Validation of the ‘Marburg bone bank system’ for thermodisinfection of allogenic femoral head transplants using selected bacteria, fungi, and spores

The Marburg Bone Bank System 'Lobator sd-2' is widely used to process human femoral heads removed during aseptic surgery by thermal disinfection. The inactivating capacity of the thermodisinfection system was validated in compliance with current standards using a newly developed femoral head model. The following micro-organisms, bacteria and fungi, taken from the American Type Culture Collection were investigated: Staphylococcus aureus, Staphyloccus epidermidis, Enterococcus faecium, Pseudomonas aeruginosa, Bacillus subtilis including spores, Clostridium sporogenes, Mycobacterium terrae, Candida albicans and Aspergillus niger spores. Highly enriched suspensions of these micro-organisms were applied to the centre of the femoral heads. The reduction in the number of micro-organisms was determined by counting the colony-forming units (cfu) before and after processing the spiked test device in the 'Lobator sd-2' system.Vegetative bacteria, fungi and fungal spores were completely inactivated (reduction factor >/=6 log(10)). The numbers of B. subtilis and C. sporogenes spores, both known to be heat-resistant, were reduced by one to two orders of magnitude. These bacteria serve as a model for spore forming pathogens which are not relevant in femoral heads from living donors. By processing human femoral heads from living donors by thermal disinfection using the Marburg Bone Banking system, a high level of safety is achieved regarding clinically relevant pathogens. To further increase the safety of the thermally treated femoral heads, we recommend that the medical history and present state of the donor, as well as the necessary serological tests should be taken into account.     

Model-based inference of neutralizing antibody avidities against influenza virus

To assess the response to vaccination, quantity (concentration) and quality (avidity) of neutralizing antibodies are the most important parameters. Specifically, an increase in avidity indicates germinal center formation, which is required for establishing long-term protection. For influenza, the classical hemagglutination inhibition (HI) assay, however, quantifies a combination of both, and to separately determine avidity requires high experimental effort. We developed from first principles a biophysical model of hemagglutination inhibition to infer IgG antibody avidities from measured HI titers and IgG concentrations. The model accurately describes the relationship between neutralizing antibody concentration/avidity and HI titer, and explains quantitative aspects of the HI assay, such as robustness to pipetting errors and detection limit. We applied our model to infer avidities against the pandemic 2009 H1N1 influenza virus in vaccinated patients (n = 45) after hematopoietic stem cell transplantation (HSCT) and validated our results with independent avidity measurements using an enzyme-linked immunosorbent assay with urea elution. Avidities inferred by the model correlated with experimentally determined avidities (ρ = 0.54, 95% CI = [0.31, 0.70], P < 10⁻⁴). The model predicted that increases in IgG concentration mainly contribute to the observed HI titer increases in HSCT patients and that immunosuppressive treatment is associated with lower baseline avidities. Since our approach requires only easy-to-establish measurements as input, we anticipate that it will help to disentangle causes for poor vaccination outcomes also in larger patient populations. This study demonstrates that biophysical modelling can provide quantitative insights into agglutination assays and complement experimental measurements to refine antibody response analyses.     

Functional gene polymorphisms in the serotonin system and traumatic life events modulate the neural basis of fear acquisition and extinction

