Search

Background Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment—modelling that in the subgingival pocket. Methods Growth and proteolytic activity of three Porphyromonas gingivalis strains in nutrient broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants of P. gingivalis was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. Results The P. gingivalis strains showed different growth rates in nutrient broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. Together in the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that community growth was facilitated by Rgp gingipain from P. gingivalis. Conclusions In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex protein substrates in serum. Whereas they are constitutively expressed by P. gingivalis in nutrient broth, gingipain expression in the model periodontal pocket environment (serum) appeared to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which then promoted growth of the whole community.

Periodontitis is an inflammatory oral disease that affects a large part of the adult population, causing significant costs and suffering. The key pathogen, Porphyromonas gingivalis, secretes gingipains, which are highly destructive proteases and the most important virulence factors in the pathogenesis of the disease. Currently, periodontitis is diagnosed mainly by mechanical manual probing and radiography, often when the disease has already progressed significantly. The possibilities of detecting gingipain activity in gingival fluid could enable early-stage diagnosis and facilitate treatment. Here, we describe a sensitive nanoparticle-based nanoplasmonic biosensor for the detection of the proteolytic activity of gingipains. Gold nanoparticles (AuNPs) were self-assembled as a submonolayer in multiwell plates and further modified with casein or IgG. The proteolytic degradation of the protein coating was tracked by monitoring the shift in the localized surface plasmon resonance (LSPR) peak position. The sensor performance was investigated using model systems with trypsin and purified gingipains (subtypes Kgp and RgpB) and further validated using supernatants from cultures of P. gingivalis. Proteolytic degradation by proteases in buffer results in a concentration- and time-dependent blueshift of the LSPR band of about 1-2 nm when using casein as a substrate. In bacterial supernatants, the degradation of the protein coating resulted in unspecific binding of proteins present in the complex sample matrix to the nanoparticles, which instead triggered a redshift of about 2 nm of the LSPR band. A significant LSPR shift was seen only in samples with gingipain activity. The sensor showed a limit of detection < 0.1 mu g/mL (4.3 nM), which is well below gingipain concentrations detected in severe chronic periodontitis cases (similar to 50 mu g/mL). This work shows the possibility of developing cost-effective nanoparticle-based biosensors for rapid detection of protease activity for chair-side periodontal diagnostics. Funding Agencies|Swedish Research Council (VR)Swedish Research Council [2016-04874, 2017-04475]; Swedish Foundation for Strategic Research (SFF)Swedish Foundation for Strategic Research [FFL15-0026]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation [KAW 2016.0231]

Nanocomposites of metal nanoparticles (NPs) and bacterial nanocellulose (BC) enable fabrication of soft and biocompatible materials for optical, catalytic, electronic, and biomedical applications. Current BC–NP nanocomposites are typically prepared by in situ synthesis of the NPs or electrostatic adsorption of surface functionalized NPs, which limits possibilities to control and tune NP size, shape, concentration, and surface chemistry and influences the properties and performance of the materials. Here a self‐assembly strategy is described for fabrication of complex and well‐defined BC–NP composites using colloidal gold and silver NPs of different sizes, shapes, and concentrations. The self‐assembly process results in nanocomposites with distinct biophysical and optical properties. In addition to antibacterial materials and materials with excellent senor performance, materials with unique mechanoplasmonic properties are developed. The homogenous incorporation of plasmonic gold NPs in the BC enables extensive modulation of the optical properties by mechanical stimuli. Compression gives rise to near‐field coupling between adsorbed NPs, resulting in tunable spectral variations and enhanced broadband absorption that amplify both nonlinear optical and thermoplasmonic effects and enables novel biosensing strategies., This work was supported by the Swedish Foundation for Strategic Research (SFF) grant no. FFL15‐0026 and framework grant RMX18‐0039 (HEALiX), the Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linköping University (Faculty Grant SFO‐Mat‐LiU no. 2009‐00971), the competence center FunMat‐II that is financially supported by Vinnova (grant no. 2016‐05156), and the Knut and Alice Wallenberg Foundation (grant no. KAW 2016.0231), the Swedish Research Council (grant Nos. 2017‐05178 and 2015‐05002) and the Spanish Ministerio de Ciencia, Innovación y Universidades (MICINN) through the MAT2016‐77391‐R. The ICN2 was supported by the Severo Ochoa Centres of Excellence programme, funded by the Spanish Research Agency (AEI, grant no. SEV‐2017‐0706).

