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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.

In this study, ginger residue from juice production was evaluated as a raw material resource for preparation of nanofiber hydrogels with multifunctional properties for advanced wound dressing applications. Alkali treatment was applied to adjust the chemical composition of ginger fibers followed by TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation prior to nanofiber isolation. The effect of alkali treatment on hydrogel properties assembled through vacuum filtration without addition of any chemical cross-linker was evaluated. An outstanding absorption ability of 6200% combined with excellent mechanical properties, tensile strength of 2.1 +/- 0.2 MPa, elastic modulus of 15.3 +/- 0.3 MPa, and elongation at break of 25.1%, was achieved without alkali treatment. Furthermore, the absorption capacity was tunable by applying alkali treatment at different concentrations and by adjusting the hydrogel grammage. Cytocompatibility evaluation of the hydrogels showed no significant effect on human fibroblast proliferation in vitro. Ginger essential oil was used to functionalize the hydrogels by providing antimicrobial activity, furthering their potential as a multifunctional wound dressing. Funding Agencies|Swedish Foundation for Strategic Research within the HEALiX projectSwedish Foundation for Strategic Research [RMX18-0039]; Bio4Energy; KAW [2018.0451]; WWSC; Kempe Foundations; Coordenacao de Aperfeicamento de Pessoal de Nivel Superior, Brasil (CAPES) [001]

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

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.

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

Background: NF-κB is a key regulator of anti-apoptotic processes and plays a role in gonad formation in mammals. Results: NF-κB activation leads to female-biased sex differentiation in zebrafish. Conclusion: Anti-apoptotic signaling during the juvenile ovary stage is needed for the maintenance of oocytes in zebrafish. Significance: Unraveling the regulation of apoptotic processes during gonadal transformation will facilitate understanding the molecular mechanism of zebrafish sex differentiation., 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-κB 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-Müllerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-κB-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-κB in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-κB 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-κB-regulated genes. To confirm the involvement of NF-κB-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-κB or NF-κB 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-κB using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-κB 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.

The issue of unfair competition lawsuit, which enables competition who encountered acts of unfair competition to get a way to preserve their competition postions and reputatior, brought the attention to the international level, and did not get much attention in the field of legal regulation in the light of domestic laws like the Iraqi law, which is not compatible with its importance in the field of protection of rights which belong to what is modern in science and technology and its effect in the scope of private international law, particularly with respect to differences in the verdict in the issue of determining the applicable law and the court specialized in disputes relating to such proceedings and of what its nature has the effect of fixing it as well about what international conventions brought and have death with such an issue, especially the trips agreement . .

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)cyclohexane (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.

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 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.

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 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 Hep G2 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.

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.

Interleukin-8 (CXCL8) is a chemokine that was originally identified as a key factor in neutrophil recruitment and activation. Numerous cell types produce CXCL8, including immune cells, mucosal epithelial cells, endothelial cells and smooth muscle cells (GarciaVicuna et al., 2004). CXCL8 is one of the important inflammatory mediators responsible for the recruitment of neutrophils and T-cells to the site of infection, therefore it is an attractive target for therapy against diseases that affect immune cells such as HIV. HIV directly targets the host’s immune system and thus reduces the ability of the innate and adaptive immune system to fight disease. As a chemokine, CXCL8 is a potential target for controling HIV infection by reducing the migration of T-cells to the site of infection. It is therefore necessary to identify the signaling pathways involved in CXCL8 regulation in order to develop viable CXCL8 based treatment strategies. In line with this, we have recent shown that CXCL8 activation in Jurkat T-cells is not primarily under the control of NFB, but that the AP-1 signaling pathway appears to be central for the regulation of CXCL8 (Khalaf et al., 2010). An understanding of the regulation and function of cytokines and chemokines, while complex, remains important for the development of new strategies in the development of HIV treatments. It is interesting to note that several lactobacilli strains are able to modulate CXCL8 expression and release (Anukam et al., 2009; Zhang et al., 2005). Disturbance of the lactobacilli flora in the vaginal tract has been shown to increase the risk of infections and acquisition of HIV type 1 (Taha et al., 1998). Several studies have shown that treatment with certain Lactobacillus species and strains have positive effects on women with HIV infections and have been successful in trials to counteract vaginal infections (Hummelen et al., 2010; Spear et al., 2007). In the present chapter we give a background to CXCL8 regulation and function as well as its involvement in HIV etiology. We also provide an overview of the available information on the possible uses of Lactobacillus species in treatment of infections with special emphasis on HIV. In addition, the effects obtained by using lactobacilli treatment together with expression of adhesion inhibitors will be discussed. The aim is to give the reader an overview of the role of CXCL8 in HIV infections and combine this with information on how lactobacilli treatment influences the chemokine levels and evaluate these systems potential in the treatment of HIV patients.

