. In contrast, the NF-κB response waspotentiated by exposure to water from both locations following activation of NF-κB bytreatment with heat-killed Escherichia coli. To determine the involvement ofpharmaceuticals in the responses, T24 cells were exposed to the pharmaceutical mixture,based on the determined levels at Jössabron. This resulted in reduction of the NF-κBresponse following exposure to the pharmaceutical mixture alone while no potentiationwas observed when cells were co-exposed to heat killed E. coli and pharmaceuticals. Theobtained results demonstrate that the identified pharmaceuticals affect the inflammatoryresponses and furthermore indicate the presence of unknown substance(s) with the abilityto potentiate inflammatory responses

  • 27.
    Klarström-Engström, Kristin
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    The platelet response to various strains of Porphyromonas gingivalisManuscript (preprint) (Other academic)
  • 28.
    Klarström-Engström, Kristin
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Kälvegren, H.
    Department of Clinical Pathology and Clinical Genetics, Linköping University Hospital, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    The role of Porphyromonas gingivalis gingipains in platelet activation and innate immune modulation2015In: Molecular Oral Microbiology, ISSN 2041-1006, E-ISSN 2041-1014, Vol. 30, no 1, p. 62-73Article in journal (Refereed)
    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.

  • 29.
    Kling, Peter
    et al.
    University of Gothenburg , Göteborg, Sweden.
    Modig, Carina
    Örebro University, School of Science and Technology.
    Mujahed, Huthayfa
    Örebro University, School of Science and Technology.
    Khalaf, Hazem
    Örebro University, School of Science and Technology.
    von Hofsten, Jonas
    Umeå University, Umeå, Sweden.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Differential regulation of the rainbow trout (Oncorhynchus mykiss) MT-A gene by nuclear factor interleukin-6 and activator protein-12013In: BMC Molecular Biology, ISSN 1471-2199, E-ISSN 1471-2199, Vol. 14, no 1, p. 28-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Previously we have identified a distal region of the rainbow trout (Oncorhynchus mykiss) metallothionein-A (rtMT-A) enhancer region, being essential for free radical activation of the rtMT-A gene. The distal promoter region included four activator protein 1 (AP1) cis-acting elements and a single nuclear factor interleukin-6 (NF-IL6) element. In the present study we used the rainbow trout hepatoma (RTH-149) cell line to further examine the involvement of NF-IL6 and AP1 in rtMT-A gene expression following exposure to oxidative stress and tumour promotion.

    RESULTS: Using enhancer deletion studies we observed strong paraquat (PQ)-induced rtMT-A activation via NF-IL6 while the AP1 cis-elements showed a weak but significant activation. In contrast to mammals the metal responsive elements were not activated by oxidative stress. Electrophoretic mobility shift assay (EMSA) mutation analysis revealed that the two most proximal AP1 elements, AP11,2, exhibited strong binding to the AP1 consensus sequence, while the more distal AP1 elements, AP13,4 were ineffective. Phorbol-12-myristate-13-acetate (PMA), a known tumor promoter, resulted in a robust induction of rtMT-A via the AP1 elements alone. To determine the conservation of regulatory functions we transfected human HepG2 cells with the rtMT-A enhancer constructs and were able to demonstrate that the cis-elements were functionally conserved. The importance of NF-IL6 in regulation of teleost MT is supported by the conservation of these elements in MT genes from different teleosts. In addition, PMA and PQ injection of rainbow trout resulted in increased hepatic rtMT-A mRNA levels.

    CONCLUSIONS: These studies suggest that AP1 primarily is involved in PMA regulation of the rtMT-A gene while NF-IL6 is involved in free radical regulation. Taken together this study demonstrates the functionality of the NF-IL6 and AP-1 elements and suggests an involvement of MT in protection during pathological processes such as inflammation and cancer.

