Vaccinia Virus Expressing Interferon Regulatory Factor 3 Induces Higher Protective Immune Responses against Lethal Poxvirus Challenge in Atopic Organism.

Vaccinia virus (VACV) is an enveloped DNA virus from the Orthopoxvirus family, various strains of which were used in the successful eradication campaign against smallpox. Both original and newer VACV-based replicating vaccines reveal a risk of serious complications in atopic individuals. VACV encodes various factors interfering with host immune responses at multiple levels. In atopic skin, the production of type I interferon is compromised, while VACV specifically inhibits the phosphorylation of the Interferon Regulatory Factor 3 (IRF-3) and expression of interferons. To overcome this block, we generated a recombinant VACV-expressing murine IRF-3 (WR-IRF3) and characterized its effects on virus growth, cytokine expression and apoptosis in tissue cultures and in spontaneously atopic Nc/Nga and control Balb/c mice. Further, we explored the induction of protective immune responses against a lethal dose of wild-type WR, the surrogate of smallpox. We demonstrate that the overexpression of IRF-3 by WR-IRF3 increases the expression of type I interferon, modulates the expression of several cytokines and induces superior protective immune responses against a lethal poxvirus challenge in both Nc/Nga and Balb/c mice. Additionally, the results may be informative for design of other virus-based vaccines or for therapy of different viral infections.

Heat-treated carbon coatings on poly (l-lactide) foils for tissue engineering.

Carbon-based materials have emerged as promising candidates for a wide variety of biomedical applications, including tissue engineering. We have developed a simple but unique technique for patterning carbon-based substrates in order to control cell adhesion, growth and phenotypic maturation. Carbon films were deposited on PLLA foils from distances of 3 to 7 cm. Subsequent heat-treatment (60 °C, 1 h) created lamellar structures with dimensions decreasing from micro- to nanoscale with increasing deposition distance. All carbon films improved the spreading and proliferation of human osteoblast-like MG 63 cells, and promoted the alignment of these cells along the lamellar structures. Similar alignment was observed in human osteoblast-like Saos-2 cells and in human dermal fibroblasts. Type I collagen fibers produced by Saos-2 cells and fibroblasts were also oriented along the lamellar structures. These structures increased the activity of alkaline phosphatase in Saos-2 cells. Carbon coatings also supported adhesion and growth of vascular endothelial and smooth muscle cells, particularly flatter non-heated carbon films. On these films, the continuity of the endothelial cell layer was better than on heat-treated lamellar surfaces. Heat-treated carbon-coated PLLA is therefore more suitable for bone and skin tissue engineering, while carbon-coated PLLA without heating is more appropriate for vascular tissue engineering.

Novel contribution to clubfoot pathogenesis: The possible role of extracellular matrix proteins.

Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro-proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non-contracted tissue (lateral side; n = 13). We used label-free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin-C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Novel injectable gellan gum hydrogel composites incorporating Zn- and Sr-enriched bioactive glass microparticles: High-resolution X-ray microcomputed tomography, antibacterial and in vitro testing.

Mineralization of hydrogel biomaterials is desirable to improve their suitability as materials for bone regeneration. In this study, gellan gum (GG) hydrogels were formed by simple mixing of GG solution with bioactive glass microparticles of 45S5 composition, leading to hydrogel formation by ion release from the amorphous bioactive glass microparticles. This resulted in novel injectable, self-gelling composites of GG hydrogels containing 20% bioactive glass. Gelation occurred within 20 min. Composites containing the standard 45S5 bioactive glass preparation were markedly less stiff. X-ray microcomputed tomography proved to be a highly sensitive technique capable of detecting microparticles of diameter approximately 8 µm, that is, individual microparticles, and accurately visualizing the size distribution of bioactive glass microparticles and their aggregates, and their distribution in GG hydrogels. The widely used melt-derived 45S5 preparation served as a standard and was compared with a calcium-rich, sol-gel derived preparation (A2), as well as A2 enriched with zinc (A2Zn5) and strontium (A2Sr5). A2, A2Zn, and A2Sr bioactive glass particles were more homogeneously dispersed in GG hydrogels than 45S5. Composites containing all four bioactive glass preparations exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. Composites containing A2Zn5 and A2Sr5 bioactive glasses supported the adhesion and growth of osteoblast-like cells and were considerably more cytocompatible than 45S5. All composites underwent mineralization with calcium-deficient hydroxyapatite upon incubation in simulated body fluid. The extent of mineralization appeared to be greatest for composites containing A2Zn5 and 45S5. The results underline the importance of the choice of bioactive glass when preparing injectable, self-gelling composites.

