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Biomedical subjects

M Rohde

Publications and source records attributed to M Rohde.

At least 19 recordsLinked to original sources
PubMed2005

Pbx1 is required for Hox D3-mediated angiogenesis.

Our previous studies showed that the homeobox (Hox) D3 transcription factor induces expression of alphavbeta3 integrin and promotes endothelial cell (EC) migration and angiogenesis. Since binding of Hox 3 factors to target DNA is enhanced by the co-factor Pbx, we investigated whether Pbx1 is also required for angiogenesis. We observed that EC predominantly express the Pbx1b isoform. Nuclear extracts from angiogenic EC express higher levels of active Pbx1 and more effectively form complexes on Pbx1/Hox consensus DNA oligonucleotides as compared to nuclear extracts from quiescent EC. Introduction of anti-sense against Pbx1 impaired the formation of Pbx1/Hox complexes on target DNA consensus in nuclear extracts from angiogenic EC. Anti-sense against Pbx1 also impaired EC migration and blocked angiogenesis induced by bFGF in vivo. Furthermore, although the levels of Hox D3 were unchanged, expression of its target gene, beta3 integrin was reduced, consistent with impaired transcriptional activation by Hox D3. Together, these studies suggest that Pbx1 is required for pro-angiogenic Hox DNA binding and transcriptional activity in endothelial cells.

Cell Line, Transformed↗
PubMed2005

Infection of human endothelial cells with Staphylococcus aureus induces transcription of genes encoding an innate immunity response.

Staphylococcus aureus is a gram-positive bacterium frequently isolated from patients with bloodstream infections. Endothelial cells (EC) play an important role in host defence against bacteria, and recent reports have shown that infection of EC with S. aureus induces expression of cytokines and cell surface receptors involved in activating the innate immune response. The ability of S. aureus to invade nonphagocytic cells, including EC, has been documented. However, the knowledge of the role of EC in pathogenesis of S. aureus infection is still limited. In this study, we investigate the gene-expression program in human EC initiated by internalized S. aureus, using microarray analysis. We found 156 genes that were differentially regulated at least threefold, using arrays representing 14,239 genes. Many of the upregulated genes code for proteins involved in innate immunity, such as cytokines, chemokines and cell adhesion proteins. Other upregulated genes encode proteins involved in antigen presentation, cell signalling and metabolism. Furthermore, intracellular bacteria survived for days without inducing EC death.

Cell Adhesion Molecules↗
PubMed2004

Effects of subinhibitory concentrations of florfenicol on morphology, growth, and viability of Staphylococcus aureus.

Staphylococcus aureus strain Newman was investigated for changes in its growth pattern, its morphology and its viability when grown in the presence of 3 microg/ml florfenicol (Ff). This concentration corresponds to the 0.75-fold strain-specific minimum inhibitory concentration (MIC). Under these conditions, S. aureus Newman showed a distinct retardation in its growth pattern and 20% dead cells were detected in a fluorescence microscopic viability assay. However, bactericidal activity - defined as a 3-log drop in the staphylococcal population - was not recorded at this Ff concentration. Further analysis of the cell wall revealed a significant increase in cell wall thickness of S. aureus Newman when grown in the presence of 3 microg/ml Ff. This might result in a compression of the protoplast with subsequent disruption of single staphylococcal cells. Accordingly, 20% of the staphylococcal cells analysed by electron microscopy proved to be disrupted. These observations suggest that Ff can cause a thickening of the cell wall accompanied by impaired viability of the staphylococcal cells.

Animals↗
PubMed2004

Non-encapsulated strains reveal novel insights in invasion and survival of Streptococcus suis in epithelial cells.

