Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix

Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered "sheet grafts" has been shown to vary extremely. Adherence of transplanted cells to the wound bed--especially in the presence of potential wound contamination-- is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal-epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.     

Interleukin-18 enhances the production of interferon-gamma (IFN-gamma) by allergen-specific and unspecific stimulated cord blood mononuclear cells

BACKGROUND: IL-18 is a pleiotropic cytokine involved in the polarisation of T-cell response. This study was performed to determine whether or not IL-18 is detectable in phytohemagglutinin (PHA) or betalactoglobulin (BLG) stimulated supernatants of cord blood mononuclear cells (CBMC) and to study the in vitro effect of IL-18 on the interferon (IFN)-gaamma and IL-13 release of CBMC of healthy neonates. METHODS: CBMC of neonates were isolated by Ficoll density centrifugation. The cytokines IFN-gamma, IL-13 and IL-18 in the cell culture supernatants were measured using the ELISA technique following stimulation with a unspecific (PHA 20 microg/ml) and an allergen-specific stimulus (BLG 25 microg/ml). In order to study the in vitro effect of IL-18, CBMC were stimulated either with medium alone or with IL-18, IL-18 + PHA and IL-18 + BLG. RESULTS: IL-18 levels in supernatants of CBMC were low and did not vary significantly between unstimulated and PHA or BLG stimulated cell cultures (median 21.4; 23.5 and 15.5 pg/ml, respectively). IFN-gamma and IL-13 levels were significantly higher in response to PHA and BLG (PHA: IFN-gamma, 6154; IL-13, 4357; BLG: IFN-gamma, 801; IL-13, 249 pg/ml) compared to unstimulated cell cultures. The addition of IL-18 to PHA or BLG stimulated CBMC significantly enhanced the IFN-gamma release (PHA: 6154; PHA + IL-18: 13474, p = 0.0001; BLG: 801; BLG + IL-18: 1077, p = 0.008). In comparison to incubation without IL-18, the release of IL-13 was invariable or even reduced, when CBMC were stimulated with PHA + IL-18 (4026, p = 0.16) or BLG + IL-18 (124, p = 0.0001) compared to stimulation of CBMC with PHA (4357 pg/ml) or BLG (249 pg/ml) alone. CONCLUSIONS: IL-18 is detectable in supernatants of CBMC. We observed a significant effect of IL-18 + PHA as well as IL-18 + BLG on IFN-gamma release in vitro. Based on our findings we conclude that IL-18 could act as a strong TH1-inducing factor on stimulated CBMC also in vivo.     

SWISS-MODEL: An automated protein homology-modeling server

SWISS-MODEL (http://swissmodel.expasy.org) is a server for automated comparative modeling of three-dimensional (3D) protein structures. It pioneered the field of automated modeling starting in 1993 and is the most widely-used free web-based automated modeling facility today. In 2002 the server computed 120 000 user requests for 3D protein models. SWISS-MODEL provides several levels of user interaction through its World Wide Web interface: in the 'first approach mode' only an amino acid sequence of a protein is submitted to build a 3D model. Template selection, alignment and model building are done completely automated by the server. In the 'alignment mode', the modeling process is based on a user-defined target-template alignment. Complex modeling tasks can be handled with the 'project mode' using DeepView (Swiss-PdbViewer), an integrated sequence-to-structure workbench. All models are sent back via email with a detailed modeling report. WhatCheck analyses and ANOLEA evaluations are provided optionally. The reliability of SWISS-MODEL is continuously evaluated in the EVA-CM project. The SWISS-MODEL server is under constant development to improve the successful implementation of expert knowledge into an easy-to-use server.     

The 1.15A crystal structure of the Staphylococcus aureus methionyl-aminopeptidase and complexes with triazole based inhibitors

Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides. There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses. Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme. Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents. Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution. The protein has a typical "pita-bread" fold as observed for the other MetAP structures. The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions. The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is.     

Identifying MHC class I epitopes by predicting the TAP transport efficiency of epitope precursors

We are able to make reliable predictions of the efficiency with which peptides of arbitrary lengths will be transported by TAP. The pressure exerted by TAP on Ag presentation thus can be assessed by checking to what extent MHC class I (MHC-I)-presented epitopes can be discriminated from random peptides on the basis of predicted TAP transport efficiencies alone. Best discriminations were obtained when N-terminally prolonged epitope precursor peptides were included and the contribution of the N-terminal residues to the score were down-weighted in comparison with the contribution of the C terminus. We provide evidence that two factors may account for this N-terminal down-weighting: 1) the uncertainty as to which precursors are used in vivo and 2) the coevolution in the C-terminal sequence specificities of TAP and other agents in the pathway, which may vary among the various MHC-I alleles. Combining predictions of MHC-I binding affinities with predictions of TAP transport efficiency led to an improved identification of epitopes, which was not the case when predictions of MHC-I binding affinities were combined with predictions of C-terminal cleavages made by the proteasome.     

