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The invasion of polarized epithelial cells by S almonella enterica requires the cooperative activity of the S almonella pathogenicity island ( SPI) 1-encoded type III secretion system ( T3 SS) and the SPI4-encoded giant non-fimbrial adhesin SiiE. SiiE is a highly repetitive protein composed of 53 bacterial Ig ( BIg) domains and mediates binding to the apical side of polarized epithelial cells. We analysed the binding properties of SiiE and observed lectin-like activity. SiiE-dependent cell invasion can be ablated by chemical or enzymatic deglycosylation. Lectin blockade experiments revealed that SiiE binding is specific for glycostructures with terminal N-acetyl-glucosamine ( GlcNAc) and/or α 2,3-linked sialic acid. In line with these data, we found that SiiE-expressing S almonella bind to the GlcNAc polymer chitin. Various recombinant SiiE fragments were analysed for host cell binding. We observed that C-terminal portions of SiiE bind to the apical side of polarized cells and the intensity of binding increases with the number of BIg domains present in the recombinant proteins. Based on these results, we propose that SiiE mediates multiple interactions per molecule with glycoproteins and/or glycosylated phospholipids present in the apical membrane of polarized epithelial cells. Thisintimate binding enables the subsequent function of the SPI1- T3 SS, resulting in host cell invasion. [ABSTRACT FROM AUTHOR]

For the aim of ex vivo engineering of functional tissue substitutes, Laser-assisted BioPrinting (LaBP) is under investigation for the arrangement of living cells in predefined patterns. So far three-dimensional (3D) arrangements of single or two-dimensional (2D) patterning of different cell types have been presented. It has been shown that cells are not harmed by the printing procedure. We now demonstrate for the first time the 3D arrangement of vital cells by LaBP as multicellular grafts analogous to native archetype and the formation of tissue by these cells. For this purpose, fibroblasts and keratinocytes embedded in collagen were printed in 3D as a simple example for skin tissue. To study cell functions and tissue formation process in 3D, different characteristics, such as cell localisation and proliferation were investigated. We further analysed the formation of adhering and gap junctions, which are fundamental for tissue morphogenesis and cohesion. In this study, it was demonstrated that LaBP is an outstanding tool for the generation of multicellular 3D constructs mimicking tissue functions. These findings are promising for the realisation of 3D in vitro models and tissue substitutes for many applications in tissue engineering. Biotechnol. Bioeng. 2012; 109:1855-1863. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Salmonella infections can be life-threatening. SiiE is a giant adhesion molecule of 5559 amino acids that is encoded in Salmonella pathogenicity island 4 (SPI4) and that promotes the initial contact between the pathogen and polarized epithelial cells in the intestine of the host. Starting from an engineered deletion version of SiiE (mini-SiiE; 97 kDa), limited proteolysis was used to reproducibly generate a 30 kDa fragment that readily crystallized. Mass spectrometry hints that this fragment spans the predicted Ig domains 50-52 of SiiE. Crystals of both native and selenomethionine-labelled protein could be obtained in space group C2 and diffraction data were recorded to a resolution of 1.85 Å. [ABSTRACT FROM AUTHOR]

The influence of oxygen on neural stem cell proliferation, differentiation, and apoptosis is of great interest for regenerative therapies in neurodegenerative disorders, such as Parkinson's disease. These oxygen depending mechanisms have to been considered for the optimization of neural cell culture conditions. In this study, we used a cell culture system with an oxygen-permeable polytetrafluorethylene (PTFE) foil to investigate the effect of oxygen on metabolism and survival of neural cell lines in vitro. Human glial astrocytoma-derived cells (GOS-3) and rat pheochromacytoma cells (PC12) were cultured on the gas-permeable PTFE foil as well as a conventional non oxygen-permeable cell culture substrate at various oxygen concentrations. Analyses of metabolic activity, gene expression of apoptotic grade, and dopamine synthesis were performed. Under low oxygen partial pressure (2%, 5%) the anaerobic metabolism and apoptotic rate of cultured cells is diminished on PTFE foil when compared with the conventional culture dishes. In contrast, under higher oxygen atmosphere (21%) the number of apoptotic cells on the PTFE foil was enhanced. This culture model demonstrates a suitable model for the improvement of oxygen dependent metabolism under low oxygen conditions as well as for induction of oxidative stress by high oxygen atmosphere without supplementation of neurotoxins. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [ABSTRACT FROM AUTHOR]