  • 30.
    Nakka, Sravya Sowdamini
    et al.
    Örebro University, School of Medical Sciences. The Institution for Protein Environmental Affinity Surveys, PEAS Institut AB, Linköping, Sweden.
    Palm, Eleonor
    Örebro University, School of Medical Sciences.
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences.
    Khalaf, Hazem
    Örebro University, School of Medical Sciences.
    Bacteriocin plantaricin NC8 αβ antagonizes Porphyromonas gingivalis infection and induces proliferation of gingival epithelial cellsManuscript (preprint) (Other academic)
  • 31.
    Nakka, Sravya Sowdamini
    et al.
    Örebro University, School of Medical Sciences. The Institution for Protein Environmental Affinity Surveys, PEAS Institut AB, Linköping, Sweden.
    Palm, Eleonor
    Örebro University, School of Medical Sciences.
    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 University, School of Medical Sciences.
    Khalaf, Hazem
    Örebro University, School of Medical Sciences.
    Effects of plantaricin NC8 αβ and antibodies on gingival epithelial cells infected by Porphyromonas gingivalisManuscript (preprint) (Other academic)
  • 32.
    Olsson, Per-Erik
    et al.
    Örebro University, School of Science and Technology.
    Khalaf, Hazem
    Örebro University, School of Science and Technology.
    Jass, Jana
    Örebro University, School of Science and Technology.
    CXCL8 regulation and function in HIV infections and potential treatment strategies2011In: HIV and AIDS: updates on biology, immunology, epidemiology and treatment strategies / [ed] Nancy Dumais, InTech, 2011, p. 327-344Chapter in book (Refereed)
  • 33.
    Palm, Eleonor
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Demirel, Isak
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    The role of toll-like and protease-activated receptors and associated intracellular signalling in Porphyromonas gingivalis-infected gingival fibroblastsManuscript (preprint) (Other academic)
  • 34.
    Palm, Eleonor
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Demirel, Isak
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    The role of toll-like and protease-activated receptors in the expression of cytokines by gingival fibroblasts stimulated with the periodontal pathogen Porphyromonas gingivalisManuscript (preprint) (Other academic)
  • 35.
    Palm, Eleonor
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Biomedicine, Örebro University Hospital, Örebro, Sweden.
    Demirel, Isak
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Biomedicine, Örebro University Hospital, Örebro, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden. Department of Biomedicine, Örebro University Hospital, Örebro, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. 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 gingivalis2015In: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 76, no 2, p. 424-432Article in journal (Refereed)
    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.

  • 36.
    Palm, Eleonor
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Porphyromonas gingivalis downregulates the immune response of fibroblasts2013In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, p. 155-Article in journal (Refereed)
    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.

  • 37.
    Palm, Eleonor
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Suppression of inflammatory responses of human gingival fibroblasts by gingipains from Porphyromonas gingivalis2015In: Molecular Oral Microbiology, ISSN 2041-1006, E-ISSN 2041-1014, Vol. 30, no 1, p. 74-85Article in journal (Refereed)
    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.

  • 38.
    Paramel Varghese, Geena
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Zhang, Boxi
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Ljungberg, Liza U.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Sirsjö, Allan
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Fransén, Karin
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Poryphyromonas gingivalis induces IL-1β in aortic smooth muscle cells: possible role of gingipains?Manuscript (preprint) (Other academic)
  • 39.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Ochsner, Scott A.
    Baylor College of Medicine, Houston, USA.
    Sreenivasan, Rajini
    Temasek Life Sciences Laboratory, Singapore, Singapore.
    Koskinen, Jarno
    Örebro University, School of Science and Technology.
    Karlsson, Marie
    Örebro University, School of Science and Technology.
    Karlsson, Jesper
    Department of Biology, School of Science and Technology, Örebro University, Örebro, Sweden.
    McKenna, Neil J.
    Baylor College of Medicine, Houston, USA.
    Orban, Laszlo
    Temasek Life Sciences Laboratory, Singapore, Singapore; National University of Singapore, Singapore, Singapore; University of Pannonia, Keszthely, Hungary.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Activation of NF-kappa B Protein Prevents the Transition from Juvenile Ovary to Testis and Promotes Ovarian Development in Zebrafish2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 45, p. 37926-37938Article in journal (Refereed)
    Abstract [en]

    Testis differentiation in zebrafish involves juvenile ovary to testis transformation initiated by an apoptotic wave. The molecular regulation of this transformation process is not fully understood. NF-kappa B is activated at an early stage of development and has been shown to interact with steroidogenic factor-1 in mammals, leading to the suppression of anti-Mullerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-kappa B-mediated induction of anti-apoptotic genes could, therefore, also play a role in zebrafish gonad differentiation. The aim of this study was to examine the potential role of NF-kappa B in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-kappa B pathways and resulted in an increased ratio of females from 30 to 85%. Microarray and quantitative real-time-PCR analysis of gonads showed elevated expression of NF-kappa B-regulated genes. To confirm the involvement of NF-kappa B-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-kappa B or NF-kappa B activation inhibitor (NAI). Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and resulted in an increased proportion of females from 25 to 45%, whereas the inhibition of NF-kappa B using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-kappa B in gonadal differentiation of zebrafish and represents an important step toward the complete understanding of the complicated process of sex differentiation in this species and possibly other cyprinid teleosts as well.

