Diamond-like carbon (DLC) thin films are promising for use in coating orthopaedic, dental and cardiovascular implants. The problem of DLC layers lies in their weak layer adhesion to metal implants. Chromium is used as a dopant for improving the adhesion of DLC films. Cr-DLC layers were prepared by a hybrid technology, using a combination of pulsed laser deposition (PLD) from a graphite target and magnetron sputtering. Depending on the deposition conditions, the concentration of Cr in the DLC layers moved from zero to 10.0 at.%. The effect of DLC layers with 0.0, 0.9, 1.8, 7.3, 7.7 and 10.0 at.% Cr content on the adhesion and osteogenic differentiation of human osteoblast-like Saos-2 cells was assessed in vitro. The DLC samples that contained 7.7 and 10.0 at.% of Cr supported cell spreading on day 1 after seeding. On day three after seeding, the most apparent vinculin-containing focal adhesion plaques were also found on samples with higher concentrations of chromium. On the other hand, the expression of type I collagen and alkaline phosphatase at the mRNA and protein level was the highest on Cr-DLC samples with a lower concentration of Cr (0-1.8 at.%). We can conclude that higher concentrations of chromium supported cell adhesion; however DLC and DLC doped with a lower concentration of chromium supported osteogenic cell differentiation.

• MeSH
• Alkaline Phosphatase metabolism   MeSH
• Coated Materials, Biocompatible MeSH
• Cell Adhesion * MeSH
• Cell Differentiation * MeSH
• Cell Line MeSH
• Chromium chemistry   MeSH
• Diamond chemistry   MeSH
• Focal Adhesions MeSH
• Collagen Type I metabolism   MeSH
• Metals chemistry   MeSH
• Lasers MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Osteoblasts cytology   MeSH
• Osteogenesis MeSH
• Surface Properties MeSH
• Gene Expression Profiling MeSH
• Talin chemistry   MeSH
• Carbon chemistry   MeSH
• Vinculin metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The adenylate cyclase toxin (CyaA) of the whooping cough agent Bordetella pertussis subverts immune functions of host myeloid cells expressing the αMβ2 integrin (CD11b/CD18, CR3 or Mac-1). CyaA delivers into cytosol of cells an extremely catalytically active adenylyl cyclase enzyme, which disrupts the innate and adaptive immune functions of phagocytes through unregulated production of the key signaling molecule cAMP. We have used phosphoproteomics to analyze cAMP signaling of CyaA in murine bone marrow-derived dendritic cells. CyaA action resulted in alterations of phosphorylation state of a number of proteins that regulate actin cytoskeleton homeostasis, including Mena, Talin-1 and VASP. CyaA action repressed mTOR signaling through activation of mTORC1 inhibitors TSC2 and PRAS40 and altered phosphorylation of multiple chromatin remodelers, including the class II histone deacetylase HDAC5. CyaA toxin action further elicited inhibitory phosphorylation of SIK family kinases involved in modulation of immune response and provoked dephosphorylation of the transcriptional coactivator CRTC3, indicating that CyaA-promoted nuclear translocation of CRTC3 may account for CyaA-induced IL-10 production. These findings document the complexity of subversive physiological manipulation of myeloid phagocytes by the CyaA toxin, serving in immune evasion of the pertussis agent.

• MeSH
• Cyclic AMP metabolism   MeSH
• Bordetella pertussis metabolism   MeSH
• Cytoskeletal Proteins metabolism   MeSH
• Dendritic Cells metabolism   MeSH
• Phosphoproteins metabolism   MeSH
• Histone Deacetylases metabolism   MeSH
• Microfilament Proteins metabolism   MeSH
• Cell Adhesion Molecules metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Whooping Cough microbiology   MeSH
• Signal Transduction physiology   MeSH
• Talin metabolism   MeSH
• Transcription Factors metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Protein-repulsive surfaces modified with ligands for cell adhesion receptors have been widely developed for controlling the cell adhesion and growth in tissue engineering. However, the question of matrix production and deposition by cells on these surfaces has rarely been addressed. In this study, protein-repulsive polydopamine-poly(ethylene oxide) (PDA-PEO) surfaces were functionalized with an RGD-containing peptide (RGD), with a collagen-derived peptide binding fibronectin (Col), or by a combination of these peptides (RGD + Col, ratio 1:1) in concentrations of 90 fmol/cm(2) and 700 fmol/cm(2) for each peptide type. When seeded with vascular endothelial CPAE cells, the PDA-PEO surfaces proved to be completely non-adhesive for cells. On surfaces with lower peptide concentrations and from days 1 to 3 after seeding, cell adhesion and growth was restored practically only on the RGD-modified surface. However, from days 3 to 7, cell adhesion and growth was improved on surfaces modified with Col and with RGD + Col. At higher peptide concentrations, the cell adhesion and growth was markedly improved on all peptide-modified surfaces in both culture intervals. However, the collagen-derived peptide did not increase the expression of fibronectin in the cells. The deposition of fibronectin on the material surface was generally very low and similar on all peptide-modified surfaces. Nevertheless, the RGD + Col surfaces exhibited the highest cell adhesion stability under a dynamic load, which correlated with the highest expression of talin and vinculin in the cells on these surfaces. A combination of RGD + Col therefore seems to be the most promising for surface modification of biomaterials, e.g. vascular prostheses.