Valve interstitial cell culture: Production of mature type I collagen and precise detection.

Collagen often acts as an extracellular and intracellular marker for in vitro experiments, and its quality defines tissue constructs. To validate collagen detection techniques, cardiac valve interstitial cells were isolated from pigs and cultured under two different conditions; with and without ascorbic acid. The culture with ascorbic acid reached higher cell growth and collagen deposition, although the expression levels of collagen gene stayed similar to the culture without ascorbic acid. The fluorescent microscopy was positive for collagen fibers in both the cultures. Visualization of only extracellular collagen returned a higher correlation coefficient when comparing the immunolabeling and second harmonic generation microscopy images in the culture with ascorbic acid. Lastly, it was proved that the hydroxyproline strongly contributes to the second-order susceptibility tensor of collagen molecules, and therefore the second harmonic generation signal is impaired in the culture without ascorbic acid.

Plasma-Activated Tropoelastin Functionalization of Zirconium for Improved Bone Cell Response.

The mechanical strength, durability, corrosion resistance, and biocompatibility of metal alloys based on zirconium (Zr) and titanium (Ti) make them desirable materials for orthopedic implants. However, as bioinert metals, they do not actively promote bone formation and integration. Here we report a plasma coating process for improving integration of such metal implants with local bone tissue. The coating is a stable carbon-based plasma polymer layer that increased surface wettability by 28%, improved surface elasticity to the range exhibited by natural bone, and additionally covalently bound the extracellular matrix protein, tropoelastin, in an active conformation. The thus biofunctionalized material was significantly more resistant to medical-grade sterilization by steam, autoclaving or gamma-ray irradiation, retaining >60% of the adhered tropoelastin molecules and preserving full bioactivity. The interface of the coating and metal was robust so as to resist delamination during surgical insertion and in vivo deployment, and the plasma process employed was utilized to also coat the complex 3D geometries typical of orthopedic implants. Osteoblast-like osteosarcoma cells cultured on the biofunctionalized Zr surface exhibited a significant 30% increase in adhesion and up to 70% improvement in proliferation. Cells on these materials also showed significant early stage up-regulation of bone marker expression (alkaline phosphatase, 1.8 fold; osteocalcin, 1.4 fold), and sustained up-regulation of these genes (alkaline phosphatase, 1.3 fold; osteocalcin, 1.2 fold) in osteogenic conditions. In addition, alkaline phosphatase production significantly increased (2-fold) on the functionalized surfaces, whereas bone mineral deposition increased by 30% above background levels compared to bare Zr. These findings have the potential to be readily translated to the development of improved Zr and Ti-based implants for accelerated bone repair.

Chitosan hydrogels enriched with polyphenols: Antibacterial activity, cell adhesion and growth and mineralization.

Injectable hydrogels for bone regeneration consisting of chitosan, sodium beta-glycerophosphate (Na-ß-GP) and alkaline phosphatase (ALP) were enriched with the polyphenols phloroglucinol (PG) and gallic acid (GA) and characterized physicochemically and biologically with respect to properties relevant for applications in bone regeneration, namely gelation kinetics, mineralizability, antioxidant properties, antibacterial activity, cytocompatibility and ability to support adhesion and growth of human osteoblast-like MG63 cells. Enrichment with PG and GA had no negative effect on gelation kinetics and mineralizability. PG and GA both enhanced antioxidant activity of unmineralized hydrogels. Mineralization reduced antioxidant activity of hydrogels containing GA. Hydrogels containing GA, PG and without polyphenols reduced colony forming ability of Escherichia coli after 1h, 3h and 6h incubation and slowed E. coli growth in liquid culture for 150min. Hydrogels containing GA were cytotoxic and supported cell growth more poorly than polyphenol-free hydrogels. PG had no negative effect on cell adhesion and growth.