  • 40.
    Selegård, Robert
    et al.
    Faculty of Medicine & Health, School of Medical Sciences, Örebro University, Örebro, Sweden.
    Musa, Amani
    Örebro University, School of Medical Sciences.
    Nyström, Pontus
    Örebro University, School of Medical Sciences.
    Aili, Daniel
    Division of Molecular Physics, Department of Physics, Chemistry & Biology (IFM), Linköping University, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences.
    Khalaf, Hazem
    Örebro University, School of Medical Sciences.
    Plantaricins markedly enhance the effects of traditional antibiotics against Staphylococcus epidermidis2019In: Future microbiology, ISSN 1746-0913, Vol. 14, no 3, p. 195-206Article in journal (Refereed)
    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.

  • 41.
    Wickham, Abeni
    et al.
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden .
    Vagin, Mikhail
    Division of Chemical and Optical Sensor Systems, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Khalaf, Hazem
    Örebro University, School of Medical Sciences. Cardiovascular Research Centre, Örebro University Hospital, Örebro, Sweden.
    Bertazzo, Sergio
    Department of Medical Physics and Biomedical Engineering, University College London, London, UK .
    Hodder, Peter
    TA Instruments Ltd., Elstree, UK .
    Dånmark, Staffan
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden .
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences. Cardiovascular Research Centre.
    Altimiras, Jordi
    Division of Biology, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden .
    Aili, Daniel
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Electroactive biomimetic collagen-silver nanowire composite scaffolds2016In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 8, no 29, p. 14146-14155Article in journal (Refereed)
    Abstract [en]

    Electroactive biomaterials are widely explored as bioelectrodes and as scaffolds for neural and cardiac regeneration. Most electrodes and conductive scaffolds for tissue regeneration are based on synthetic materials that have limited biocompatibility and often display large discrepancies in mechanical properties with the surrounding tissue causing problems during tissue integration and regeneration. This work shows the development of a biomimetic nanocomposite material prepared from self-assembled collagen fibrils and silver nanowires (AgNW). Despite consisting of mostly type I collagen fibrils, the homogeneously embedded AgNWs provide these materials with a charge storage capacity of about 2.3 mC cm(-2) and a charge injection capacity of 0.3 mC cm(-2), which is on par with bioelectrodes used in the clinic. The mechanical properties of the materials are similar to soft tissues with a dynamic elastic modulus within the lower kPa range. The nanocomposites also support proliferation of embryonic cardiomyocytes while inhibiting the growth of both Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermidis. The developed collagen/AgNW composites thus represent a highly attractive bioelectrode and scaffold material for a wide range of biomedical applications.

  • 42.
    Zhang, Boxi
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Elmabsout, Ali Ateia
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Basic, Vladimir T.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Jayaprakash, Kartheyaene
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Kruse, Robert
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Sirsjö, Allan
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    The periodontal pathogen Porphyromonas gingivalis changes the gene expression in vascular smooth muscle cells involving the TGFbeta/Notch signalling pathway and increased cell proliferation2013In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 14, p. 770-Article in journal (Refereed)
    Abstract [en]

    Background: Porphyromonas gingivalis is a gram-negative bacterium that causes destructive chronic periodontitis. In addition, this bacterium is also involved in the development of cardiovascular disease. The aim of this study was to investigate the effects of P. gingivalis infection on gene and protein expression in human aortic smooth muscle cells (AoSMCs) and its relation to cellular function.

    Results: AoSMCs were exposed to viable P. gingivalis for 24 h, whereafter confocal fluorescence microscopy was used to study P. gingivalis invasion of AoSMCs. AoSMCs proliferation was evaluated by neutral red assay. Human genome microarray, western blot and ELISA were used to investigate how P. gingivalis changes the gene and protein expression of AoSMCs. We found that viable P. gingivalis invades AoSMCs, disrupts stress fiber structures and significantly increases cell proliferation. Microarray results showed that, a total of 982 genes were identified as differentially expressed with the threshold log2 fold change >|1| (adjust p-value <0.05). Using bioinformatic data mining, we demonstrated that up-regulated genes are enriched in gene ontology function of positive control of cell proliferation and down-regulated genes are enriched in the function of negative control of cell proliferation. The results from pathway analysis revealed that all the genes belonging to these two categories induced by P. gingivalis were enriched in 25 pathways, including genes of Notch and TGF-beta pathways.

