Tilapia Lake Virus-Induced Neuroinflammation in Zebrafish: Microglia Activation and Sickness Behavior.

In mammals, the relationship between the immune system and behavior is widely studied. In fish, however, the knowledge concerning the brain immune response and behavioral changes during brain viral infection is very limited. To further investigate this subject, we used the model of tilapia lake virus (TiLV) infection of zebrafish (Danio rerio), which was previously developed in our laboratory. We demonstrated that TiLV persists in the brain of adult zebrafish for at least 90 days, even when the virus is not detectable in other peripheral organs. The virions were found in the whole brain. During TiLV infection, zebrafish displayed a clear sickness behavior: decreased locomotor activity, reduced food intake, and primarily localizes near the bottom zone of aquaria. Moreover, during swimming, individual fish exhibited also unusual spiral movement patterns. Gene expression study revealed that TiLV induces in the brain of adult fish strong antiviral and inflammatory response and upregulates expression of genes encoding microglia/macrophage markers. Finally, using zebrafish larvae, we showed that TiLV infection induces histopathological abnormalities in the brain and causes activation of the microglia which is manifested by changes in cell shape from a resting ramified state in mock-infected to a highly ameboid active state in TiLV-infected larvae. This is the first study presenting a comprehensive analysis of the brain immune response associated with microglia activation and subsequent sickness behavior during systemic viral infection in zebrafish.

Type I interferon-dependent response of zebrafish larvae during tilapia lake virus (TiLV) infection.

Tilapia lake virus (TiLV; genus: Tilapinevirus, family: Amnoonviridae) is a recently characterised enveloped virus with a linear, negative-sense single-stranded RNA genome, which causes high mortality in tilapia species. In the present study, we demonstrated that zebrafish (Danio rerio) larvae are susceptible to TiLV infection upon systemic injection. TiLV replicated in zebrafish larvae and caused their high mortality (of about 70%). Histopathological examination revealed that TiLV infection caused pathological abnormalities in zebrafish larvae that were well visible within the brain. Moreover, gene expression analysis revealed that TiLV infection induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I (ddx58), tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), and pro-inflammatory cytokine (il-1ß). We also demonstrated the protective role of the recombinant zebrafish IFNϕ1 on the survival of zebrafish larvae during TiLV infection. Our results show the importance of type I IFN response during TiLV infection in zebrafish larvae and demonstrate that zebrafish is a good model organism to study interactions between TiLV - a newly emerging in aquaculture virus, and fish host.

Mechanisms of vascular damage by systemic dissemination of the oral pathogen Porphyromonas gingivalis.

Several studies have shown a clear association between periodontal disease and increased risk of cardiovascular disease. Porphyromonas gingivalis (Pg), a key oral pathogen, and its cell surface-expressed gingipains, induce oedema in a zebrafish larvae infection model although the mechanism of these vascular effects is unknown. Here, we aimed to determine whether Pg-induced vascular damage is mediated by gingipains. In vitro, human endothelial cells from different vascular beds were invaded by wild-type (W83) but not gingipain-deficient (ΔK/R-ab) Pg. W83 infection resulted in increased endothelial permeability as well as decreased cell surface abundance of endothelial adhesion molecules PECAM-1 and VE-cadherin compared to infection with ΔK/R-ab. In agreement, when transgenic zebrafish larvae expressing fluorescently labelled PECAM-1 or VE-cadherin were systemically infected with W83 or ΔK/R-ab, a significant reduction in adhesion molecule fluorescence was observed specifically in endothelium proximal to W83 bacteria through a gingipain-dependent mechanism. Furthermore, this was associated with increased vascular permeability in vivo when assessed by dextran leakage microangiography. These data are the first to show that Pg directly mediates vascular damage in vivo by degrading PECAM-1 and VE-cadherin. Our data provide a molecular mechanism by which Pg might contribute to cardiovascular disease.

Antiviral response of adult zebrafish (Danio rerio) during tilapia lake virus (TiLV) infection.