Development of thermosensitive hydrogels of chitosan, sodium and magnesium glycerophosphate for bone regeneration applications.

Thermosensitive injectable hydrogels based on chitosan neutralized with sodium beta-glycerophosphate (Na-ß-GP) have been studied as biomaterials for drug delivery and tissue regeneration. Magnesium (Mg) has been reported to stimulate adhesion and proliferation of bone forming cells. With the aim of improving the suitability of the aforementioned chitosan hydrogels as materials for bone regeneration, Mg was incorporated by partial substitution of Na-ß-GP with magnesium glycerophosphate (Mg-GP). Chitosan/Na-ß-GP and chitosan/Na-ß-GP/Mg-GP hydrogels were also loaded with the enzyme alkaline phosphatase (ALP) which induces hydrogel mineralization. Hydrogels were characterized physicochemically with respect to mineralizability and gelation kinetics, and biologically with respect to cytocompatibility and cell adhesion. Substitution of Na-ß-GP with Mg-GP did not negatively influence mineralizability. Cell biological testing showed that both chitosan/Na-ß-GP and chitosan/Na-ß-GP/Mg-GP hydrogels were cytocompatible towards MG63 osteoblast-like cells. Hence, chitosan/Na-ß-GP/Mg-GP hydrogels can be used as an alternative to chitosan/Na-ß-GP hydrogels for bone regeneration applications. However the incorporation of Mg in the hydrogels during hydrogel formation did not bring any appreciable physicochemical or biological benefit.

Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films.

Nanocrystalline diamond (NCD) films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination) or oxygen atoms (O-termination). Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers - type I collagen, alkaline phosphatase, and osteocalcin - was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix mineralization, and this is promising for better osteoconductivity of potential bone implant coatings.

Injectable self-gelling composites for bone tissue engineering based on gellan gum hydrogel enriched with different bioglasses.

Hydrogels of biocompatible calcium-crosslinkable polysaccharide gellan gum (GG) were enriched with bioglass particles to enhance (i) mineralization with calcium phosphate (CaP); (ii) antibacterial properties and (iii) growth of bone-forming cells for future bone regeneration applications. Three bioglasses were compared, namely one calcium-rich and one calcium-poor preparation both produced by a sol-gel technique (hereafter referred to as A2 and S2, respectively) and one preparation of composition close to that of the commonly used 45S5 type (hereafter referred to as NBG). Incubation in SBF for 7 d, 14 d and 21 d caused apatite formation in bioglass-containing but not in bioglass-free samples, as confirmed by FTIR, XRD, SEM, ICP-OES, and measurements of dry mass, i.e. mass attributable to polymer and mineral and not water. Mechanical testing revealed an increase in compressive modulus in samples containing S2 and NBG but not A2. Antibacterial testing using biofilm-forming meticillin-resistant staphylococcus aureus (MRSA) showed markedly higher antibacterial activity of samples containing A2 and S2 than samples containing NBG and bioglass-free samples. Cell biological characterization using rat mesenchymal stem cells (rMSCs) revealed a stimulatory effect of NBG on rMSC differentiation. The addition of bioglass thus promotes GG mineralizability and, depending on bioglass type, antibacterial properties and rMSC differentiation.

Heme arginate potentiates latent HIV-1 reactivation while inhibiting the acute infection.