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-κB in IL-1β 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-1β and CXCL8 expression by P. gingivalis. Gingipains hydrolyse IL-1β and CXCL8, which is more evident for IL-1β accumulation at 24 h. Inhibition of PKC (protein kinase C), p38 and ERK (extracellular signal-regulated kinases) partially reduced P. gingivalis-induced IL-1β at 6 h, whereas PKC and ERK reduced CXCL8 at both 6 and 24 h. Following NF-κB inhibition, P. gingivalis-induced IL-1β and CXCL8 were completely suppressed to basal levels. Overall, TLRs, PARs and NOD possibly act in synergy with PKC, MAPK ERK/p38 and NF-κB in P. gingivalis-induced IL-1β and CXCL8 release from THP1 cells. These pro-inflammatory cytokines could affect leucocytes in circulation and exacerbate other vascular inflammatory conditions such as atherosclerosis.

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 alpha beta were investigated against different Staphylococcus spp. The peptide sequences of PLNC8 alpha and beta were modified, either through truncation or replacement of all L-amino acids with D-amino acids. Both L- and D-PLNC8 alpha beta 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, beta 1-22, beta 7-34 and beta 1-20 retained an inhibitory activity. The peptides diffused rapidly (2min) through the bacterial cell wall and permeabilized the cell membrane, causing swelling with a disorganized peptidoglycan layer. Interestingly, sub-MIC concentrations of PLNC8 alpha beta substantially enhanced the effects of different antibiotics in an additive or synergistic manner. This study shows that PLNC8 alpha beta 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. Funding Agencies|Foundation of Magnus Bergvalls [2015-00823]; Knowledge Foundation [20150244, 20150086]; Swedish Research CouncilSwedish Research Council [2016-04874, 2017-04475]; Swedish Cancer SocietySwedish Cancer Society [17 0532]; Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linkoping University [2009-00971]; Swedish Foundation for Strategic ResearchSwedish Foundation for Strategic Research [RMX18-0039]; orebro University

The plant microbiome is an important factor for plant health and productivity. While the impact of nitrogen (N) availability for plant growth and development is well established, its influence on the microbial phyllosphere community structure is unknown. We hypothesize that nitrogen impacts the growth and abundance of several microorganisms on the leaf surface. The bacterial and fungal communities of baby leaf spinach (Spinacia oleracea), and rocket (Diplotaxis tenuifolia) were investigated in a field trial for two years in a commercial setting. Nitrogen fertilizer was tested in four doses (basic nitrogen, basic + suboptimal, basic + commercial, basic + excess) with six replicates in each. Culture-independent (Illumina sequencing) and culture-dependent (viable count and identification of bacterial isolates) community studies were combined with monitoring of plant physiology and site weather conditions. This study found that alpha diversity of bacterial communities decreased in response to increasing nitrogen fertilizer dose, whereas viable counts showed no differences. Correspondingly, fungal communities of the spinach phyllosphere showed a decreasing pattern, whereas the decreasing diversity of fungal communities of rocket was not significant. Plant species and effects of annual variations on microbiome structure were observed for bacterial and fungal communities on both spinach and rocket. This study provides novel insights on the impact of nitrogen fertilizer regime on a nutrient scarce habitat, the phyllosphere.

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-1β 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-1β accumulation. Oxidized LDL (oxLDL) induced IL-1β release and CD36 expression on THP1 cells. Modified LDL co-stimulated with ATCC33277 exhibited regulatory effects on caspase 1 activity, IL-1β 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.

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.

Periodontal pathogens, including Porphyromonas 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-β1 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.

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.

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 expre ...

