Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection

We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8pos and also CD4pos cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection

The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence

Trost E, Ott L, Schneider J, et al. The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence. BMC Genomics. 2010;11(1): 728

Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis

Risk of Transmission of MRSA on Contact Surfaces in Ambulance

The gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of treatment-associated nosocomial infections. The incidence of MRSA among the population and in hospitalised patients is growing worldwide. Ambulance service is an interface between the enviroment, outpatient treatment and inpatient treatment of patients. What is the probability that MRSA is applied by the patient on contact surfaces in ambulance vehicles in compliance with an infection control concept and what are risk contact surfaces in the ambulance vehicle? We studied defined contact surfaces in 30 ambulances after transport of a MRSA positive patients. In the control group 15 ambulances were examined after transport with unknown MRSA status. The sampling was carried out before the final disinfection, genotyping of MRSA strains were carried out within the Euregio MRSA-net project. In three transports with known MRSA status of the patient we found on four contact surfaces in the vehicles a nosocomial MRSA strain with the same genotype of previously transported patient. In the control group, we detected an incidental finding of a nosocomial MRSA strain on the disinfectant dispenser. The risk areas identified were all close to the patient and all hand-related areas of the staff. All sampling was carried out before the final disinfection. In 10% MRSA is transmitted from patient to near patient and all hand contact surfaces in the ambulance vehicle. A targeted disinfection, as well as an infection control concept are sufficient to eliminate MRSA transmission on contact surfaces in the ambulance vehicle

Digital transition in rural emergency medicine: Impact of job satisfaction and workload on communication and technology acceptance.

BackgroundTele-emergency physicians (TEPs) take an increasingly important role in the need-oriented provision of emergency patient care. To improve emergency medicine in rural areas, we set up the project 'Rural|Rescue', which uses TEPs to restructure professional rescue services using information and communication technologies (ICTs) in order to reduce the therapy-free interval. Successful implementation of ICTs relies on user acceptance and knowledge sharing behavior.MethodWe conducted a factorial design with active knowledge transfer and technology acceptance as a function of work satisfaction (high vs. low), workload (high vs. low) and point in time (prior to vs. after digitalization). Data were collected via machine readable questionnaires issued to 755 persons (411 pre, 344 post), of which 304 or 40.3% of these persons responded (194 pre, 115 post).ResultsTechnology acceptance was higher after the implementation of TEP for nurses but not for other professions, and it was higher when the workload was high. Regarding active communication and knowledge sharing, employees with low work satisfaction are more likely to share their digital knowledge as compared to employees with high work satisfaction. This is an effect of previous knowledge concerning digitalization: After implementing the new technology, work satisfaction increased for the more experienced employees, but not for the less experienced ones.ConclusionOur research illustrates that employees' workload has an impact on the intention of using digital applications. The higher the workload, the more people are willing to use TEPs. Regarding active knowledge sharing, we see that employees with low work satisfaction are more likely to share their digital knowledge compared to employees with high work satisfaction. This might be attributed to the Dunning-Kruger effect. Highly knowledgeable employees initially feel uncertain about the change, which translates into temporarily lower work satisfaction. They feel the urge to fill even small knowledge gaps, which in return leads to higher work satisfaction. Those responsible need to acknowledge that digital change affects their employees' workflow and work satisfaction. During such times, employees need time and support to gather information and knowledge in order to cope with digitally changed tasks

Supplementary Material for: High Proportions of CD4+ T Cells among Residual Bone Marrow T Cells in Childhood Acute Lymphoblastic Leukemia Are Associated with Favorable Early Responses

