In the healthy intestinal mucosa, homeostasis between the immune system and commensal microflora prevents detrimental inflammatory responses. Infection with acute enteropathogens such as Salmonella enterica serovar Typhimurium disturbs this homeostasis and triggers inflammation, but the underlying mechanisms are poorly understood. We found that bacterial delivery or ectopic expression of the S. Typhimurium type III effector protein SopE, a known activator of host cellular Rho GTPases, led to proinflammatory caspase-1 activation and consequent maturation and secretion of the cytokine IL-1beta. In vivo, SopE triggered mucosal inflammation in wild-type but not caspase-1(-/-), IL-1R(-/-), or IL-18(-/-) mice. Bone marrow chimeras indicated that caspase-1 was more important in stromal cells, most likely enterocytes, than in bone marrow-derived cells. SopE-mediated caspase-1 activation in vitro was mediated by cellular Rho GTPases Rac-1 and Cdc42. These findings implicate SopE-driven Rho GTPase-mediated caspase-1 activation in stromal cells as a mechanism eliciting mucosal inflammation during S. Typhimurium infection.

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Tumor progression is controlled by signals from cellular and extra-cellular microenvironment including stromal cells and the extracellular matrix. Consequently, three-dimensional in vitro tumor models are essential to study the interaction of tumor cells with their microenvironment appropriately in a biologically relevant manner. We have previously used organotypic co-cultures to analyze the malignant growth of human squamous cell carcinoma (SCC) cell lines on a stromal equivalent in vitro. In this model, SCC cell lines are grown on a collagen-I gel containing fibroblasts. Since macrophages play a critical role in the progression of many tumor types, we now have expanded this model by integrating macrophages into the collagen gel of these organotypic tumor co-cultures. This model was established as a murine and a human system of skin SCCs. The effect of macrophages on tumor progression depends on their polarization. We demonstrate that macrophage polarization in organotypic co-cultures can be modulated towards and M1 or an M2 phenotype by adding recombinant IFN-γ and LPS or IL-4 respectively to the growth medium. IL-4 stimulation of macrophage-containing cultures resulted in enhanced tumor cell invasion evidenced by degradation of the basement membrane, enhanced collagenolytic activity and increased MMP-2 and MMP-9. Interestingly, extended co-culture with tumor cells for three weeks resulted in spontaneous M2 polarization of macrophages without IL-4 treatment. Thus, we demonstrate that macrophages can be successfully integrated into organotypic co-cultures of murine or human skin SCCs and that this model can be exploited to analyze macrophage activation towards a tumor supporting phenotype.

Highly porous carbide-derived carbon (CDC) mesofoams (DUT-70) are prepared by nanocasting of mesocellular silica foams with a polycarbosilane precursor. Ceramic conversion followed by silica removal and high-temperature chlorine treatment yields CDCs with a hierarchical micro-mesopore arrangement. This new type of polymer-based CDC is characterized by specifi c surface areas as high as 2700 m 2 g −1 , coupled with ultrahigh microand mesopore volumes up to 2.6 cm 3 g −1 . The relationship between synthesis conditions and the properties of the resulting carbon materials is described in detail, allowing precise control of the properties of DUT-70. Since the hierarchical pore system ensures both effi cient mass transfer and high capacities, the novel CDC shows outstanding performance as an electrode material in electrochemical double-layer capacitors (EDLCs) with specifi c capacities above 240 F g −1 when measured in a symmetrical two-electrode confi guration. Remarkable capacities of 175 F g −1 can be retained even at high current densities of 20 A g −1 as a result of the enhanced ion-transport pathways provided by the cellular mesostructure. Moreover, DUT-70 can be infi ltrated with sulfur and host the active material in lithium-sulfur battery cathodes. Reversible capacities of 790 mAh g −1 are achieved at a current rate of C/10 after 100 cycles, which renders DUT-70 an ideal support material for electrochemical energy-storage applications.