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 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.

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 Pam3 CSK4 , 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 Pam3 CSK4 -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.

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-β1 (TGF-β1 ), CXCL8, secretory leucocyte protease inhibitor (SLPI), c-Jun and indoleamine 2,3-dioxygenase (IDO). HGFs were inoculated for 6 and 24 h 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-β1 in HGFs, and the kgp mutant gave significantly higher immune response with increased CXCL8 (P < 0.001) and low levels of TGF-β1 . 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.

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-β1 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-β1 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-β1, in saliva and GCF may serve to predict the progression of periodontitis and associated systemic inflammatory diseases.

High levels of hepatocyte growth factor (HGF), a healing factor with regenerative and cytoprotective effects, are associated with inflammatory diseases, including periodontitis. HGF biologic activity requires binding to its receptors, the proto-oncogene c-Met and heparan sulfate proteoglycan (HSPG). This study investigates HGF expression and its relationship to subgingival microbiota in medically healthy individuals with and without periodontitis.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 and binding affinity for HSPG and c-Met using surface plasmon resonance. 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.Patients with periodontitis showed higher HGF concentrations in saliva, GCF, and serum (P0.001); however, the binding affinities for HSPG and c-Met were reduced in GCF and saliva (P0.002). In contrast to the controls, saliva from patients showed no significant regenerative effect over time on gingival epithelial cells. Compared with controls, patients had a higher prevalence of periodontally related bacteria.Higher circulatory HGF levels indicate a systemic effect of periodontitis. However, the HGF biologic 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.

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 NFκB (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-κB, PKC, ERK, p38 and JNK.

Porphyromonas gingivalis, which is considered a keystone agent in periodontitis, has evolved elaborate mechanisms to grow and survive in a hostile milieu. The gingival fibroblast is the major cell type in the gingiva and is considered to be important in the periodontitis-associated inflammation. As a part of the innate immune response, they produce cytokines such as CXCL8 and interleukin (IL)-6 which are believed to contribute to the destruction of the tooth-supporting tissues. This study investigates how the expression of protease-activated receptors (PAR1, PAR2) and toll-like receptors (TLR2, TLR4) changes with P. gingivalis exposure and how silencing of one receptor affects the expression of the other receptors. The importance of protein kinase C (PKC) and p38 in the regulation of CXCL8 and IL-6 was also examined. Receptors were knockdown with small-interfering RNA. PKC or p38 was blocked prior to stimulation with P. gingivalis. Fibroblasts were able to compensate for PAR1 knockdown with increased expression of PAR2. PKC and p38 were involved in the regulation of P. gingivalis-induced CXCL8 and IL-6. Our results indicate that PAR1 and PAR2 could be implicated in periodontitis and that PKC and P38 play a role in the inflammatory response in P. gingivalis-infected gingival fibroblasts.

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.

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. Funding Agencies|Linkoping University; Swedish Foundation for Strategic Research (SSF).The previous status of this article was Manuscript and the working title was Collagen-Silver Nanowire Composites as Electrically Activeand Antibacterial Scaffolds for Embryonic Cardiac Cell Proliferation

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.

Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling. funding agencies|strategic areas Cardiovascular Inflammation Research Centre (CIRC)||Material in Medicine, the Heart- and Lung foundation||County of Ostergotlands Lan||STIFUD||Olle E||Swedish Research Council

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.

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.

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 A 1 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.

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 MRI. The relaxivity is compared to commercially available Gd based chelates. The synthesis, PEGylation, and dialysis of small (3-5 nm) gadolinium oxide (DEG-Gd(2)O(3)) 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)-Gd(2)O(3) nanoparticles compared to commercial Gd-DTPA. A slight decrease of the relaxivity for as-synthesized DEG-Gd(2)O(3) 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 Gd(2)O(3). 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.