Streptococcus suis is a porcine and human pathogen causing invasive diseases, such as meningitis or septicaemia. Host cell interactions of S. suis have been studied mainly with serotype 2 strains, but multiple capsular serotypes as well as non-typeable strains exist with diverse virulence features. At present, S. suis is considered an extracellular pathogen. However, whether or not it can also invade host cells is a matter of controversial discussions. We have assessed adherence and invasion of S. suis for HEp-2 epithelial cells by comparing 10 serotype 2 strains and four non-typeable (NT) strains. Only the NT strains and a non-encapsulated serotype 2 mutant strain, but none of the serotype 2 strains, adhered strongly and were invasive. Invasion seemed to be affected by environmental signals, as suggested from comparison of strains grown in different media. Further phenotypic and genotypic characterization revealed a high diversity among the different strains. Electron microscopic analysis of invasion of selected invasive NT strains indicated different uptake mechanisms. One strain induced large invaginations comparable to those seen in 'caveolae' mediated uptake, whereas invasion of the other strains was accompanied by formation of filipodia-like membrane protrusions. Invasion of all strains, however, was similarly susceptible to hypertonic sucrose, which inhibits receptor-mediated endocytosis. Irrespective of the uptake pathway, streptococci resided in acidified phago-lysosome like vacuoles. All strains, except one, survived intracellularly as well as extracellular acidic conditions. Survival seemed to be associated with the AdiS protein, an environmentally regulated arginine deiminase of S. suis. Concluding, invasion and survival of NT strains of S. suis in epithelial cells revealed novel evidence that S. suis exhibits a broad variety of virulence-associated features depending on genetic variation and regulation.

Acids↗
PubMed2003

Improved bounce-back methods for no-slip walls in lattice-Boltzmann schemes: theory and simulations.

A detailed analysis is presented for the accuracy of several bounce-back methods for imposing no-slip walls in lattice-Boltzmann schemes. By solving the lattice-BGK (Bhatnagar-Gross-Krook) equations analytically in the case of plane Poiseuille flow, it is found that the volumetric approach by Chen et al. is first-order accurate in space, and the method of Bouzidi et al. second-order accurate in space. The latter method, however, is not mass conservative because of errors associated with interpolation of densities residing on grid nodes. Therefore, similar interpolations are applied to Chen's volumetric scheme, which indeed improves the accuracy in the case of plane Poiseuille flow with boundaries parallel to the underlying grid. For skew boundaries, however, it is found that the accuracy remains first order. An alternative volumetric approach is proposed with a more accurate description of the geometrical surface. This scheme is demonstrated to be second-order accurate, even in the case of skew channels. The scheme is mass conservative in the propagation step because of its volumetric description, but still not in the collision step. However, the deviation in the mass is, in general, found to be small and proportional to the second-order terms in the standard BGK equilibrium distribution. Consequently, the scheme is a priori mass conservative for Stokes flow.

Journal Article↗
PubMed2003

Fatal outcome of lung transplantation in cystic fibrosis patients due to small-colony variants of the Burkholderia cepacia complex.

The aim of this study was to investigate the possible role of small-colony variant morphotypes of Burkholderia cepacia-like organisms in infectious complications in cystic fibrosis patients following lung transplantation. Respiratory tract specimens from 470 cystic fibrosis patients were screened over a 22-month period for Burkholderia cepacia-like organisms. Nineteen patients were positive for these organisms, eight of whom harboured small-colony-variant morphotypes. Three patients underwent bilateral lung transplantation during the study, two of whom harboured small-colony variants in addition to clonally identical wildtypes of Burkholderia multivorans and Burkholderia cepacia genomovar III prior to lung transplantation. Both patients developed fatal systemic infections post transplantation due to small-colony variants. In vitro testing revealed that small-colony variants exhibited increased serum resistance in comparison to wildtypes. The results of this study indicate that diagnostic efforts should be undertaken to carefully identify small-colony variants of Burkholderia cepacia complex, since they might be an indicator of poor post-transplantation outcome in patients with cystic fibrosis.

Adolescent↗
PubMed2003

Oceanisphaera litoralis gen. nov., sp. nov., a novel halophilic bacterium from marine bottom sediments.

A polyphasic taxonomic study was performed to characterize a new bacterial isolate, designated KMM 3654(T), from a marine bottom sand sample. The strain was Gram-negative, encapsulated, aerobic, moderately halophilic and grew between 0.5 and 10 % NaCl and at 4-42 degrees C. Its DNA G+C content was 56.4 mol%. Isolate KMM 3654(T) was phylogenetically closely related to members of the genus Oceanimonas, showing 96.7 and 95.6 % 16S rRNA gene sequence similarity to Oceanimonas doudoroffii DSM 7028(T) and Oceanimonas baumannii ATCC 700832(T), respectively. Strain KMM 3654(T) shared some physiological and chemotaxonomic properties with these two Oceanimonas species, but differed from them in morphology, growth at 4 degrees C, urease activity, weak phenol degradation and utilization of phenylacetate. On the basis of phenotypic and phylogenetic evidence, Oceanisphaera litoralis gen. nov., sp. nov. is proposed, with the type strain KMM 3654(T) (=DSM 15406(T)).