• MeSH
• Adsorption MeSH
• Biomimetics * MeSH
• Cell Adhesion * MeSH
• Gene Expression MeSH
• Fibronectins chemistry   genetics   MeSH
• Indoles chemistry   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Oligopeptides chemistry   MeSH
• Polyethylene Glycols chemistry   MeSH
• Polymers chemistry   MeSH
• Surface Properties MeSH
• Amino Acid Sequence MeSH
• Talin genetics   MeSH
• Vinculin genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Immune evasion genes help human cytomegalovirus (HCMV) establish lifelong persistence. Without immune pressure, laboratory-adapted HCMV strains have undergone genetic alterations. Among these, the deletion of the UL/b' domain is associated with loss of virulence. In a screen of UL/b', we identified pUL135 as a protein responsible for the characteristic cytopathic effect of clinical HCMV strains that also protected from natural killer (NK) and T cell attack. pUL135 interacted directly with abl interactor 1 (ABI1) and ABI2 to recruit the WAVE2 regulatory complex to the plasma membrane, remodel the actin cytoskeleton and dramatically reduce the efficiency of immune synapse (IS) formation. An intimate association between F-actin filaments in target cells and the IS was dispelled by pUL135 expression. Thus, F-actin in target cells plays a critical role in synaptogenesis, and this can be exploited by pathogens to protect against cytotoxic immune effector cells. An independent interaction between pUL135 and talin disrupted cell contacts with the extracellular matrix.

• MeSH
• Adaptor Proteins, Signal Transducing metabolism   MeSH
• Killer Cells, Natural immunology   virology   MeSH
• CD8-Positive T-Lymphocytes immunology   virology   MeSH
• Cytomegalovirus immunology   MeSH
• Cytoskeletal Proteins metabolism   MeSH
• Immunological Synapses virology   MeSH
• Immunomodulation MeSH
• Host-Pathogen Interactions MeSH
• Humans MeSH
• Actin Cytoskeleton metabolism   MeSH
• Wiskott-Aldrich Syndrome Protein Family metabolism   MeSH
• Talin metabolism   MeSH
• Viral Proteins physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Bordetella adenylate cyclase toxin (CyaA) binds the alpha(M)beta(2) integrin (CD11b/CD18, Mac-1, or CR3) of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC) enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin 'translocation intermediate', which can be 'locked' in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the 'intermediate' permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca(2+) influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the 'translocation intermediate' promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol.

• MeSH
• Adenylate Cyclase Toxin chemistry   metabolism   MeSH
• CD11b Antigen metabolism   MeSH
• CD18 Antigens metabolism   MeSH
• Bordetella enzymology   MeSH
• Cell Membrane enzymology   microbiology   MeSH
• Cholesterol metabolism   MeSH
• Cytosol enzymology   MeSH
• Extracellular Space metabolism   MeSH
• Humans MeSH
• Macrophage-1 Antigen metabolism   MeSH
• Macrophages metabolism   microbiology   MeSH
• Membrane Microdomains enzymology   microbiology   MeSH
• Mice MeSH
• Talin metabolism   MeSH
• Protein Structure, Tertiary MeSH
• U937 Cells MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Micropatterned surfaces have been used as a tool for controlling the extent and strength of cell adhesion, the direction of cell growth and the spatial distribution of cells. In this study, chemically micropatterned surfaces were prepared by successive plasma polymerization of acrylic acid (AA) and 1,7-octadiene (OD) through a mask. Rat vascular smooth muscle cells (VSMC), bovine endothelial cells (EC), porcine mesenchymal stem cells (MSC) or human skeletal muscle cells (HSKMC) were seeded on these surfaces in densities from 9,320 cells/cm2 to 31,060 cells/cm2. All cell types adhered and grew preferentially on the strip-like AA domains. Between day 1 and 7 after seeding, the percentage of cells on AA domains ranged from 84.5 to 63.3 % for VSMC, 85.3 to 73.5 % for EC, 98.0 to 90.0 % for MSC, and 93.6 to 55.0 % for HSKMC. The enzyme-linked immunosorbent assay (ELISA) revealed that the concentration of alpha-actin per mg of protein was significantly higher in VSMC on AA. Similarly, immunofluorescence staining of von Willebrand factor showed more apparent Weibel-Palade bodies in EC on AA domains. MSC growing on AA had better developed beta-actin cytoskeleton, although they were less stained for hyaluronan receptor (CD44). In accordance with this, MSC on AA contained a higher concentration of beta-actin, although the concentration of CD44 was lower. HSKMC growing on AA had a better developed alphaactin cytoskeleton. These results based on four cell types suggest that plasma polymerization is a suitable method for producing spatially defined patterned surfaces for controlled cell adhesion, proliferation and maturation.