Objective: Several studies support an association between periodontal disease and atherosclerosis with a crucial role for the pathogen Porphyromonas gingivalis. This study aims to investigate the proteolytic and oxidative activity of P. gingivalis on LDL in a whole blood system by using a proteomic approach and analyze the effects of P. gingivalis-modifed LDL on cell proliferation. Methods: The cellular effects of P. gingivalis in human whole blood were assessed using lumi-aggregometry analyzing reactive oxygen species (ROS) production and aggregation. Blood was incubated for 30 min with P. gingivalis, whereafter LDL was isolated and a proteomic approach was applied to examine protein expression. LDL-oxidation was determined by analyzing the formation of protein carbonyls. The effects of P. gingivalis-modifed LDL on fibroblast proliferation were studied using the MTS-assay. Results: Incubation of whole blood with P. gingivalis caused an extensive aggregation and ROS-production, indicating platelet and leukocyte activation. LDL prepared from the bacteria-exposed blood showed an increased protein oxidation, elevated levels of apoM and formation of two apoB-100 N-terminal fragments. P. gingivalis-modified LDL markedly increased the growth of fibroblasts. Inhibition of gingipain R suppressed the modification of LDL by P. gingivalis. Conclusions: The ability of P. gingivalis to change the protein expression and the proliferative capacity of LDL may represent a crucial event in periodontitis-associated atherosclerosis. The definitive version is available at www.blackwell-synergy.com: Torbjörn Bengtsson, Helen Karlsson, Patrik Gunnarsson, Caroline Skoglund, Charlotte Elison, Per Leanderson and Mats Lindahl, The periodontal pathogen Porphyromonas gingivalis cleaves apoB-100 and increases the expression of apoM in LDL in whole blood leading to cell proliferation, 2008, Journal of Internal Medicine, (263), 5, 558-571. http://dx.doi.org/10.1111/j.1365-2796.2007.01917.x. Copyright: Blackwell Publishing www.blackwell-synergy.com

Background There is a rapid emergence of multiple resistant gram-negative bacteria due to overuse of antibiotics in the treatment of infections. Biofilms consist of polymicrobial communities that survive the host’s defense system. The key bacteria in biofilms are slow growing and support an attachment and rapid growth of other microorganisms. Current antimicrobial strategies often fail due to poor diagnosis of key pathogens in biofilms. The study aims to develop anti-bacterial human antibodies in vitro from patients who had recently undergone a systemic infection by pathogenic bacteria and to use these antibodies as a tool for detecting bacteria in biofilms. Methods Lymphocytes were separated from whole blood of patients (n = 10) and stimulated with heat-killed bacteria to produce antibodies in vitro. The specificity of antibodies in recognizing the bacteria against which they were directed was evaluated by surface plasmon resonance system (SPR) and electron microscopy. The ulcer secretions from patients with chronic and acute leg ulcers and healthy controls were analyzed by the SPR system and the results were compared with culture studies. Results The produced antibodies recognized bacteria with high sensitivity (SPR). The antibodies against Enterococcus fecalis bound specifically to the microorganism in a bacterial co-culture that was visualized by electron microscopy. Conclusion In the present work, a method for producing specific antibodies against bacteria is introduced to recognize bacterial components in body fluids of patients suffering from pathogenic biofilms. This diagnostic technique may be most useful in clinical microbiology and in the choice of antibiotics in the treatment of serious infections. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0821-5) contains supplementary material, which is available to authorized users.

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-β1 (TGF-β1) 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-β1. 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.

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.