Aeromonadaceae↗
PubMed2003

Structural and functional cellular changes induced by Burkholderia pseudomallei rhamnolipid.

In this study we report that extracellular Burkholderia pseudomallei rhamnolipid induced cytopathic changes characterized by retraction, rounding up, and, finally, detachment in phagocytic and nonphagocytic cell lines. These changes were due to a progressive reorganization of the F-actin network resulting in impaired cell cycle progression and a reduced phagocytic function of macrophages.

Actins↗
PubMed2002

Volumetric method for calculating the flow around moving objects in lattice-Boltzmann schemes.

A method for calculating the fluid flow around moving objects is presented, based on a volumetric representation of the lattice-Boltzmann scheme and surfaces defined by facets. It enables us to move objects of arbitrary shape and orientation independent of the position of the grid nodes. To represent the motion of the object, additional momentum is added to the reflected particles from each facet in the propagation step. These particles are redistributed on nodes in the vicinity of the surface, depending on the position and orientation of the facet. Because the surface is considered to be closed, additional techniques need to be used to guarantee the conservation of mass. The flow field of a moving periodic cubic array of cubes at two Re numbers (Re=0.5 and Re=50) is compared with that of a fixed array. For Re=0.5, no significant deviations are found for the velocity field, pressure field, and the drag force. For Re=50, the drag and pressure field exhibit small fluctuations that relate to the position of the surface relative to the position of the grid. However, the influence of the pressure fluctuations on the velocity field is very small. Results on the velocity for a moving array of cubes show second-order accuracy in the lattice spacing. For physical consistency, the drag force on a periodic cubic array of moving spheres at Re=0.5 is compared with Hasimoto's analytical solution. The dependence on the grid spacing, the resolution of the surface of the object, and the viscosity have been studied. The discrepancies between simulations and the analytical results are smaller than 1.5%. For Re=50, the drag force, the streamline pattern, and the pressure field around a moving sphere in a large periodic domain showed good agreement with data from literature on a single sphere in an infinitely large flow field.

Journal Article↗
PubMed2002

Halomonas halocynthiae sp. nov., isolated from the marine ascidian Halocynthia aurantium.

The marine bacterium strain KMM 1376T was isolated from gill tissue of the ascidian Halocynthia aurantium, an inhabitant of the coastal waters of the Sea of Japan. Strain KMM 1376T is an aerobic, gram-negative, non-motile, encapsulated, non-pigmented, slightly halophilic bacterium that is characterized by fimbria-like structures, growth in 0.5-15% NaCl at 7-35 degrees C and absence of acid production from many carbohydrates. The DNA G+C content is 54 mol%. The main fatty acids are C16:0, C16.1omega7c and C18:1omega9c. Comparative 16S rDNA sequence analysis of strain KMM 1376T revealed 92.7-95.3% sequence similarity to members of Halomonas. Based on phenotypic and phylogenetic characteristics, it is proposed that the novel isolate be classified as Halomonas halocynthiae sp. nov., with the type strain KMM 1376T (= DSM 14573T).

Animals↗
PubMed2001

Genetic control of susceptibility to group A streptococcal infection in mice.

The influence of genetic background on the ability to control infection with group A streptococci was investigated in different inbred strains of mice. Whereas BALB/c, C57BL/10, and DBA/2 mice were the most resistant strains, with lower bacteria loads and higher survival times, C3H/HeN and CBA/J mice exhibited substantially higher bacterial growth and 100% mortality. Differences in susceptibility were not dependent on the inoculum size. Resistance was influenced by sex, with males being much more susceptible than females. B cell- and T cell-deficient mice from the resistant background were as resistant to infection as were immunocompetent mice, which suggests that the effector mechanisms are independent of adaptive immunity. These results demonstrate for the first time the influence of genetic background and sex on susceptibility to infection with Streptococcus pyogenes in mice. The use of this mouse model of group A streptococcal infection will allow for a better definition of parameters involved in the outcome of the disease.

Animals↗
PubMed2001

TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice.

