Free-standing multilayer films as growth factor reservoirs for future wound dressing applications.

Chronic skin wounds place a high burden on patients and health care systems. The use of angiogenic and mitogenic growth factors can facilitate the healing but growth factors are quickly inactivated by the wound environment if added exogenously. Here, free-standing multilayer films (FSF) are fabricated from chitosan and alginate as opposing polyelectrolytes in an alternating manner using layer-by-layer technique. One hundred bilayers form an about 450 µm thick, detachable free-standing film that is subsequently crosslinked by either ethyl (dimethylaminopropyl) carbodiimide combined with N-hydroxysuccinimide (E-FSF) or genipin (G-FSF). The characterization of swelling, oxygen permeability and crosslinking density shows reduced swelling and oxygen permeability for both crosslinked films compared to non-crosslinked films (N-FSF). Loading of fibroblast growth factor 2 (FGF2) into the films results in a sustained release from crosslinked FSF in comparison to non-crosslinked FSF. Biocompatibility studies in vitro with human dermal fibroblasts cultured underneath the films demonstrate increased cell growth and cell migration for all films with and without FGF2. Especially G-FSF loaded with FGF2 greatly increases cell proliferation and migration. In vivo biocompatibility studies by subcutaneous implantation in mice show that E-FSF causes an inflammatory tissue response that is absent in the case of G-FSF. N-FSF also represents a biocompatible film but shows early degradation. All FSF possess antibacterial properties against gram+ and gram- bacteria demonstrated by an agar diffusion disc assay. In summary, FSF made of alginate and chitosan crosslinked with genipin can act as a reservoir for the sustained release of FGF2, possessing high biocompatibility in vitro and in vivo. Moreover, G-FSF promotes growth and migration of human dermal fibroblasts and has antibacterial properties, which makes it an interesting candidate for bioactive wound.

Helicobacter pylori dupA is polymorphic, and its active form induces proinflammatory cytokine secretion by mononuclear cells.

BACKGROUND: Infection with Helicobacter pylori possessing a newly described virulence factor--duodenal ulcer-promoting gene A (dupA)--has been associated with duodenal ulceration and increased gastric inflammation. METHODS: The dupA locus of 34 strains was sequenced. A panel of dupA mutants was generated and cocultured with human gastric epithelial cells and peripheral blood mononuclear cells; proinflammatory cytokine release was measured. IL8 expression was measured in human gastric biopsy specimens and related to the dupA and cagA status of infecting strains. RESULTS: Most H. pylori strains had a dupA allele that was longer (1884 bp; dupA1) than previously described dupA alleles, although some had truncated versions (dupA2). Unlike the best-characterized H. pylori virulence determinant, the cag pathogenicity island (cag PaI), neither dupA type induced release of interleukin (IL)-8 from gastric epithelial cells. However, infections due to dupA-positive strains were associated with higher-level mucosal IL-8 messenger RNA expression in the human stomach than were infections due to dupA-negative strains. To explain this paradox, we found that dupA1 (but not dupA2 or the cag PaI) substantially increased H. pylori-induced IL-12p40 and IL-12p70 production from CD14(+) mononuclear cells. Other T helper 1-associated cytokines were also modestly induced. CONCLUSION: We suggest that virulent H. pylori strains cause inflammation by stimulating epithelial cells through cag-encoded proteins and mononuclear inflammatory cells through dupA1 products.

Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants.

Recombinant microbial transglutaminase (rMTG), an enzyme useful for the cross-linking or the posttranslational modification of (therapeutic) proteins, was optimized by random mutagenesis for the first time. A screening method was developed which, in addition to state-of-the-art procedures, includes a proteolytic activation step of the expressed soluble pro-enzyme. The library of 5,500 clones was screened for variants with increased thermostability and heat-sensitivity, respectively. Mutant enzymes were overproduced, isolated and characterized. After just one round of mutagenesis, nine variants with a single amino acid exchange showed a remarkably increased thermostability at 60 degrees C. The exchange of a serine residue close to the N-terminus against proline resulted in an rMTG mutant (S2P) with 270% increased half-life. Seven variants exhibited an increased heat-sensitivity at 60 degrees C of which one mutant (G25S) retained its specific activity between 10 and 40 degrees C. The mutations responsible for the increased thermostability and the heat-sensitivity were identified and assigned to the three-dimensional (3D) structure. All single point mutations related to changed thermal properties of rMTG are located in the N-terminal domain (i.e. the left side wall of the active site cleft of the front view of the MTG as defined by the literature [Kashiwagi, T., Yokoyama, K., Ishikawa, K., Ono, K., Ejima, D., Matsui, H., Suzuki, E., 2002. Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J. Biol. Chem. 277, 44252-44260] showing the importance of this part of the protein.