Tilapia lake virus (TiLV) is a novel enveloped orthomyxo-like virus with a genome of 10 segments of linear negative-sense single-stranded RNA. It causes massive mortality of wild and farmed tilapia species and because of its spread in Asia, Africa, South and North America, it is considered a threat to tilapia aquaculture. Here, we have evaluated the possible use of zebrafish (Danio rerio) to study immune response and host-pathogen interactions during an infection with TiLV. Adult zebrafish were infected with TiLV by intraperitoneal (i.p) injection or by cohabitation. Increased viral load was observed in liver, spleen and kidney of i.p. injected fish at 1, 3, 6, and 14 days post infection (dpi) but not in fish from the cohabitation group (only liver was tested). We also demonstrated that in spleen and kidney i.p. injection of TiLV induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I, tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), pro-inflammatory (il-1ß, tnf-α, il-8, ifnγ1-2) and anti-inflammatory (il-10) cytokines, CD4 markers (cd4-1, cd4-2), and IgM (igm). Moreover, tissue tropism of TiLV and histopathological changes were analyzed in selected organs of i.p. injected zebrafish. Our results indicate that zebrafish is a good model to study mechanisms of the TiLV infection and to follow antiviral responses.

Incorporation of Ca ions into anodic oxide coatings on the Ti-13Nb-13Zr alloy by plasma electrolytic oxidation.

The present work concerns the surface modification of The Ti-13Nb-13Zr alloy by electropolishing and plasma electrolytic oxidation (PEO) process in Ca-containing electrolytes: calcium formate and calcium lactate solutions (0.1-1.0 mol dm-3) under voltages of 200 and 400 V. As a result of the PEO process, a porous oxide layer containing incorporated calcium compounds was formed on the Ti-13Nb-13Zr alloy surface. The morphology and chemical composition of the modified Ti-13Nb-13Zr alloy were investigated using scanning electron microscopy (SEM + EDS), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). An increase in the applied voltage caused an increase in the number of pores and an increase in the amount of calcium incorporated in the oxide layer. Analysis showed that all samples were covered by titanium oxide, which was present in the form of anatase and/or rutile. In course of the experiments, it was showed that the proposed procedure has a positive effect on the overall bioactivity of the Ti-13Nb-13Zr alloy. Bioactivity investigations using simulated body fluid (SBF) confirmed the formation of apatite on the anodized surfaces. The cell adhesion results obtained by the use of human bone marrow mesenchymal stem cells (hBMSC) demonstrated that the PEO coatings on the Ti-13Nb-13Zr alloy remarkably enhanced the cytocompatibility of the substrate, indicating a potential application in orthopedic surgeries. The incorporation of Ca into the oxide layer proceeded to a higher extent when the PEO treatment was performed in the calcium lactate bath. The oxide layers formed in the calcium lactate solution exhibited also superior biological behavior towards hBMSC. This can be ascribed to the presence of carboxylic groups onto coatings' surface (as identified by XPS), which facilitated the anchoring of cells and tissues.

Role of OmpA2 surface regions of Porphyromonas gingivalis in host-pathogen interactions with oral epithelial cells.

Outer membrane protein A (OmpA) is a key outer membrane protein found in Gram-negative bacteria that contributes to several crucial processes in bacterial virulence. In Porphyromonas gingivalis, OmpA is predicted as a heterotrimer of OmpA1 and OmpA2 subunits encoded by adjacent genes. Here we describe the role of OmpA and its individual subunits in the interaction of P. gingivalis with oral cells. Using knockout mutagenesis, we show that OmpA2 plays a significant role in biofilm formation and interaction with human epithelial cells. We used protein structure prediction software to identify extracellular loops of OmpA2, and determined these are involved in interactions with epithelial cells as evidenced by inhibition of adherence and invasion of P. gingivalis by synthetic extracellular loop peptides and the ability of the peptides to mediate interaction of latex beads with human cells. In particular, we observe that OmpA2-loop 4 plays an important role in the interaction with host cells. These data demonstrate for the first time the important role of P. gingivalis OmpA2 extracellular loops in interaction with epithelial cells, which may help design novel peptide-based antimicrobial therapies for periodontal disease.

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two ß-propeller domains and a C-terminal ß-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

Zebrafish as a new model to study effects of periodontal pathogens on cardiovascular diseases.

Porphyromonas gingivalis (Pg) is a keystone pathogen in the aetiology of chronic periodontitis. However, recent evidence suggests that the bacterium is also able to enter the bloodstream, interact with host cells and tissues, and ultimately contribute to the pathogenesis of cardiovascular disease (CVD). Here we established a novel zebrafish larvae systemic infection model showing that Pg rapidly adheres to and penetrates the zebrafish vascular endothelium causing a dose- and time-dependent mortality with associated development of pericardial oedemas and cardiac damage. The in vivo model was then used to probe the role of Pg expressed gingipain proteases using systemically delivered gingipain-deficient Pg mutants, which displayed significantly reduced zebrafish morbidity and mortality compared to wild-type bacteria. In addition, we used the zebrafish model to show efficacy of a gingipain inhibitor (KYT) on Pg-mediated systemic disease, suggesting its potential use therapeutically. Our data reveal the first real-time in vivo evidence of intracellular Pg within the endothelium of an infection model and establishes that gingipains are crucially linked to systemic disease and potentially contribute to CVD.