Abnormal accumulation of the amyloid-beta peptide (Abeta) in the brain appears crucial to pathogenesis in all forms of Alzheimer disease (AD), but the underlying mechanisms in the sporadic forms of AD remain unknown. Transforming growth factor beta1 (TGF-beta1), a key regulator of the brain's responses to injury and inflammation, has been implicated in Abeta deposition in vivo. Here we demonstrate that a modest increase in astroglial TGF-beta1 production in aged transgenic mice expressing the human beta-amyloid precursor protein (hAPP) results in a three-fold reduction in the number of parenchymal amyloid plaques, a 50% reduction in the overall Abeta load in the hippocampus and neocortex, and a decrease in the number of dystrophic neurites. In mice expressing hAPP and TGF-beta1, Abeta accumulated substantially in cerebral blood vessels, but not in parenchymal plaques. In human cases of AD, Abeta immunoreactivity associated with parenchymal plaques was inversely correlated with Abeta in blood vessels and cortical TGF-beta1 mRNA levels. The reduction of parenchymal plaques in hAPP/TGF-beta1 mice was associated with a strong activation of microglia and an increase in inflammatory mediators. Recombinant TGF-beta1 stimulated Abeta clearance in microglial cell cultures. These results demonstrate that TGF-beta1 is an important modifier of amyloid deposition in vivo and indicate that TGF-beta1 might promote microglial processes that inhibit the accumulation of Abeta in the brain parenchyma.

Aged↗
PubMed2001

The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix.

Production of cellulose has been thought to be restricted to a few bacterial species such as the model organism Acetobacter xylinus. We show by enzymatic analysis and mass spectrometry that, besides thin aggregative fimbriae, the second component of the extracellular matrix of the multicellular morphotype (rdar) of Salmonella typhimurium and Escherichia coli is cellulose. The bcsA, bcsB, bcsZ and bcsC genes responsible for cellulose biosynthesis are not regulated by AgfD, the positive transcriptional regulator of the rdar morphotype. Transcription of the bcs genes was not co-expressed with the rdar morphotype under any of the environmental conditions examined. However, cellulose biosynthesis was turned on by the sole expression of adrA, a gene encoding a putative transmembrane protein regulated by agfD, indicating a novel pathway for the activation of cellulose synthesis. The co-expression of cellulose and thin aggregative fimbriae leads to the formation of a highly hydrophobic network with tightly packed cells aligned in parallel in a rigid matrix. As the production of cellulose would now appear to be a property widely distributed among bacteria, the function of the cellulose polymer in bacteria will have to be considered in a new light.

Arabidopsis Proteins↗
PubMed2001

The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells.

Group A streptococci (GAS) specifically attach to and internalize into human epithelial host cells. In some GAS isolates, fibronectin-binding proteins were identified as being responsible for these virulence traits. In the present study, the previously identified global negative regulator Nra was shown to control the binding of soluble fibronectin probably via regulation of protein F2 and/or SfbII expression in the serotype M49 strain 591. According to results from a conventional invasion assay based on the recovery of viable intracellular bacteria, the increased fibronectin binding did not affect bacterial adherence to HEp-2 epithelial cells, but was associated with a reduction in the internalization rates. However, when examined by confocal and electron microscopy techniques, the nra-mutant bacteria were shown to exhibit higher adherence and internalization rates than the corresponding wild type. The mutant bacteria escaped from the phagocytic vacuoles much faster, promoting consistent morphological changes which resulted in severe host cell damage. The apoptotic and lytic processes observed in nra-mutant infected host cells were correlated with an increased expression of the genes encoding superantigen SpeA, the cysteine protease SpeB, and streptolysin S in the nra-mutant bacteria. Adherence and internalization rates of a nra/speB-double mutant at wild-type levels indicated that the altered speB expression in the nra mutant contributed to the observed changes in both processes. The Nra-dependent effects on bacterial virulence were confined to infections carried out with stationary growth phase bacteria. In conclusion, the obtained results demonstrated that the global GAS regulator Nra modulates virulence genes, which are involved in host cell damage. Thus, by helping to achieve a critical balance of virulence factor expression that avoids the injury of target cells, Nra may facilitate GAS persistence in a safe intracellular niche.

Bacterial Adhesion↗
PubMed2001

alpha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface.