Human immunodeficiency virus-1 (HIV-1) successfully escapes from host immune surveillance, vaccines and antiretroviral agents. The available antiretroviral compounds can only control viremia, but it is impossible to eliminate the virus from the organism, namely because HIV-1 provirus persists in the reservoir cells from which the virus repeatedly disseminates into new cells. Current therapeutic approaches, however, do not specifically address the stage of virus reactivation. Heme has been demonstrated as very efficient in inhibiting HIV-1 reverse transcription, while its derivative hemin ameliorated HIV-1 infection via induction of heme oxygenase-1. Normosang (heme arginate; HA) is a human hemin-containing compound used to treat acute porphyria. In this work, we studied the effects of HA in HIV-1-acutely infected T-cell lines, and in cell lines harboring either a complete HIV-1 provirus (ACH-2 cells) or an HIV-1 "mini-virus" (Jurkat clones expressing EGFP under control of HIV LTR). We demonstrate that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA alone stimulated the reactivation of HIV-1 "mini-virus" and synergized with phorbol ester or TNF-α in the reactivation of HIV-1 provirus. The stimulatory effects of HA were inhibited by N-acetyl cysteine, suggesting an increased redox stress and activation of NF-κB. Further, HA induced expression of heme oxygenase-1 (HO-1) in ACH-2 cells, while HO-1 was found expressed in untreated Jurkat clones. Inhibitor of HO-1 activity, tin protoporphyrin IX, further increased HA-mediated reactivation of HIV-1 "mini-virus" in Jurkat clones, and this effect was also inhibited by N-acetyl cysteine. The stimulatory effects of HA on HIV-1 reactivation thus seem to involve HO-1 and generation of free radicals. Additionally, the effective concentrations of HA did neither affect normal T-cell activation with PMA nor induce activation of the unstimulated cells. In conclusion, HA appears to possess a combination of unique properties that could help to decrease the pool of latently infected reservoir cells, while simultaneously inhibiting HIV-1 replication in newly infected cells. Our results thus suggest a new direction to explore in treatment of HIV/AIDS disease.

Apoptosis and necrosis in vaccinia virus-infected HeLa G and BSC-40 cells.

In most cells, vaccinia virus (VACV) infection is considered to cause a lytic cell death, an equivalent of necrosis. However, upon infection of the epithelial cell lines HeLa G and BSC-40 with VACV strain Western Reserve (WR), we have previously observed an increased activation of and activity attributable to caspases, a typical sign of apoptosis. In this paper, we have further analyzed the type of cell death in VACV-infected cells HeLa G and BSC-40. In a cell-based flow cytometric assay, we showed a specific activation of caspase-2 and 4 in HeLa G and BSC-40 cells infected with VACV, strain WR, while we did not find any effects of inhibitors of calpain and cathepsin D and E. The actual activity of the two caspases, but also of caspase-3, was then confirmed in lysates of infected HeLa G, but not in BSC-40 cells. Accordingly, poly(ADP)-ribose polymerase (PARP) cleavage was found increased only in infected HeLa G cells. Consequently, we have determined morphological features of apoptosis and/or activity of the executioner caspase-3 in infected HeLa G cells in situ, while only a background apoptosis was observed in infected BSC-40 cells. Finally, vaccination strains Dryvax and Praha were found to induce apoptosis in both HeLa G and BSC-40 cells, as characterized morphologically and by PARP cleavage. These findings may be important for understanding the differences in VACV-host interactions and post-vaccination complications in different individuals.

Lytic infection with vaccinia virus activates caspases in a Bcl-2-inhibitable manner.

Vaccinia virus (VV) is considered to cause lytic infection of most cells, with lysis being regarded equivalent to necrosis. Activation of caspases has not been associated with necrosis. However, we observed the activation and activity of caspases in epithelial cells HeLa G and BSC-40 lytically infected with VV. Using three different flow-cytometric approaches, we characterized the distinct stages of caspase cascade in VV-infected cells: a cleaved, activated form of caspases detected using a fluorescent pan-caspase inhibitor; caspase activity assayed by cleavage of a non-fluorescent substrate into a fluorescent product; caspase-specific cleavage of death substrates characterized by a fluorescent antibody detecting a neo-epitope in cytokeratin-18. All of these approaches yielded an increased fluorescent signal in VV-infected cells compared to mock-infected controls. Additionally, the signal was decreased by the expression of Bcl-2. The cleavage of cytokeratin-18 was confirmed by western blotting, but another key protein involved in apoptosis, PARP, was not cleaved in VV-infected lytic cells. The necrotic phenotype of the cells was confirmed by increased cell membrane permeability and/or decreased mitochondrial membrane potential. In conclusion, our data suggest that VV infection of the epithelial cells HeLa G and BSC-40 initiates the apoptotic program, however, apoptosis is not completed and switches into necrosis.