Fear acquisition and extinction are crucial mechanisms in the etiology and maintenance of anxiety disorders. Moreover, they might play a pivotal role in conveying the influence of genetic and environmental factors on the development of a (more or less) stronger proneness for, or resilience against psychopathology. There are only few insights in the neurobiology of genetically and environmentally based individual differences in fear learning and extinction. In this functional magnetic resonance imaging study, 74 healthy subjects were investigated. These were invited according to 5-HTTLPR/rs25531 (S+ vs. L(A)L(A); triallelic classification) and TPH2 (G(-703)T) (T+ vs. T-) genotype. The aim was to investigate the influence of genetic factors and traumatic life events on skin conductance responses (SCRs) and neural responses (amygdala, insula, dorsal anterior cingulate cortex (dACC) and ventromedial prefrontal cortex (vmPFC)) during acquisition and extinction learning in a differential fear conditioning paradigm. Fear acquisition was characterized by stronger late conditioned and unconditioned responses in the right insula in 5-HTTLPR S-allele carriers. During extinction traumatic life events were associated with reduced amygdala activation in S-allele carriers vs. non-carriers. Beyond that, T-allele carriers of the TPH2 (G(-703)T) polymorphism with a higher number of traumatic life events showed enhanced responsiveness in the amygdala during acquisition and in the vmPFC during extinction learning compared with non-carriers. Finally, a combined effect of the two polymorphisms with higher responses in S- and T-allele carriers was found in the dACC during extinction. The results indicate an increased expression of conditioned, but also unconditioned fear responses in the insula in 5-HTTLPR S-allele carriers. A combined effect of the two polymorphisms on dACC activation during extinction might be associated with prolonged fear expression. Gene-by-environment interactions in amygdala and vmPFC activation may reflect a neural endophenotype translating genetic and adverse environmental influences into vulnerability for or resilience against developing affective psychopathology.     

The genes encoding E-selectin (SELE) and lymphotactin (SCYC1) lie on separate chicken chromosomes although they are closely linked in human and mouse

Three differentially expressed selectin genes (SELE, SELP, and SELL), important in the initial stages of leukocyte extravasation, have been reported in mammals. All three genes map close to the chemokine SCYC1 (small inducible cytokine subfamily C, member 1) in a large conserved chromosomal segment that extends from RXRG (retinoic acid receptor, gamma) to TNNT2 (troponin T2) on Chromosome (Chr) 1 in both human and mouse. In the mouse, we demonstrate that Sele is flanked by Prrx1 (paired-related homeobox gene 1) and Scyc1 and define the order of, and distances between, loci as centromere-Prrx1-(0.7+/-0.7 cM)-Sele-(1.2+/-0.9 cM)-Scyc1-telomere. In the chicken, we isolated BAC clones containing PRRX1, SELE, and SCYC1 and positioned them by fluorescent in situ hybridization. SELE and PRRX1 mapped to the short arm of chicken Chr 8 and SCYC1 mapped to the region equivalent to 1q11-1q13 on the long arm of chicken Chr 1. The location of SELE on chicken Chr 8 was independently established by linkage analysis of COM0185, an (AT)16 microsatellite locus identified in a BAC clone that contained SELE. COM0185 was linked to several loci that mapped to one end of chicken Chr 8, with the order of loci, and genetic distances (in cM) between them defined as MSU0435, MSU0325-(7.8+/-3.7)-COM0185-(5.8+/-3.2)-ROS0338-(9.6+/-4.0)-ABR0322-(3.8+/-2.6)-GLUL. We have therefore positioned an evolutionary breakpoint in mammals and chickens between SELE and SCYC1. Furthermore, comparative mapping analysis of the RXRG-TNNT2 chromosomal segment that is conserved on human and mouse Chr 1 indicates that it is divided into four segments in the chicken, each of which maps to a different chromosome.     

Sudemycin E influences alternative splicing and changes chromatin modifications

Sudemycin E is an analog of the pre-messenger RNA splicing modulator {"type":"entrez-nucleotide","attrs":{"text":"FR901464","term_id":"525229801","term_text":"FR901464"}}FR901464 and its derivative spliceostatin A. Sudemycin E causes the death of cancer cells through an unknown mechanism. We found that similar to spliceostatin A, sudemycin E binds to the U2 small nuclear ribonucleoprotein (snRNP) component SF3B1. Native chromatin immunoprecipitations showed that U2 snRNPs physically interact with nucleosomes. Sudemycin E induces a dissociation of the U2 snRNPs and decreases their interaction with nucleosomes. To determine the effect on gene expression, we performed genome-wide array analysis. Sudemycin E first causes a rapid change in alternative pre-messenger RNA splicing, which is later followed by changes in overall gene expression and arrest in the G2 phase of the cell cycle. The changes in alternative exon usage correlate with a loss of the H3K36me3 modification in chromatin encoding these exons. We propose that sudemycin E interferes with the ability of U2 snRNP to maintain an H3K36me3 modification in actively transcribed genes. Thus, in addition to the reversible changes in alternative splicing, sudemycin E causes changes in chromatin modifications that result in chromatin condensation, which is a likely contributing factor to cancer cell death.     

Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane

Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol.     

Phosphorylation of Serine 774 of the Neural Cell Adhesion Molecule (NCAM) Is Involved in the Interaction with Collapsin Response Mediator Protein-2

The neural cell adhesion molecule NCAM is a major adhesion receptor involved in the development and regeneration of the nervous system. It is expressed in three major isoforms of which two have large intracellular domains of different lengths (NCAM140 and NCAM180). Several intracellular ligands of NCAM have been described. One of them is the collapsin response mediator protein-2 (CRMP-2), which is known to be involved in cell differentiation and axonal growth. The cytoplasmic domains of NCAM contain up to 49 phosphorylation sites and it has been demonstrated recently that the phosphorylation of serine 774 is crucial for NCAM-mediated signal transduction and neurite outgrowth. Here we analyzed the interaction of NCAM with CRMP-2 in more detail using a biochemical approach. We found that CRMP-2 binds specifically to NCAM180 in a sequence between amino acid 788 and 819. In addition we could demonstrate that serine 774, which has been shown previously to be phosphorylated and involved in neurite outgrowth, is also important for the interaction of CRMP-2 with NCAM.     

Anti-inflammatory effects of Saccharomyces boulardii mediated by myeloid dendritic cells from patients with Crohn's disease and ulcerative colitis

Saccharomyces boulardii (Sb) is a probiotic yeast that has demonstrated efficacy in pilot studies in patients with inflammatory bowel disease (IBD). Microbial antigen handling by dendritic cells (DC) is believed to be of critical importance for immunity and tolerance in IBD. The aim was to characterize the effects of Sb on DC from IBD patients. Highly purified (>95%), lipopolysaccharide-stimulated CD1c(+)CD11c(+)CD123(-) myeloid DC (mDC) from patients with ulcerative colitis (UC; n = 36), Crohn's disease (CD; n = 26), or infectious controls (IC; n = 4) were cultured in the presence or absence of fungal supernatant from Sb (SbS). Phenotype and cytokine production and/or secretion of IBD mDC were measured by flow cytometry and cytometric bead arrays, respectively. T cell phenotype and proliferation were assessed in a mixed lymphocyte reaction (MLR) with allogenic CD4(+)CD45RA(+) na?ve T cells from healthy donors. Mucosal healing was investigated in epithelial wounding and migration assays with IEC-6 cells. SbS significantly decreased the frequency of CD40-, CD80-, and CD197 (CCR7; chemokine receptor-7)-expressing IBD mDC and reduced their secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-6 while increasing IL-8. In the MLR, SbS significantly inhibited T cell proliferation induced by IBD mDC. Moreover, SbS inhibited T(H)1 (TNF-α and interferon-γ) polarization induced by UC mDC and promoted IL-8 and transforming growth factor-β-dependent mucosal healing. In summary, we provide novel evidence of synergistic mechanisms how Sb controls inflammation (inhibition of T cell costimulation and inflammation-associated migration and mobilization of DC) and promotes epithelial restitution relevant in IBD.     

Rewiring two-component signal transduction with small RNAs

Bacterial two-component systems (TCSs) and small regulatory RNAs (sRNAs) form densely interconnected networks that integrate and transduce information from the environment into fine-tuned changes of gene expression. Many TCSs control target genes indirectly through regulation of sRNAs, which in turn regulate gene expression by base-pairing with mRNAs or targeting a protein. Conversely, sRNAs may control TCS synthesis, thereby recruiting the TCS regulon to other regulatory networks. Several TCSs control expression of multiple homologous sRNAs providing the regulatory networks with further flexibility. These sRNAs act redundantly, additively or hierarchically on targets. The regulatory speed of sRNAs and their unique features in gene regulation make them ideal players extending the flexibility, dynamic range or timing of TCS signaling.