The cytotoxic necrotizing factors CNF1 and CNF2 produced by pathogenic Escherichia coli strains and CNF Y of Yersinia pseudotuberculosis constitutively activate small GTPases of the Rho family. They deamidate a glutamine (Gln63 in RhoA), which is crucial for GTP hydrolysis. CNF1 and CNF Y exhibit 61% identity on the amino acid level, with equal distribution over the whole molecule. Although the two toxins are homologous in the receptor binding domain, we show that they bind to different cellular receptors. CNF Y does not enter Caco-2 and CHO-K1 cells, which are responsive to CNF1. In contrast, HeLa, Hep-2, and HEK 293 cells do respond to both toxins. Competition studies with catalytically inactive mutants of the toxins revealed that binding of CNF1 has no influence on the uptake of CNF Y into HeLa cells. In contrast, uptake of CNF1 is retarded after preincubation of HeLa cells with the catalytically inactive mutant of CNF Y , suggesting that the toxin receptors overlap. Moreover, we compared the pathways of the toxins from receptor binding into the cytosol and showed that both toxins are taken up independent of the presence of clathrin or lipid rafts and are released into the cytosol from acidified endosomes.Cytotoxic necrotizing factor 1 (CNF1) is an AB-type toxin expressed by pathogenic Escherichia coli strains which cause urinary tract infections and neonatal meningitis. CNF1 is a 115-kDa single-chain molecule comprising an N-terminal receptor binding domain and a C-terminal catalytic domain, which contains deamidase activity (for a review, see reference 9). These two domains are separated by a putative translocation domain, which contains two hydrophobic helices involved in membrane translocation (16). CNF1 deamidates small GTPbinding proteins of the Rho family at glutamine 63/61, leading to constitutive activation of the small GTPases by blocking their intrinsic and GTPase activating protein-stimulated GTP hydrolysis (5, 17).CNF1 has been shown to enter cells by receptor-mediated endocytosis independent of clathrin and independent of sphingolipid-cholesterol-rich membrane microdomains (lipid rafts), including caveolae (4). Based on interaction studies, using a yeast two-hybrid system, it has been suggested that cell binding of the toxin is mediated by the laminin receptor precursor p37, a subunit of the mature nonintegrin 67-kDa laminin receptor (3, 11). Moreover, it was shown that CNF1 contributes to E. coli K1 invasion of human brain microvascular endothelial cells via the 67-kDa laminin receptor. Following internalization, CNF1 is delivered to late endosomes by microtubule-dependent transport. From there, it is released into the cytosol in an acidic pH-dependent manner. Specific acidic residues located in the hydrophilic loop, connecting the two hydrophobic helices of the translocation domain, appear to be involved in the translocation process (16).More recently, a cytotoxic necrotizing factor (CNF) produced by Yersinia pseudotuberculosis (CNF Y ) was discovered, which catalyzes the same deamidation reac...

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Silica nanospheres are used as templates for the generation of carbide-derived carbons with monodisperse spherical mesopores (d=20-40 nm) and microporous walls. The nanocasting approach with a polycarbosilane precursor and subsequent pyrolysis, followed by silica template removal and chlorine treatment, results in carbide-derived carbons DUT-86 (DUT=Dresden University of Technology) with remarkable textural characteristics, monodisperse, spherical mesopores tunable in diameter, and very high pore volumes up to 5.0 cm3 g(-1). Morphology replication allows these nanopores to be arranged in a nanostructured inverse opal-like structure. Specific surface areas are very high (2450 m2 g(-1)) due to the simultaneous presence of micropores. Testing DUT-86 samples as cathode materials in Li-S batteries reveals excellent performance, and tailoring of the pore size allows optimization of cell performance, especially the active center accessibility and sulfur utilization. The outstanding pore volumes allow sulfur loadings of 80 wt %, a value seldom achieved in composite cathodes, and initial capacities of 1165 mAh gsulfur(-1) are reached. After 100 cycle capacities of 860 mAh gsulfur(-1) are retained, rendering DUT-86 a high-performance sulfur host material.