Entwicklung und Charakterisierung eines iPSZ basierten Differenzierungsprotokolls zu olfaktorischen Rezeptorneuronen

Das Riechsystem ist einzigartig in unserem Organismus, da etwa fünf Prozent unserer Gene dem olfaktorischen System, vor allem den jeweiligen Odorant-Rezeptoren der Rezeptorneurone, zugeordnet werden können. Jedes Gen detektiert für einen anderen Rezeptor, jedes Rezeptorneuron exprimiert dabei nur einen einzigen Rezeptortyp und jeder Rezeptor reagiert auf eine andere Kombination und Intensität von Duftstoffen. Bisher sind nur sehr wenige Zuordnungen zwischen Duftstoff und Rezeptor bekannt. Dies ist nur einer von vielen Punkten, der wiederspiegelt, wie wichtig der Riechsinn für den Menschen ist. Auf den ersten Blick erscheint er als Sinn eher untergeordent, jedoch nimmt er unterbewusst einen sehr großen Stellenwert ein. Zudem ist der primäre (Seh-) Sinn weit besser untersucht und spielt in der aktuellen Forschungslandschaft eine deutlich höhere Rolle. Deshalb widmet sich diese Doktorarbeit einem noch rudimentär in der Forschung beleuchteten Sinnessystem unseres Körpers. Hierbei soll ein olfaktorisches Differenzierungsprotokoll, ausgehend von pluripotenten Stammzellen entwickelt und anschließend näher charakterisiert werden. Das Ziel ist es ein System zu entwickeln, mit dem man die in vivo Bedingungen des olfaktorischen Systems leicht zugänglich nachahmen kann und weitere Forschungsfragen damit beantworten kann. Im Rahmen dieser Doktorarbeit wurden dafür folgende Arbeitsschritte experimentell bearbeitet: I. Generierung von induziert pluripotenten Stammzellen aus humanen Keratinozyten Es wurden von drei unterschiedlichen, freiwilligen Spendern Haarproben entnommen und kultiviert. Daraus konnten primäre Keratinozyten gewonnen werden, die erfolgreich zu iPSZ lentiviral reprogrammiert wurden. Die generierten Stammzellen sind pluripotent und können in alle drei Keimblätter differenzieren. Sie bilden somit die Grundlage für die Etablierung des olfaktorischen Differenzierungsprotokolls. II. Etablierung eines Differenzierungsprotokolls zur Gewinnung von olfaktorischen Rezeptorneuronen Die generierten iPSZ dienten als Ausgangsmaterial für die Entwicklung eines gerichteten, olfaktorischen Differenzierungsprotokolls. Hierbei stand die Anreicherung von OMP positiven Neuronen in der neuronalen Mischkultur im Fokus. Dies wurde durch die gezielte Verbesserung eines neuro-ektodermalen Protokolls durch die Zugabe von Wachstumsfaktoren zu bestimmten Differenzierungsstadien erreicht. Zusätzliche Variationen in den Zeitpunkten und Zeitabständen der einzelnen Differenzierungsschritte führte ebenso zu einer höheren Ausbeute an OMP positiven Zellen. Es konnte im Neuronen Stadium an Tag 74 eine 58-fach signifikant erhöhte OMP Expression im Vergleich zu Stammzellen gemessen werden. III. Charakterisierung des gerichteten Protokolls anhand von detaillierten Genexpressionsanalysen Im letzten Arbeitsschritt wurde das entwickelte Protokoll mithilfe einer detaillierten Genexpressionsanalyse näher charakterisiert. Dabei wurden zu verschiedenen Differenzierungstadien Markergene untersucht, die spezifisch für die jeweilige embryonale olfaktorische Entwicklung sind. Zusätzlich wurde die Marker Expression auch auf Proteinebene überprüft. Es konnte so ein Schema erstellt werden, das die einzelnen Differenzierungsstadien mit den olfaktorischen Entwicklungsschritten in vivo in direkten Zusammenhang bringt. Signalkaskaden, die in der olfaktorischen Entwicklung und Neurogenese auftreten stimmen teilweise mit denen Verlaufskurven in vitro überein. Insgesamt lässt sich zusammengefasst sagen, dass das generierte Differenzierungsprotokoll zu großen Teilen die embryonale olfaktorische Entwicklung in vitro wiederspiegelt