The pseudorabies virus UL11 protein is a virion component involved in secondary envelopment in the cytoplasm

Homologs of the small tegument protein encoded by the UL11 gene of herpes simplex virus type 1 are conserved throughout all herpesvirus subfamilies. However, their function during viral replication has not yet been conclusively shown. Using a monospecific antiserum and an appropriate viral deletion and rescue mutant, we identified and functionally characterized the UL11 protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL11 encodes a protein with an apparent molecular mass of 10 to 13 kDa that is primarily detected at cytoplasmic membranes during viral replication. In the absence of the UL11 protein, viral titers were decreased approximately 10-fold and plaque sizes were reduced by 60% compared to wild-type virus. Intranuclear capsid maturation and nuclear egress resulting in translocation of DNA-containing capsids into the cytoplasm were not detectably affected. However, in the absence of the UL11 protein, intracytoplasmic membranes were distorted. Moreover, in PrV-DeltaUL11-infected cells, capsids accumulated in the cytoplasm and were often found associated with tegument in aggregated structures such as had previously been demonstrated in cells infected with a PrV triple-mutant virus lacking glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Thus, the PrV UL11 protein, like glycoproteins E, I, and M, appears to be involved in secondary envelopment.     

Demography and natural selection have shaped genetic variation in Drosophila melanogaster: a multi-locus approach

Demography and selection have been recognized for their important roles in shaping patterns of nucleotide variability. To investigate the relative effects of these forces in the genome of Drosophila melanogaster, we used a multi-locus scan (105 fragments) of X-linked DNA sequence variation in a putatively ancestral African and a derived European population. Surprisingly, we found evidence for a recent size expansion in the African population, i.e., a significant excess of singletons at a chromosome-wide level. In the European population, such an excess was not detected. In contrast to the African population, we found evidence for positive natural selection in the European sample: (i) a large number of loci with low levels of variation and (ii) a significant excess of derived variants at the low-variation loci that are fixed in the European sample but rare in the African population. These results are consistent with the hypothesis that the European population has experienced frequent selective sweeps in the recent past during its adaptation to new habitats. Our study shows the advantages of a genomic approach (over a locus-specific analysis) in disentangling demographic and selective forces.     

PGE(2) increases release of substance P from renal sensory nerves by activating the cAMP-PKA transduction cascade

Increasing renal pelvic pressure increases afferent renal nerve activity (ARNA) by a PGE(2)-mediated release of substance P (SP) from renal pelvic nerves. The role of cAMP activation in the PGE(2)-mediated release of SP was studied by examining the effects of the adenylyl cyclase (AC) activator forskolin and AC inhibitor dideoxyadenosine (DDA). Forskolin enhanced the bradykinin-mediated release of SP from an isolated rat renal pelvic wall preparation, from 7.3 +/- 1.3 to 15.6 +/- 3.0 pg/min. PGE(2) at a subthreshold concentration for SP release mimicked the effects of forskolin. The EP(2) receptor agonist butaprost, 15 microM, and PGE(2), 0.14 microM, produced similar increases in SP release, from 5.8 +/- 0.8 to 17.0 +/- 2.3 pg/min and from 8.0 +/- 1.3 to 21.6 +/- 2.7 pg/min. DDA blocked the SP release produced by butaprost and PGE(2). The PGE(2)-induced release of SP was also blocked by the PKA inhibitors PKI(14-22) and H-89. Studies in anesthetized rats showed that renal pelvic administration of butaprost, 10 microM, and PGE(2), 0.14 microM, resulted in similar ARNA responses, 1,520 +/- 390 and 1,170 +/- 270%. s (area under the curve of ARNA vs. time) that were blocked by DDA. Likewise, the ARNA response to increased renal pelvic pressure, 7,180 +/- 710%. s, was blocked by DDA. In conclusion, PGE(2) activates the cAMP-PKA pathway leading to a release of SP and activation of renal pelvic mechanosensory nerve fibers.