BackgroundActivator protein (AP)-1 and nuclear factor (NF)-κB largely control T-cell activation, following binding of foreign antigens to the T-cell receptor leading to cytokine secretion. Elevated levels of pro-inflammatory cytokines and chemokines 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.ResultsPhorbol myristate acetate (PMA) exposure resulted in an up-regulation of AP-1 and down-regulation of NF-κB activity, however, exposure to heat killed (HK)Escherichia. coliMG1655 resulted in a dose-dependent increase in NF-κB activity 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 HKE. coli, while the levels of the anti-inflammatory cytokine IL-10 were not affected by PMA but were significantly down-regulated by HKE. coli. AP-1 activation was significantly increased 2 h after PMA exposure and continued to increase thereafter. In contrast, NF-κB responded 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 HKE. coli. In order to further study NF-κB activation, we considered two up-stream signalling proteins, PKC and Bcl10. Phosphorylated-PKC levels increased in response to PMA and HKE. coli, while Bcl10 levels significantly decreased following PMA treatment. Using an NF-κB activation inhibitor, we observed complete inhibition of IL-6 expression while CXCL8 levels only decreased by 40% at the 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.ConclusionThe present study shows that NF-κB regulated IL-6 but not CXCL8. This complex regulation of CXCL8 suggests that there is a need to further evaluate the signalling pathways in order to develop new treatment for diseases with elevated CXCL8 levels, such as AIDS and autoimmune diseases.

Pharmaceuticals are regularly released into the environment; in particular non-steroidal anti-inflammatory drugs (NSAIDs) and antibiotics. Erythromycin, naproxen, furosemide and atenolol are reported to be stable for up to 1 year in the environment, which increases the risk for accumulation. In the present study we have measured the occurrence and concentration of pharmaceuticals in river Viskan (Jossabron) downstream of a sewage treatment plant in Boras, Sweden. Pharmaceuticals and water samples were tested for potential human risk by evaluating inflammatory responses (NF-kappaB and AP-1) using human T24 bladder epithelial cells and Jurkat T-cells. NF-kappaB activity in T24 cells was significantly reduced by all NSAIDs analysed (diclofenac, ketoprofen, naproxen, ibuprophen and dextropropoxyphene), but also by trimethoprim, using environmentally relevant concentrations. NF-kappaB and AP-1 activation was further analysed in response to water samples collected from different locations in Sweden. Dose-dependent down-regulation of AP-1 activity in Jurkat cells was observed at all locations. At two locations (Jossabron and Almenas) down-regulation of NF-kappaB was observed. In contrast, the NF-kappaB response was potentiated by exposure to water from both locations following activation of NF-kappaB by treatment with heat-killed Escherichia coli. To determine the involvement of pharmaceuticals in the responses, T24 cells were exposed to the pharmaceutical mixture, based on the determined levels at Jossabron. This resulted in reduction of the NF-kappaB response following exposure to the pharmaceutical mixture alone while no potentiation was observed when cells were co-exposed to heat killed E. coli and pharmaceuticals. The obtained results demonstrate that the identified pharmaceuticals affect the inflammatory responses and furthermore indicate the presence of unknown substance(s) with the ability to potentiate inflammatory responses.

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.

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-α (TNF- α) 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-α-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.