Binding of human plasminogen to Streptococcus pneumoniae and its subsequent activation promotes penetration of bacteria through reconstituted basement membranes. In this study, we have characterized a novel pneumococcal surface protein with a molecular mass of 47 kDa, designated Eno, which specifically binds human plasmin(ogen), exhibits alpha-enolase activity and is necessary for viability. Using enzyme assays, we have confirmed the alpha-enolase activity of both pneumococcal surface-displayed Eno and purified recombinant Eno protein. Immunoelectron microscopy indicated the presence of Eno in the cytoplasm as well as on the surface of encapsulated and unencapsulated pneumococci. Plasminogen-binding activity was demonstrated with whole pneumococcal cells and purified Eno protein. Binding of activated plasminogen was also shown for Eno; however, the affinity for plasmin is significantly reduced compared with plasminogen. Results from competitive inhibition assays indicate that binding is mediated through the lysine binding sites in plasmin(ogen). Carboxypeptidase B treatment and amino acid substitutions of the C-terminal lysyl residues of Eno indicated that the C-terminal lysine is pivotal for plasmin(ogen)-binding activity. Eno is ubiquitously distributed among pneumococcal serotypes, and binding experiments suggested the reassociation of secreted Eno to the bacterial cell surface. The reassociation was also confirmed by immunoelectron microscopy. The results suggest a mechanism of plasminogen activation for human pathogens that might contribute to their virulence potential in invasive infectious processes.

Amino Acid Sequence↗
PubMed2001

The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence.

Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins mediating these reactions are largely uncharacterized. In this report we describe a novel pneumococcal protein PavA, which binds fibronectin and is associated with pneumococcal adhesion and virulence. The pavA gene, present in 64 independent isolates of S. pneumoniae tested, encodes a 551 amino acid residue polypeptide with 67% identical amino acid sequence to Fbp54 protein in Streptococcus pyogenes. PavA localized to the pneumococcal cell outer surface, as demonstrated by immunoelectron microscopy, despite lack of conventional secretory or cell-surface anchorage signals within the primary sequence. Full-length recombinant PavA polypeptide bound to immobilized human fibronectin in preference to fluid-phase fibronectin, in a heparin-sensitive interaction, and blocked binding of wild-type pneumococcal cells to fibronectin. However, a C-terminally truncated PavA' polypeptide (362 aa residues) failed to bind fibronectin or block pneumococcal cell adhesion. Expression of pavA in Enterococcus faecalis JH2-2 conferred > sixfold increased cell adhesion levels to fibronectin over control JH2-2 cells. Isogenic mutants of S. pneumoniae, either abrogated in PavA expression or producing a 42 kDa C-terminally truncated protein, showed up to 50% reduced binding to immobilized fibronectin. Inactivation of pavA had no effects on growth rate, cell morphology, cell-surface physico-chemical properties, production of pneumolysin, autolysin, or surface proteins PspA and PsaA. Isogenic pavA mutants of encapsulated S. pneumoniae D39 were approximately 104-fold attenuated in virulence in the mouse sepsis model. These results provide evidence that PavA fibronectin-binding protein plays a direct role in the pathogenesis of pneumococcal infections.

Adhesins, Bacterial↗
PubMed2001

Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells.

The facultative intracellular, Gram-positive bacterium Listeria monocytogenes invades phagocytic and non-phagocytic cells from the tissues and organs of a wide variety of animals and humans. Here, we report the use of these bacteria as vehicles for gene transfer. Eukaryotic expression plasmids were introduced into the nucleus of host cells following lysis of the intracytosolic, plasmid-carrying bacteria with antibiotics. Cell lines of different tissues and species could be transfected in this way. We examined bacterial properties required for delivery of the expression plasmids and found that this was strictly dependent on the ability of these bacteria to both invade eukaryotic cells and egress from the vacuole into the cytosol of the infected host cells. Macrophage-like cell lines or primary, peritoneal macrophages proved to be almost refractory to Listeria-mediated gene transfer. Thus, attenuated L. monocytogenes represents a serious candidate for consideration as a DNA-transfer vehicle for in vivo somatic gene therapy. The potential for oral administration of L. monocytogenes and the ease in producing and cultivating recombinant strains are further attributes that make its use as a gene transfer vehicle attractive.

Animals↗