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

SummaryThere is increasing evidence that Chlamydia pneumoniae is linked to atherosclerosis and thrombosis. In this regard, we have recently shown that C. pneumoniae stimulates platelet aggregation and secretion, which may play an important role in the progress of atherosclerosis and in thrombotic vascular occlusion. The aims of the present study were to investigate the effects of C. pneumoniae on platelet-mediated formation of reactive oxygen species (ROS) and oxidation of low-density lipoprotein (LDL) in vitro. ROS production was registered as changes in 2‘,7’-dichlorofluorescin- fluorescence in platelets with flow cytometry. LDL-oxidation was determined by measuring thiobarbituric acid reactive substances (TBARs). We found that C. pneumoniae stimulated platelet production of ROS. Polymyxin B treatment of C. pneumoniae, but not elevated temperature, abolished the stimulatory effects on platelet ROS- production, which suggests that chlamydial lipopolysaccharide has an important role. Inhibition of nitric oxide synthase with nitro-L-arginine, lipoxygenase with 5,8,11-eicosatriynoic acid and protein kinase C with GF 109203X significantly lowered the production of radicals. In contrast, inhibition of NADPH-oxidase with di-phenyleneiodonium (DPI) did not affect the C. pneumoniae induced ROS-production. These findings suggest that the activities of nitric oxide synthase and lipoxygenase are the sources for ROS and that the generation is dependent of the activity of protein kinase C. The C. pneumoniae-induced ROS-production in platelets was associated with an extensive oxidation of LDL, which was significantly higher compared to the effect obtained by separate exposure of LDL to C. pneumoniae or platelets. In conclusion, C. pneumoniae interaction with platelets leading to aggregation, ROS-production and oxidative damage on LDL, may play a crucial role in the development of atherosclerotic cardiovascular disease.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple organ involvement, production of a wide range of autoantibodies and by formation and tissue deposition of immune complexes in the inflamed organs. In contrast to most systemic inflammatory conditions, and despite raised levels of pro-inflammatory cytokines, SLE flares are rarely reflected by elevated C-reactive protein (CRP), an important acute-phase reactant in man with homologues in vertebrates and several invertebrates. Normally, the circulating concentration of liver-derived CRP rises rapidly in response to infections and tissue injury, and CRP measurement is widely employed as a marker of ongoing inflammation. With sensitive methods, detection of small elevations of CRP is also valuable as a prognostic marker in cardiovascular disease. As a part of the innate immune system, CRP binds certain molecules exposed on the surface of dying cells/apoptotic bodies and on the surface of pathogens and mediates their elimination by uptake in the reticuloendothelial system. CRP also interacts with IgG-containing immune complexes, binds Fc-receptors and activates the complement system via C1q. In murine lupus, CRP has been found to decrease autoantibody levels and increase the survival rates. In this review we discuss possible explanations for, and consequences of, the relative CRP failure in SLE, as well as pathogenetic implications of anti-CRP autoantibodies.

A growing amount of evidence suggests that reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, regulate intracellular signalling and have a role in cell proliferation. In the present study, we show that platelets increase the mitogenic rate in human fibroblasts and that this effect was inhibited by the intracellular antioxidant N-acetyl-L-cysteine (NAC) and the NADPH-oxidase inhibitor diphenyleneiodonium chloride (DPI). The mitogenic effects of platelets were mimicked by the platelet factors platelet-derived growth factor BB-isoform (PDGF-BB), transforming growth factor beta1 (TGF-beta1) and sphingosine-1-phosphate (S1P). The sphingosine kinase inhibitor DL-threo-dihydrosphingosine (DL-dihydro) abrogated the platelet-induced growth, while antibodies directed against PDGF or TGF-beta had modest effects. Exposure of fibroblasts to platelets, PDGF-BB, TGF-beta1 or S1P caused an extensive intracellular ROS production, measured as changes in dichlorofluorescein fluorescence. This ROS production was totally inhibited by NAC, pyrrolidinethiocarbamate (PDTC), DPI and apocynin. In conclusion, the results presented are indicative of a crucial role of ROS in the platelet-mediated regulation of fibroblast proliferation.

