Charakterisierung von Tumor-induzierten Memory-like NK Zellen

Natürliche Killerzellen (NK Zellen) gehören zur angeborenen Immunabwehr und sind als wichtige Effektorlymphozyten an der Abwehr virusinfizierter und maligner Zellen beteiligt. Obwohl das immunologische Gedächtnis normalerweise durch Zelltypen des adaptiven Immunsystems vermittelt wird, konnten in den letzten Jahren auch Memory-Eigenschaften für NK Zellen beschrieben werden. In der Arbeitsgruppe André und in der vorliegenden Arbeit konnte gezeigt werden, dass ein vorausgehender Kontakt mit bestrahlten Tumorzellen, ein sogenanntes Priming, zur Ausbildung Tumor-induzierter Memory-like (TIML) NK Zellen führt. Diese zeichnen sich durch eine gesteigerte Funktionalität verglichen mit unstimulierten NK Zellen aus. In vitro konnte belegt werden, dass diese TIML-NK Zellen tumorspezifisch sind, d.h. die erhöhte Funktionalität äußert sich nur gegen jene Tumorentität, welche auch im Primingprozess beteiligt war. Primingexperimenten mit Tumorfragmenten zeigten, dass ein direkter Kontakt zwischen Rezeptoren und Liganden der Tumor- und NK Zellen nötig ist. Neu exprimierte Rezeptoren auf der Oberfläche der Tumorzellen sowie sezernierte Cytokine führen nicht zu einer Konvertierung in TIML-NK Zellen. Diese Daten werden von TranswellExperimenten, in welchen NK Zellen und Tumorzellen während des Primingprozesses keinen direkten Kontakt miteinander eingehen konnten, bestätigt. Durch weitere Primingversuche in Anwesenheit des blockierenden anti-NKG2D Antikörpers, wurde die Bedeutung der NKG2D/IL15 Achse für die Ausbildung von TIML-NK Zellen untersucht. Die Ergebnisse zeigen jedoch, dass die NKG2D/IL15 Achse keine Rolle bei der Konvertierung in Memory-like NK Zellen spielt. Neben ihrer cytotoxischen Aktivität sind NK Zellen wichtige immunregulatorische Zellen. Sie verbinden unter anderem durch Sekretion von Cytokinen sowie Chemokinen die angeborene und adaptive Immunantwort. Im zweiten Teil der vorliegenden Arbeit wurde ein Kokulturmodell mit NK Zellen und PBMCs etabliert, um den Einfluss naiver und TIMLNK Zellen auf die T Zellantwort zu untersuchen. Diese Analysen könnten Aufschluss über eine mögliche schädliche Graft versus Host Disease (GvHD) oder verstärkte Graft versus Leukämie (GvL) Effekte durch T Zellen geben. Bestehende adoptive NK Zelltransferprotokolle könnten durch die Verwendung von TIML-NK Zellen und ihren adaptiven Eigenschaften somit verbessern werden, was einen weiteren Schritt in Richtung individualisierte Behandlungsstrategie für den Patienten bedeutet

Chronic thoracic hemisection spinal cord injury in adult rats induces a progressive decline in transmission from uninjured fibers to lumbar motoneurons

Although most spinal cord injuries are anatomically incomplete, only limited functional recovery has been observed in people and rats with partial lesions. To address why surviving fibers cannot mediate more complete recovery, we evaluated the physiological and anatomical status of spared fibers after unilateral hemisection (HX) of thoracic spinal cord in adult rats. We made intracellular and extracellular recordings at L5 (below HX) in response to electrical stimulation of contralateral white matter above (T6) and below (L1) HX. Responses from T6 displayed reduced amplitude, increased latency and elevated stimulus threshold in the fibers across from HX, beginning 1-2 weeks after HX. Ultrastructural analysis revealed demyelination of intact axons contralateral to the HX, with a time course similar to the conduction changes. Behavioral studies indicated partial recovery which arrested when conduction deficits began. These findings suggest a chronic pathological state in intact fibers and necessity for prompt treatment to minimize it

Dynamic clonal progression in xenografts of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21