• MeSH
• Acrylates pharmacology   chemistry   MeSH
• Actins metabolism   MeSH
• Alkenes pharmacology   chemistry   MeSH
• Hyaluronan Receptors metabolism   MeSH
• Cell Adhesion drug effects   MeSH
• Cell Culture Techniques MeSH
• Endothelial Cells metabolism   drug effects   MeSH
• Financing, Organized MeSH
• Fluorescent Antibody Technique MeSH
• Collagen Type IV chemistry   MeSH
• Muscle Fibers, Skeletal metabolism   drug effects   MeSH
• Rats MeSH
• Humans MeSH
• Mesenchymal Stem Cells metabolism   drug effects   MeSH
• Myocytes, Smooth Muscle metabolism   drug effects   MeSH
• Polymers pharmacology   chemistry   MeSH
• Swine MeSH
• Cell Proliferation drug effects   MeSH
• Cattle MeSH
• Talin metabolism   MeSH
• Tissue Adhesives pharmacology   chemistry   MeSH
• Water chemistry   MeSH
• von Willebrand Factor metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Cattle MeSH
• Animals MeSH

Nanocomposite Ti/hydrocarbon plasma polymer (Ti/ppCH) films were deposited by DC magnetron sputtering of titanium target in n-hexane, argon, or a mixture of these two gases. The resultant films were heterogeneous, with inorganic regions of nanometer scale distributed within a plasma polymer matrix. The titanium content was controlled by adjusting the argon/n-hexane ratio in the working gas. In the pure n-hexane atmosphere, the Ti concentration was found to be below 1 at %, whereas in pure argon it reached 20 at %, as measured by Rutherford backscattering spectroscopy and elastic recoil detection analysis (RBS/ERDA). A high level of titanium oxidation is detected with TiO(2), substoichiometric titania, and titanium carbide, composing an inorganic phase of the composite films. In addition, high hydrogen content is detected in films rich with titanium. Ti-deficient and Ti-rich films proved equally good substrates for adhesion and growth of cultured human osteoblast-like MG 63 cells. In these cells, the population densities on days 1, 3, and 7 after seeding, spreading area on day 1, formation of talin-containing focal adhesion plaques as well as concentrations of talin and osteocalcin (per mg of protein) were comparable to the values obtained in cells on the reference cell culture materials, represented by microscopic glass coverslips or a polystyrene dish. An interesting finding was made when the Ti/ppCH films were seeded with calf pulmonary artery endothelial cells of the line CPAE. The cell population densities, the spreading area and also the concentration of von Willebrand factor, a marker of endothelial cell maturation, were significantly higher on Ti-rich than on Ti-deficient films. On Ti-rich films, these parameters were also higher or similar in comparison with the reference cell culture materials. Thus, both types of films could be used for coating bone implants, of which the Ti-rich film remains effective in enhancing the endothelialization of blood contacting artificial materials.

• MeSH
• Biocompatible Materials chemistry   MeSH
• Cell Adhesion MeSH
• Cell Differentiation MeSH
• Cell Line MeSH
• Endothelial Cells cytology   physiology   MeSH
• Financing, Organized MeSH
• Humans MeSH
• Magnetics MeSH
• Nanocomposites chemistry   MeSH
• Osteoblasts cytology   physiology   MeSH
• Osteocalcin metabolism   MeSH
• Surface Properties MeSH
• Cattle MeSH
• Talin metabolism   MeSH
• Materials Testing MeSH
• Titanium chemistry   MeSH
• Hydrocarbons chemistry   MeSH
• von Willebrand Factor metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Cattle MeSH
• Animals MeSH