    Conclusions: This study demonstrates that P. gingivalis is able to invade AoSMCs and stimulate their proliferation. The activation of TGF-beta and Notch signaling pathways may be involved in the bacteria-mediated proliferation of AoSMCs. These findings further support the association between periodontitis and cardiovascular diseases.

  • 43.
    Zhang, Boxi
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Sirsjö, Allan
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Gingipains from the Periodontal Pathogen Porphyromonas gingivalis Play a Significant Role in Regulation of Angiopoietin 1 and Angiopoietin 2 in Human Aortic Smooth Muscle Cells2015In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 83, no 11, p. 4256-4265Article in journal (Refereed)
    Abstract [en]

    Angiopoietin 1 (Angpt1) and angiopoietin 2 (Angpt2) are the ligands of tyrosine kinase (Tie) receptors, and they play important roles in vessel formation and the development of inflammatory diseases, such as atherosclerosis. Porphyromonas gingivalis is a Gram-negative periodontal bacterium that is thought to contribute to the progression of cardiovascular disease. The aim of this study was to investigate the role of P. gingivalis infection in the modulation of Angpt1 and Angpt2 in human aortic smooth muscle cells (AoSMCs). We exposed AoSMCs to wild-type (W50 and 381), gingipain mutant (E8 and K1A), and fimbrial mutant (DPG-3 and KRX-178) P. gingivalis strains and to different concentrations of tumor necrosis factor (TNF). The atherosclerosis risk factor TNF was used as a positive control in this study. We found that P. gingivalis (wild type, K1A, DPG3, and KRX178) and TNF upregulated the expression of Angpt2 and its transcription factor ETS1, respectively, in AoSMCs. In contrast, Angpt1 was inhibited by P. gingivalis and TNF. However, the RgpAB mutant E8 had no effect on the expression of Angpt1, Angpt2, or ETS1 in AoSMCs. The results also showed that ETS1 is critical for P. gingivalis induction of Angpt2. Exposure to Angpt2 protein enhanced the migration of AoSMCs but had no effect on proliferation. This study demonstrates that gingipains are crucial to the ability of P. gingivalis to markedly increase the expressed Angpt2/Angpt1 ratio in AoSMCs, which determines the regulatory role of angiopoietins in angiogenesis and their involvement in the development of atherosclerosis. These findings further support the association between periodontitis and cardiovascular disease.

  • 44.
    Zhang, Boxi
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Sirsjö, Allan
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Transcriptional profiling of human smooth muscle cells infected with gingipain and fimbriae mutants of Porphyromonas gingivalisManuscript (preprint) (Other academic)
  • 45.
    Zhang, Boxi
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Sirsjö, Allan
    Örebro University, School of Medicine, Örebro University, Sweden. Sch Hlth Sci, Dept Clin Med, Univ Orebro, Orebro, Sweden.
    Khalaf, Hazem
    Örebro University, School of Medicine, Örebro University, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Transcriptional profiling of human smooth muscle cells infected with gingipain and fimbriae mutants of Porphyromonas gingivalis2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 21911Article in journal (Refereed)
    Abstract [en]

    Porphyromonas gingivalis (P. gingivalis) is considered to be involved in the development of atherosclerosis. However, the role of different virulence factors produced by P. gingivalis in this process is still uncertain. The aim of this study was to investigate the transcriptional profiling of human aortic smooth muscle cells (AoSMCs) infected with wild type, gingipain mutants or fimbriae mutants of P. gingivalis. AoSMCs were exposed to wild type (W50 and 381), gingipain mutants (E8 and K1A), or fimbriae mutants (DPG-3 and KRX-178) of P. gingivalis. We observed that wild type P. gingivalis changes the expression of a considerable larger number of genes in AoSMCs compare to gingipain and fimbriae mutants, respectively. The results from pathway analysis revealed that the common differentially expressed genes for AoSMCs infected by 3 different wild type P. gingivalis strains were enriched in pathways of cancer, cytokine-cytokine receptor interaction, regulation of the actin cytoskeleton, focal adhesion, and MAPK signaling pathway. Disease ontology analysis showed that various strains of P. gingivalis were associated with different disease profilings. Our results suggest that gingipains and fimbriae, especially arginine-specific gingipain, produced by P. gingivalis play important roles in the association between periodontitis and other inflammatory diseases, including atherosclerosis.

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