Intrachromosomal amplification of chromosome 21 is a heterogeneous chromosomal rearrangement occurring in 2% of childhood precursor B-cell acute lymphoblastic leukemia. There are no cell lines with iAMP21 and these abnormalities are too complex to faithfully engineer in animal models. As a resource for future functional and pre-clinical studies, we have created xenografts from intrachromosomal amplification of chromosome 21 leukemia patient blasts and characterised them by in-vivo and ex-vivo luminescent imaging, FLOW immunophenotyping, and histological and ultrastructural analysis of bone marrow and the central nervous system. Investigation of up to three generations of xenografts revealed phenotypic evolution, branching genomic architecture and, compared with other B-cell acute lymphoblastic leukemia genetic subtypes, greater clonal diversity of leukemia initiating cells. In support of intrachromosomal amplification of chromosome 21 as a primary genetic abnormality, it was always retained through generations of xenografts, although we also observed the first example of structural evolution of this rearrangement. Clonal segregation in xenografts revealed convergent evolution of different secondary genomic abnormalities implicating several known tumour suppressor genes and a region, containing the B-cell adaptor, PIK3AP1, and nuclear receptor co-repressor, LCOR, in the progression of B-ALL. Tracking of mutations in patients and derived xenografts provided evidence for co-operation between abnormalities activating the RAS pathway in B-ALL and for their aggressive clonal expansion in the xeno-environment. Bi-allelic loss of the CDKN2A/B locus was recurrently maintained or emergent in xenografts and also strongly selected as RNA sequencing demonstrated a complete absence of reads for genes associated with the deletions

Design and functionality of a prototype for cold needle perforation of wheat

Wheat is a vital crop in global food security, but up to 25% of the wheat harvested is contaminated with mycotoxins that have detrimental effects on human health. To address this issue, biological detoxification strategies have been developed using microorganisms and enzymes. Perforating the whole wheat kernel using cold needle perforation (CNP) followed by a detoxification step could be a promising approach to reduce cross-contamination during the milling of mycotoxin-containing wheat. In this study, a pilot-scale CNP prototype was developed to perforate wheat kernels, and its effectiveness was evaluated. The height-adjustable perforation unit consists of 3120 needles. The throughput of the CNP prototype was adjusted to 6 kg/h, and the kernels were perforated for 1, 5, or 10 cycles. The results show that the CNP prototype effectively perforates wheat kernels, as evidenced by the significant increase in pore count. Fluorescence microscopy confirmed the penetration of particles in the size range of enzymes and microorganisms into the kernel. This study demonstrates the successful scale-up of CNP for wheat kernel perforation and highlights the potential of CNP as a cost-effective and efficient method for the biological detoxification of mycotoxin-contaminated wheat

Differential effects of anti-Nogo-A antibody treatment and treadmill training in rats with incomplete spinal cord injury

Locomotor training on treadmills can improve recovery of stepping in spinal cord injured animals and patients. Likewise, lesioned rats treated with antibodies against the myelin associated neurite growth inhibitory protein, Nogo-A, showed increased regeneration, neuronal reorganization and behavioural improvements. A detailed kinematic analysis showed that the hindlimb kinematic patterns that developed in anti-Nogo-A antibody treated versus treadmill trained spinal cord injured rats were significantly different. The synchronous combined treatment group did not show synergistic effects. This lack of synergistic effects could not be explained by an increase in pain perception, sprouting of calcitonin gene-related peptide (CGRP) positive fibres or by interference of locomotor training with anti-Nogo-A antibody induced regeneration and sprouting of descending fibre tracts. The differential mechanisms leading to behavioural recovery during task-specific training and in regeneration or plasticity enhancing therapies have to be taken into account in designing combinatorial therapies so that their potential positive interactive effects can be fully expresse

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Înjured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration

Effect of fruiting-related genes on the formation of volatile organic compounds in the mushroom Schizophyllum commune