In vitro bioactivity investigations of Ti-15Mo alloy after electrochemical surface modification.

Titanium and its aluminum and vanadium-free alloys have especially great potential for medical applications. Electrochemical surface modification improves their surface bioactivity and stimulates osseointegration process. In this work, the effect of plasma electrolytic oxidation of the ß-type alloy Ti-15Mo surface on its bioactivity is presented. Bioactivity of the modified alloy was investigated by immersion in simulated body fluid (SBF). Biocompatibility of the modified alloys were tested using human bone marrow stromal cells (hBMSC) and wild intestinal strains (DV/A, DV/B, DV/I/1) of Desulfovibrio desulfuricans bacteria. The particles of apatite were formed on the anodized samples. Human BMSC cells adhered well on all the examined surfaces and expressed ALP, collagen, and produced mineralized matrix as determined after 10 and 21 days of culture. When the samples were inoculated with D. desulfuricans bacteria, only single bacteria were visible on selected samples. There were no obvious changes in surface morphology among samples. Colonization and bacterial biofilm formation was observed on as-ground sample. In conclusion, the surface modification improved the Ti-15Mo alloy bioactivity and biocompatibility and protected surface against colonization of the bacteria. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 903-913, 2016.

Bioactivity of coatings formed on Ti-13Nb-13Zr alloy using plasma electrolytic oxidation.

In this work, we investigated the bioactivity of anodic oxide coatings on Ti-13Nb-13Zr alloy by plasma electrolytic oxidation (PEO) in solutions containing Ca and P. The bioactive properties of the films were determined by immersion in simulated body fluid (SBF), and their biocompatibility was examined using adult human bone marrow derived mesenchymal stem cells (hBMSCs). The oxide layers were characterised based on their surface morphology (SEM, AFM, profilometry) as well as on their chemical and phase compositions (EDX, XRF, XRD, XPS). We report that anodic oxidation of Ti-13Nb-13Zr led to the development of relatively thick anodic oxide films that were enriched in Ca and P in the form of phosphate compounds. Furthermore, the treatment generated rough surfaces with a significant amount of open pores. The surfaces were essentially amorphous, with small amounts of crystalline phases (anatase and rutile) being observed, depending on the PEO process parameters. SBF soaking led to the precipitation of small crystals after one week of experiment. During culturing of hBMSCs on the bioactive Ti-13Nb-13Zr surfaces the differentiation of human mesenchymal stem cells toward osteoblasts was promoted, which indicated a potential of the modified materials to improve implant osseointegration.

New sol-gel bioactive glass and titania composites with enhanced physico-chemical and biological properties.

We developed TiO2 matrix composites modified by sol-gel bioactive glasses (SBG) of either high CaO content (A2) or high SiO2 content (S2). The latter were mixed with titanium dioxide (TiO2) at 75:25, 50:50, and 25:75 weight ratios and sintered at 1250°C for 2 h. We examined the effects of various types (A2 or S2) and compositional TiO2 :SBG ratios on the mechanical properties of resulting composites, their bioactivity and human bone marrow mesenchymal stem cells (MSC) response. The chemistry of SBGs influenced the phase composition, mechanical and biological properties of the composites. Rutile and titanite prevailed in A2-TiO2 composites, and rutile and crystobalite in S2-TiO2 composites. Compressive strength increased significantly for 25A2-TiO2 composites (140 MPa) compared to matrix TiO2 (58 MPa). Composites containing 50-75 wt % of either SBG displayed bioactive properties as determined by simulated body fluid test. Compared to TiO2, human bone marrow stromal cell (BMSC) viability was enhanced on the composites containing 25 wt % of either SBG, whereas the composites modified by 25 wt % of S2 enhanced alkaline phosphatase activity and mineralization in cultures treated with osteogenic inducers-dexamethasone (Dex) or bone morphogenetic protein. Increasing amounts of A2 in TiO2 matrix decreased cell viability but increased collagen deposition and mineralized matrix production by BMSC. Considering the physico-chemical and biological properties of the presented composites, the modification of TiO2 with SBG may prove useful strategy in several bone tissue related regeneration strategies.