Chlamydiaceae are obligate intracellular bacteria that cause endemic trachoma, sexually transmitted diseases and respiratory infections. The course of the diseases is determined by local inflammatory immune responses and the propensity of the pathogen to replicate within infected host cells. Both features require energy which is inseparably coupled to oxygen availability in the microenvironment. Hypoxia-inducible factor-1 (HIF-1) regulates crucial genes involved in the adaptation to low oxygen concentrations, cell metabolism and the innate immune response. Here we report that Chlamydia pneumoniae directly interferes with host cell HIF-1α regulation in a biphasic manner. In hypoxia, C. pneumoniae infection had an additive effect on HIF-1α stabilization resulting in enhanced glucose uptake during the early phase of infection. During the late phase of intracellular chlamydial replication, host cell adaptation to hypoxia was actively silenced by pathogen-induced HIF-1α degradation. HIF-1α was targeted by the chlamydial protease-like activity factor, which was secreted into the cytoplasm of infected cells. Direct interference with HIF-1α stabilization was essential for efficient C. pneumoniae replication in hypoxia and highlights a novel strategy of adaptive pathogen–host interaction in chlamydial diseases. [ABSTRACT FROM AUTHOR]

The enteric pathogen Salmonella typhimurium co-ordinates the expression of virulence determinants in response to environmental cues from the host organism. S. typhimurium possesses Salmonella pathogenicity island 2 (SPI2), a large virulence locus encoding a type III secretion system for virulence determinants required for systemic infections and accumulation inside host cells. We have generated transcriptional fusions to SPI2 genes to analyse expression and used antibodies against recombinant SPI2 proteins to monitor levels of SPI2 proteins under various conditions. Here, we demonstrate that SPI2 gene expression is induced by Mg2+ deprivation and phosphate starvation. These conditions are likely to represent the environmental cues encountered by S. typhimurium inside the phagosome of infected host cells. The induction of SPI2 gene expression is modulated by the global regulatory system PhoPQ and is dependent on SsrAB, a two-component regulatory system encoded by SPI2. [ABSTRACT FROM AUTHOR]

The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for bacterial replication inside macrophages. SseB has been considered a putative target of the secretion system on the basis of its similarity with EspA, a protein secreted by the type III secretion system of enteropathogenic Escherichia coli (EPEC). EspA forms a filamentous structure on the bacterial cell surface and is involved in translocation of proteins into the eukaryotic cytosol. In this paper, we show that SseB is a secreted protein that associates with the surface of the bacterial cell and might, therefore, also be required for delivery of SPI-2 effector proteins to the eukaryotic cell cytosol. SseB begins to accumulate inside the bacterial cell when the culture enters early stationary phase. However, SseB is only secreted if the bacteria are grown at low pH or if the pH is shifted after growth from 7.0 to below pH 5.0. The secretion occurs within minutes of acidification and is totally dependent on a functional SPI-2 type III secretion system. As the pH of the Salmonella -containing vacuole inside host cells has been shown to acidify to between pH 4.0 and 5.0, and as SPI-2 gene expression occurs inside host cells, low pH might be a physiological stimulus for SPI-2-mediated secretion in vivo . [ABSTRACT FROM AUTHOR]

In animal models of organ transplantation, infusion of donor-derived leucocytes or bone marrow cells can support tolerance induction. To date, little is known about the suppressive effects of human allogeneic mononuclear cells on alloreactivity in the human system. To study this, mixed leucocyte cultures (MLC) were incubated in the presence and absence of viable allogeneic mononuclear cells (MNC) (modulator cells) of stimulator/donor origin, and the cytotoxic and proliferative potential of the resulting effector cells was determined. The experiments showed that: viable allogeneic MNC from bone marrow and from lymph nodes and peripheral blood (PBMC) were able to suppress allospecific cytotoxicity by an average of 60%; that allospecific as well as non-specific inhibitory effects could be observed with unseparated PBMC; that CD2+ PMNC showed predominantly allospecific inhibition of cytotoxicity with little effect on proliferation whereas CD2- PBMC showed non-specific inhibitory effects (both for cytotoxicity and proliferation), which could be eliminated by indomethacin; that addition of interleukin-2 (IL-2) up to 50 U/ml to the MLC could not reverse the inhibitory effect; and that selective removal of CD8+ cells from the CD2+ modulator population diminished the specific inhibitory effect only partially. These findings demonstrate that viable human MNC from different compartments can have a marked suppressive effect on alloreactivity in vitro. For peripheral blood mononuclear cells (PBMC) the data suggest that various mechanisms can contribute to allosuppression, including specific suppressive veto effects by CD2+ cells. Such inhibitory effects might be applicable in vivo for down-regulating allospecific cytotoxicity and to facilitate the acceptance of allografts. [ABSTRACT FROM AUTHOR]