In fungi, little is known about connections between volatile organic compound (VOC) formation and developmental stages that are amongst others triggered by fruiting-related genes (FRGs). We analysed the volatilomes of Schizophyllum commune during different developmental stages in a variety of FRG-deletion strains and wild-type strains. The deletion strains Δtea1Δtea1, Δwc-2Δwc-2 and Δhom2Δhom2 were unable to develop fruiting bodies, and Δfst4Δfst4 formed only rudimentary fruiting body structures. Early developmental stages of these strains were dominated by esters, including methyl 2-methylbutanoate, ethyl 2-methylbutanoate, isobutyl 2-methylpropionate, and 2-methylbutyl acetate, of which the last three were not found in the headspace (HS) of the wild-type samples. Compared to the wild type, in the HS of hom2con samples, that are able to form fruiting bodies, methyl 2-methylbutanoate was the most abundant substance at early stages (68–81% of the total peak area). In contrast to fruiting body forming strains, Δtea1Δtea1, Δwc-2Δwc-2, Δhom2Δhom2 and Δfst4Δfst4 showed less sesquiterpenes in the HS. However, the sesquiterpenes found in the HS of FRG-deletion strains, namely, (E)-nerolidol, δ-cadinene, L-calamenene, α-bisabolol and β-bisabolene, were not present in hom2con or wild-type strains that mainly formed fruiting bodies and barely mycelium. Several sesquiterpenes, including α-guaiene, chamigrene and γ-gurjunene, were only found in presence of fruiting bodies. Our results show remarkable connections between FRGs, fruiting body development and VOC production in S. commune, especially counting for sesquiterpenes. Future studies are needed to reveal whether FRGs directly regulates VOC formation or indirectly by changing the phenotype

Corticosteroids as risk factor for COVID-19-associated pulmonary aspergillosis in intensive care patients

Purpose: Corticosteroids, in particular dexamethasone, are one of the primary treatment options for critically ill COVID-19 patients. However, there are a growing number of cases that involve COVID-19-associated pulmonary aspergillosis (CAPA), and it is unclear whether dexamethasone represents a risk factor for CAPA. Our aim was to investigate a possible association of the recommended dexamethasone therapy with a risk of CAPA. Methods: We performed a study based on a cohort of COVID-19 patients treated in 2020 in our 13 intensive care units at Charite Universitatsmedizin Berlin. We used ECMM/ISHM criteria for the CAPA diagnosis and performed univariate and multivariable analyses of clinical parameters to identify risk factors that could result in a diagnosis of CAPA. Results: Altogether, among the n = 522 intensive care patients analyzed, n = 47 (9%) patients developed CAPA. CAPA patients had a higher simplified acute physiology score (SAPS) (64 vs. 53, p < 0.001) and higher levels of IL-6 (1,005 vs. 461, p < 0.008). They more often had severe acute respiratory distress syndrome (ARDS) (60% vs. 41%, p = 0.024), renal replacement therapy (60% vs. 41%, p = 0.024), and they were more likely to die (64% vs. 48%, p = 0.049). The multivariable analysis showed dexamethasone (OR 3.110, CI95 1.112-8.697) and SAPS (OR 1.063, CI95 1.028-1.098) to be independent risk factors for CAPA. Conclusion: In our study, dexamethasone therapy as recommended for COVID-19 was associated with a significant three times increase in the risk of CAPA

A Practical Guide to the Automated Analysis of Vascular Growth, Maturation and Injury in the Brain

The distinct organization of the brain’s vasculature ensures the adequate delivery of oxygen and nutrients during development and adulthood. Acute and chronic pathological changes of the vascular system have been implicated in many neurological disorders including stroke and dementia. Here, we describe a fast, automated method that allows the highly reproducible, quantitative assessment of distinct vascular parameters and their changes based on the open source software Fiji (ImageJ). In particular, we developed a practical guide to reliably measure aspects of growth, repair and maturation of the brain’s vasculature during development and neurovascular disease in mice and humans. The script can be used to assess the effects of different external factors including pharmacological treatments or disease states. Moreover, the procedure is expandable to blood vessels of other organs and vascular in vitro models. © Copyright © 2020 Rust, Kirabali, Grönnert, Dogancay, Limasale, Meinhardt, Werner, Laviña, Kulic, Nitsch, Tackenberg and Schwab

Development of a GEM-TPC prototype

The use of GEM foils for the amplification stage of a TPC instead of a con- ventional MWPC allows one to bypass the necessity of gating, as the backdrift is suppressed thanks to the asymmetric field configuration. This way, a novel continuously running TPC, which represents one option for the PANDA central tracker, can be realized. A medium sized prototype with a diameter of 300 mm and a length of 600 mm will be tested inside the FOPI spectrometer at GSI using a carbon or lithium beam at intermediate energies (E = 1-3AGeV). This detector test under realistic experimental conditions should allow us to verify the spatial resolution for single tracks and the reconstruction capability for displaced vertexes. A series of physics measurement implying pion beams is scheduled with the FOPI spectrometer together with the GEM-TPC as well.Comment: 5 pages, 4 figures, Proceedings for 11th ICATTP conference in como (italy