The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen.

The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10−/− mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10−/− mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.

Sensorineural hearing loss (SNHL) can be overcome by electrical stimulation of spiral ganglion neurons (SGNs) via a cochlear implant (CI). Restricted CI performance results from the spatial gap between the SGNs and the electrode, but the efficacy of CI is also limited by the degeneration of SGNs as one consequence of SHNL. In the healthy cochlea, the survival of SGNs is assured by endogenous neurotrophic support. Several applications of exogenous neurotrophic supply have been shown to reduce SGN degeneration in vitro and in vivo. In the present study, nanoporous silica nanoparticles (NPSNPs), with an approximate diameter of <100 nm, were loaded with the brain-derived neurotrophic factor (BDNF) to test their efficacy as long-term delivery system for neurotrophins. The neurotrophic factor was released constantly from the NPSNPs over a release period of 80 days when the surface of the nanoparticles had been modified with amino groups. Cell culture investigations with NIH3T3 fibroblasts attest a good general cytocompatibility of the NPSNPs. In vitro experiments with SGNs indicate a significantly higher survival rate of SGNs in cell cultures that contained BDNF-loaded nanoparticles compared to the control culture with unloaded NPSNPs (p<0.001). Importantly, also the amounts of BDNF released up to a time period of 39 days increased the survival rate of SGNs. Thus, NPSNPs carrying BDNF are suitable for the treatment of inner ear disease and for the protection and the support of SGNs. Their nanoscale nature and the fact that a direct contact of the nanoparticles and the SGNs is not necessary for neuroprotective effects, should allow for the facile preparation of nanocomposites, e.g., with biocompatible polymers, to install coatings on implants for the realization of implant-based growth factor delivery systems.

Hyperbaric oxygen therapy (HBOT) is a noninvasive widely applied treatment that increases the oxygen pressure in tissues. In cochlear implant (CI) research, intracochlear application of neurotrophic factors (NTFs) is able to improve survival of spiral ganglion neurons (SGN) after deafness. Cell-based delivery of NTFs such as brain-derived neurotrophic factor (BDNF) may be realized by cell-coating of the surface of the CI electrode. Human mesenchymal stem cells (MSC) secrete a variety of different neurotrophic factors and may be used for the development of a biohybrid electrode in order to release endogenously-derived neuroprotective factors for the protection of residual SGN and for a guided outgrowth of dendrites in the direction of the CI electrode. HBOT could be used to influence cell behaviour after transplantation to the inner ear. The aim of this study was to investigate the effect of HBOT on the proliferation, BDNF-release and secretion of neuroprotective factors. Thus, model cells (an immortalized fibroblast cell line (NIH3T3)–native and genetically modified) and MSCs were repeatedly (3 x – 10 x) exposed to 100% oxygen at different pressures. The effects of HBO on cell proliferation were investigated in relation to normoxic and normobaric conditions (NOR). Moreover, the neuroprotective and neuroregenerative effects of HBO-treated cells were analysed by cultivation of SGN in conditioned medium. Both, the genetically modified NIH3T3/BDNF and native NIH3T3 fibroblasts, showed a highly significant increased proliferation after five days of HBOT in comparison to normoxic controls. By contrast, the number of MSCs was decreased in MSCs treated with 2.0 bar of HBO. Treating SGN cultures with supernatants of fibroblasts and MSCs significantly increased the survival rate of SGN. HBO treatment did not influence (increase / reduce) this effect. Secretome analysis showed that HBO treatment altered the protein expression pattern in MSCs.