Residual nonmalignant T cells in the bone marrow of patients with acute leukemias may be involved in active immune responses to leukemic cells. Here, we investigated the phenotypic signature of T cells present at diagnosis in 39 pediatric patients with acute lymphoblastic leukemia (ALL) treated within standardized ALL-BFM study protocols. Previously described age associations of lymphocyte subpopulations in the peripheral blood of healthy children were reproduced in leukemic bone marrow. Analysis of individual lymphocyte parameters and risk-associated variables using univariate linear regression models revealed a correlation of higher CD4/CD8 ratios at diagnosis with a favorable bone marrow response on day 15. Separate analysis of CD4+ cells with the CD4+CD25^hiFoxP3+ T_reg cell phenotype showed that the association was caused by non-T_reg CD4+ cells. The association of higher CD4/CD8 ratios with a favorable bone marrow response on day 15 of treatment persisted in a cohort extended to 69 patients. We conclude that CD4+ non-T_reg cells in leukemic bone marrow at diagnosis may have a role in early response to treatment. Prospective analysis of the CD4/CD8 ratio in a large cohort of pediatric patients is now needed. Moreover, future experiments will establish the functional role of the individual T cell subsets in immune control in pediatric ALL

EMMA 2-A MAGE-compliant system for the collaborative analysis and integration of microarray data

Dondrup M, Albaum S, Griebel T, et al. EMMA 2-A MAGE-compliant system for the collaborative analysis and integration of microarray data. BMC Bioinformatics. 2009;10(1): 50.Background: Understanding transcriptional regulation by genome-wide microarray studies can contribute to unravel complex relationships between genes. Attempts to standardize the annotation of microarray data include the Minimum Information About a Microarray Experiment (MIAME) recommendations, the MAGE-ML format for data interchange, and the use of controlled vocabularies or ontologies. The existing software systems for microarray data analysis implement the mentioned standards only partially and are often hard to use and extend. Integration of genomic annotation data and other sources of external knowledge using open standards is therefore a key requirement for future integrated analysis systems. Results: The EMMA 2 software has been designed to resolve shortcomings with respect to full MAGE-ML and ontology support and makes use of modern data integration techniques. We present a software system that features comprehensive data analysis functions for spotted arrays, and for the most common synthesized oligo arrays such as Agilent, Affymetrix and NimbleGen. The system is based on the full MAGE object model. Analysis functionality is based on R and Bioconductor packages and can make use of a compute cluster for distributed services. Conclusion: Our model-driven approach for automatically implementing a full MAGE object model provides high flexibility and compatibility. Data integration via SOAP-based web-services is advantageous in a distributed client-server environment as the collaborative analysis of microarray data is gaining more and more relevance in international research consortia. The adequacy of the EMMA 2 software design and implementation has been proven by its application in many distributed functional genomics projects. Its scalability makes the current architecture suited for extensions towards future transcriptomics methods based on high-throughput sequencing approaches which have much higher computational requirements than microarrays

Smartphone-based dispatch of community first responders to out-of-hospital cardiac arrest-statements from an international consensus conference

Background: Over the past decade Smartphone-based activation (SBA) of Community First Responders (CFR) to out-of-hospital cardiac arrests (OHCA) has gained much attention and popularity throughout Europe. Various programmes have been established, and interestingly there are considerable differences in technology, responder spectrum and the degree of integration into the prehospital emergency services. It is unclear whether these dissimilarities affect outcome. This paper reviews the current state in five European countries, reveals similarities and controversies, and presents consensus statements generated in an international conference with the intention to support public decision making on future strategies for SBA of CFR. Methods: In a consensus conference a three-step approach was used: (i) presentation of current research from five European countries; (ii) workshops discussing evidence amongst the audience to generate consensus statements; (iii) anonymous real-time voting applying the modified RAND-UCLA Appropriateness method to adopt or reject the statements. The consensus panel aimed to represent all stakeholders involved in this topic. Results: While 21 of 25 generated statements gained approval, consensus was only found for 5 of them. One statement was rejected but without consensus. Members of the consensus conference confirmed that CFR save lives. They further acknowledged the crucial role of emergency medical control centres and called for nationwide strategies. Conclusions: Members of the consensus conference acknowledged that smartphone-based activation of CFR to OHCA saves lives. The statements generated by the consensus conference may assist the public, healthcare services and governments to utilise these systems to their full potential, and direct the research community towards fields that still need to be addressed