The interaction between neutrophil granulocytes and platelets is considered to play an important role in the inflammatory process induced by an implanted foreign material. However, the cellular mechanisms involved remain incompletely understood. We used a luminol-dependent chemiluminescence (CL) technique to analyze the generation of reactive oxygen species (ROS) in human neutrophils interacting with different plasma protein-coated surfaces in the presence or absence of unstimulated or stimulated platelets. The role of tyrosine phosphorylation in the regulation of NADPH oxidase activity was evaluated with quantitative fluorescence microscopy and the specific tyrosine kinase inhibitor genistein. We found that the ROS-production is 2 to 3 times higher in neutrophils on immunoglobulin G (IgG)-coated surfaces than in cells interacting with albumin- or fibrinogen-coated surfaces. Incubation with superoxide dismutase and catalase revealed that about 45% of the ROS was released extracellularly on IgG surfaces whereas corresponding values were 90% and 85% in neutrophils interacting with albumin and fibrinogen, respectively. The presence of platelets markedly increased the extracellular generation of ROS, mainly in neutrophils interacting with IgG- or fibrinogen-coated surfaces whereas the intracellular production was only modestly affected. Quantitative fluorescence microscopy of neutrophils stained with FITC-conjugated anti-phosphotyrosine antibodies showed a correlation between tyrosine phosphorylation, cell spreading, and ROS production. Platelets markedly amplified the anti-phosphotyrosine staining on both fibrinogen- and IgG-coated surfaces whereas the low level of tyrosine phosphorylation in neutrophils on albumin-coated surfaces was not further elevated by platelets. Furthermore, the tyrosine kinase inhibitor genistein inhibited both extra- and intracellular ROS production in neutrophils regardless of the presence of platelets. We demonstrate that plasma protein coating and the presence of platelets are crucial for the inflammatory response of adhering neutrophils and that the oxidative response correlates with the extent of tyrosine phosphorylation of proteins in focal contacts.

Objective—Evidence linkingChlamydia pneumoniaeto atherosclerotic cardiovascular disease is expanding. Platelets are considered to play an essential role in cardiovascular diseases; however, so far platelets have not been associated with an infectious cause of atherosclerosis. This study aims to clarify the interaction betweenC pneumoniaeand platelets and possibly present a novel mechanism in the pathogenesis of atherosclerosis.Methods and Results—The effects ofC pneumoniaeon platelet aggregation and secretion were assessed with lumiaggregometry, and the ability ofC pneumoniaeto bind to platelets and stimulate expression of P-selectin was analyzed with flow cytometry. We found thatC pneumoniae, at a chlamydia:platelet ratio of 1:15, adheres to platelets and triggers P-selectin expression after 1 minute and causes an extensive aggregation and ATP secretion after 20 minutes of incubation. Inhibition of glycoprotein IIb/IIIa with Arg-Gly-Asp-Ser or abciximab markedly reducedC pneumoniae-induced platelet aggregation. Exposure ofC pneumoniaeto polymyxin B, but not elevated temperature, abolished the stimulatory effects on platelet activation, suggesting that chlamydial lipopolysaccharide has an active role. In contrast, other tested bacteria had no or only moderate effects on platelet functions.Conclusion—Our findings demonstrate a new concept of howC pneumoniaeactivates platelets and thereby may cause atherosclerosis and thrombotic vascular occlusion.

Actin exists as globular (G) monomers or polymeric filaments (F) in the cytoplasm of eukaryotic cells, mediating cell morphologic changes and motility. Large amounts of this protein may be released ...

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

The effects of exogenous and endogenous adenosine on the production of oxygen metabolites in neutrophils triggered by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) or immunoglobulin G (IgG)-opsonized yeast particles, were investigated. By using luminol-enhanced chemiluminescence, we found that adenosine A1 receptor activation did not affect, whereas adenosine A receptor activation, through a mechanism involving the cyclic AMP (cAMP)-protein kinase A signalling pathway, both inhibited the fMLP- and IgG-triggered respiratory burst. The adenosine-induced inhibition was however more pronounced after exposure to fMLP than to IgG-yeast. Stimulation with fMLP caused an extracellular accumulation of endogenous adenosine, which indicates that this event is a negative-feedback mechanism preventing an uncontrolled activation of chemoattractant-stimulated neutrophils. On the contrary, exposure of neutrophils to IgG-yeast did not appear to accumulate extracellular adenosine, probably due to increased adenosine deaminase activity during phagocytosis. In conclusion, this work accentuates the importance of adenosine, both exogenously applied and endogenously formed, as an inflammatory agent modulating the respiratory burst during the different phases in neutrophil activation.