Successful long-term treatment of persistent pulmonary air leak in pneumocystis jirovecii pneumonia by unidirectional endobronchial valves

Spontaneous pneumothorax is a rare complication of pneumocystis jirovecii pneumonia. We report a patient with pneumocystis jirovecii pneumonia and therapy-refractory, right-sided pneumothorax due to persistent air leak (PAL) despite prolonged chest tube placement and multiple pleurodesis attempts. Due to the patient's morbidity, we evaluated if the PAL can be sealed by unidirectional endobronchial valves (EBVs). After occlusion of the right upper lobe by a balloon catheter, the air leak flow-rate decreased from 800 ml/min to 250 ml/min. Zephyr EBVs (ZEBVs) were placed in the segmental right upper lobe bronchi and subsequently, a complete resolution of the pneumothorax was noted. During 30 months of follow-up, neither recurrence of pneumothorax nor any adverse events of EBV treatment were noted. We conclude that ZEBV placement might be an effective and well-tolerated treatment option for PAL secondary to pneumocystis jirovecii pneumonia with promising long-term results

Rationale and design of the German-speaking myeloma multicenter group (GMMG) trial HD6: a randomized phase III trial on the effect of elotuzumab in VRD induction/consolidation and lenalidomide maintenance in patients with newly diagnosed myeloma

Background: Despite major advances in therapy, multiple myeloma is still an incurable malignancy in the majority of patients. To increase survival, deeper remissions (i.e. CR) translating into longer PFS need to be achieved. Incorporation of new drugs (i.e. bortezomib and lenalidomide) as induction and maintenance treatment in an intensified treatment concept, including high dose melphalan (200 mg/m2), has resulted in increased CR rates, and is considered the standard of care for younger patients. Elotuzumab in combination with lenalidomide and dexamethasone has given better results as lenalidomide and dexamethasone alone in a phase III trial. The GMMG-HD6 trial will be the first phase III trial investigating the role of elotuzumab in combination with bortezomib, lenalidomide and dexamethasone (VRD) induction/consolidation and lenalidomide maintenance within a high dose concept. Methods: GMMG-HD6 is a randomized, open, multicenter phase III trial. The planned recruitment number is 564 NDMM patients. All patients will receive 4 VRD cycles as induction and undergo peripheral blood stem cell mobilization and harvesting. Thereafter they will be treated with high dose melphalan therapy plus autologous stem cell transplantation followed by 2 cycles of VRD consolidation and lenalidomide maintenance. Patients in arm B1 + B2 will additionally receive elotuzumab in the induction phase, whereas patients in A2 + B2 will be treated with elotuzumab added to consolidation and maintenance. The primary endpoint of the trial is PFS. Secondary objectives and endpoints are OS, CR rates after induction therapy comparing the two arms VRD (A1 + A2) vs VRD + elotuzumab (B1 + B2), CR rates after consolidation treatment, best response to treatment during the study, time to progression (TTP), duration of response (DOR), toxicity and quality of life. Results: Since this is the publication of a study protocol of an ongoing study, no results can be presented. Discussion: This phase III trial is designed to evaluate whether the addition of elotuzumab to an intensified treatment concept with high dose melphalan chemotherapy plus autologous stem cell transplantation and induction, consolidation and maintenance treatment with bortezomib and lenalidomide is able to improve PFS compared to the same concept without elotuzumab. Trial registration: NCT02495922 on June 24th, 2015

Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury

Neutrophils trigger inflammation-induced acute kidney injury (AKI), a frequent and potentially lethal occurrence in humans. Molecular mechanisms underlying neutrophil recruitment to sites of inflammation have proved elusive. In this study, we demonstrate that SLP-76 (SH2 domain–containing leukocyte phosphoprotein of 76 kD) and ADAP (adhesion and degranulation promoting adaptor protein) are involved in E-selectin–mediated integrin activation and slow leukocyte rolling, which promotes ischemia-reperfusion–induced AKI in mice. By using genetically engineered mice and transduced Slp76(−/−) primary leukocytes, we demonstrate that ADAP as well as two N-terminal–located tyrosines and the SH2 domain of SLP-76 are required for downstream signaling and slow leukocyte rolling. The Tec family kinase Bruton tyrosine kinase is downstream of SLP-76 and, together with ADAP, regulates PI3Kγ (phosphoinositide 3-kinase–γ)- and PLCγ2 (phospholipase Cγ2)-dependent pathways. Blocking both pathways completely abolishes integrin affinity and avidity regulation. Thus, SLP-76 and ADAP are involved in E-selectin–mediated integrin activation and neutrophil recruitment to inflamed kidneys, which may underlie the development of life-threatening ischemia-reperfusion–induced AKI in humans

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

A Comparative View on Human Somatic Cell Sources for iPSC Generation

The breakthrough of reprogramming human somatic cells was achieved in 2006 by the work of Yamanaka and Takahashi. From this point, fibroblasts are the most commonly used primary somatic cell type for the generation of induced pluripotent stem cells (iPSCs). Various characteristics of fibroblasts supported their utilization for the groundbreaking experiments of iPSC generation. One major advantage is the high availability of fibroblasts which can be easily isolated from skin biopsies. Furthermore, their cultivation, propagation, and cryoconservation properties are uncomplicated with respect to nutritional requirements and viability in culture. However, the required skin biopsy remains an invasive approach, representing a major drawback for using fibroblasts as the starting material. More and more studies appeared over the last years, describing the reprogramming of other human somatic cell types. Cells isolated from blood samples or urine, as well as more unexpected cell types, like pancreatic islet beta cells, synovial cells, or mesenchymal stromal cells from wisdom teeth, show promising characteristics for a reprogramming strategy. Here, we want to highlight the advantages of keratinocytes from human plucked hair as a widely usable, noninvasive harvesting method for primary material in comparison with other commonly used cell types

Conventional cell culture media do not adequately supply cells with antioxidants and thus facilitate peroxide-induced genotoxicity

Commercially available calf serum did not supply the cultured murine fibroblast cell line L929 with amounts of selenium and α-tocopherol sufficient to protect against peroxide damage. Supplementation of the culture medium with 30μM α-tocopherol or 50 nM sodium selenite led to a substantial increase of cellular α-tocopherol concentrations from 18 ± 3.0 to 3179 ±93.0 pmol/106 cells or cellular selenium concentrations from 0.17 ±0.02 to 1.75 ±0.16 ng/106 cells, respectively. L929 fibroblasts grown in selenite-containing medium also had markedly raised activities of both cytosolic glutathione peroxidase (from 11 ±0.9 to 67.2 ±4.2 mU/107 cells) and phospholipid hydroperoxide glutathione peroxidase (from 0.2 to 9.5 ±0.9 mU/107 cells). Supplementation with α-tocopherol inhibited single-strand breaks induced by low concentrations of H2O2 only, whereas an adequate selenium supply almost completely inhibited single-strand breaks induced by up to 30μM H2O2 and also significantly reduced H2O2induced cell death. An inadequate selenium supply and corresponding increase of GPx activity upon selenite supplementation was also observed with other cell lines, for instance, D10N, ECV-304, HepG2, and THP-1. Our data strengthen the relevance of standardized and adequate supplementation of tissue culture media with antioxidants to improve viability and genetic stability of cultured cells in general and in particular, if they are oxidatively challenged