Summary The mechanisms by which adenosine regulates the inflammatory reaction are poorly characterized. In this study, we investigated the effects of adenosine on neutrophil actin polymerization elicited by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) or IgG-opsonized yeast particles. We used bodipy-phallacidin staining in combination with flow cytometry and found that adenosine markedly reduced actin polymerization triggered by IgG-yeast, whereas the effect on the fMLP-response was less pronounced. Similar or even more pronounced effects were obtained with the adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), suggesting an A2 receptor-mediated mechanism. The following observations indicate that the A2 receptor-induced effects involve the cAMP-protein kinase A (PKA) signaling pathway: (1) a combination of NECA and the cAMP-specific phosphodiesterase (PDE) inhibitor Ro 20-1724 raised the cAMP content in both unstimulated and stimulated neutrophils and also further inhibited the actin dynamics; (2) the PKA inhibitor H89 reversed the inhibitory effects of NECA on the actin dynamics; (3) Ro 20-1724, isoproterenol and dibutyryl cAMP (DBcAMP) reduced actin polymerization in almost the same way as NECA did. NECA together with Ro 20-1724 impaired the fMLP-induced shape changes and cortical accumulation of actin filaments. In contrast, H89 potentiated the fMLP-induced formation of a submembranous ring of actin filaments. Neutrophils phagocytosing yeast particles in the presence of NECA and Ro 20-1724 were predominantly round in shape, and their ability to extend actin-rich pseudopods around the prey was reduced. These effects were partly antagonized by H89. In correlation with the effects on actin polymerization, NECA more effectively diminished IgG-induced upregulation of the s2 integrin CDllb/CD18 than such upregulation induced by fMLP. The inhibitory effects of A2-receptor activation on actin dynamics and (J2 integrin expression in neutrophils exposed to IgG-yeast were also associated with a cAMP-dependent reduction of the phagocytic capacity. In conclusion, we show that adenosine inhibits actin dynamics and shape changes in neutrophils via a cAMP-dependent pathway. This finding further characterizes the mechanisms by which adenosine functions as an important modulator of the inflammatory response.

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 Glanzmanns 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.

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

Background: Hepatocyte growth factor (HGF) acts as an acute phase protein with regenerative properties. HGF is produced systemically and locally during inflammation but exhibits decreased binding affinity to heparan sulphate proteoglycan (HSPG)/glycosaminoglycan during chronic inflammation. We previously observed a high faecal concentration and binding affinity of HGF to HSPG during acute gastroenteritis. High faecal concentrations of calprotectin and HGF have been reported in chronic inflammatory bowel disease (IBD). Methods: Stool samples from patients with ulcerative colitis in remission (n = 11) or exacerbation (n = 5), microscopic colitis (n = 11), colon cancer (n = 6), or acute gastroenteritis caused by Clostridium difficile (n = 20), as well as healthy controls (n = 7), were analysed for the presence of HGF by ELISA, surface plasmon resonance, SDS-PAGE, and Western blot. Then in two patients with ulcerative colitis exacerbation and C. difficile infection, the expression of HGF and calprotectin was studied in colonic biopsies. Results: The faecal concentration of HGF was significantly higher in patients with ulcerative colitis compared to the other groups. The binding affinity to dextran was lower in all groups compared to acute inflammation. HGF receptor binding was similar across groups. In a patient with concomitant C. difficile infection and distal ulcerative colitis, HGF was highly expressed in the part of the bowel unaffected by ulcerative colitis, but no expression was found at the site of chronic inflammation. In the patient with total colitis the biopsies showed low expression of HGF. The areas with chronic inflammation exhibited infiltrating calprotectin-stained neutrophils. Conclusion: HGF is produced locally during inflammation of the bowel. The HGF produced during acute inflammation or exacerbations of chronic inflammation by the unaffected area shows binding affinity to glucosaminoglycans. Measuring HGF binding in faeces and biopsies may be a tool for differentiating between acute and chronic bowel inflammation, which should be assessed thoroughly in future studies.