Hypergravity attenuates Reactivity in Primary Murine Astrocytes

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2g and 10g). We revealed spreading rates, migration velocities, and stellation to be diminished under 2g hypergravity. In contrast, proliferation and apoptosis rates were not affected. In particular, hypergravity attenuated reactivity induction. We observed cytoskeletal remodeling of actin filaments and microtubules under hypergravity. Hence, the reorganization of these key elements of cell structure demonstrates that fundamental mechanisms on shape and mobility of astrocytes are affected due to altered gravity conditions. In future experiments, potential target molecules for pharmacological interventions that attenuate astrocytic reactivity will be investigated. The ultimate goal is to enhance neuronal regeneration for novel therapeutic approache

Hypergravity attenuates Reactivity in Primary Murine Astrocytes

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning and memory as well as motion. Alterations or even disruptions of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we use increased gravitational (mechanical) loading induced by hypergravity to identify a potential method to modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the reactivity phenotype in vivo on custom-built centrifuges for cultivation as well as for livecell imaging under hypergravity conditions in a physiological range (2g and 10g). This resulted in significant changes to astrocyte morphology, behavior and reactivity phenotypes, with the ultimate goal being to enhance neuronal regeneration for novel therapeutic approaches

Microelectrode Array Electrophysiological Recording of Neuronal Network Activity during a Short-Term Microgravity Phase

During spaceflight, humans are subjected to a variety of environmental factors which deviate from Earth conditions. Especially the lack of gravity poses a big challenge to the human body and has been identified as a major trigger of many detrimental effects observed in returning astronauts but also in participants of spaceflight-analog studies. Structural alterations within the brain as well as declines in cognitive performance have been reported, which has brought the topic of brain health under microgravity into the focus of space research. However, the physiological mechanisms underlying these observations remain elusive. Every aspect of human cognition, behavior and psychomotor function is processed by the brain based on electro-chemical signals of billions of neurons, which relay information via neuronal networks throughout the body. Alterations in neuronal activity are the main cause of a variety of mental disorders and changed neuronal transmission may also lead to diminished human performance in space. Thus, understanding the functioning of these fundamental processes under the influence of altered gravity conditions on a cellular level is of high importance for any manned space mission. Previous electrophysiological experiments using patch clamp have shown that propagation velocity of action potentials (APs) is dependent on gravity. With this project, we aim to advance the electrophysiological approach from a single-cell level to a complex network level by employing Microelectrode array (MEA) technology. MEAs feature the advantage of real-time electrophysiological recording of a complex and mature neuronal network in vitro, without the need for invasive patch clamp insertion into cells. Using a custom-built pressure chamber, we were able to integrate and conduct our experiment on the ZARM Drop Tower platform, exposing the entire system to 4.7 s of high-quality microgravity (10-6 to 10-5 x g0). With this setup we were able to evaluate the functional activity patterns of iPSC-derived neuronal networks subjected to microgravity, while keeping them under controlled and stable temperature and pressure conditions. Activity data was acquired constantly - immediately before the drop, during the free-fall (microgravity) phase and during a subsequent post-drop recording phase. For neuronal activity analysis the action potential frequency in each experiment phase was calculated for the single electrodes. We found that during the 4.7 s lasting microgravity phase the mean action potential frequency across the neuronal networks was significantly elevated. Additionally, electrical activity readapted back to baseline level within 10 minutes of post-drop recordings. Our preliminary data shows that real-time, electrophysiological recording of neuronal network activity based on MEA technology is possible under altered gravity conditions and that differences in activity can be detected already in very short time frames in the second range. Furthermore, the observation that microgravity has an effect on the electrophysiological activity of neuronal networks is in line with previously published findings in single neurons and poses further questions with regards to astronaut brain health on manned space missions. The MEA payload system was approved for autonomous recording of redundant cellular electrophysiological data in the Drop Tower. It will be applied on other microgravity platforms such as sounding rockets and parabolic flights and thus increased experimental time. Apart from neurons, various other electrically active cellular systems such as myocytes or myotubes could be examined using this hardware

Classification of current anticancer immunotherapies

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into “passive” and “active” based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Hypergravity Reduces Astrocyte Migration by Altering Cytoskeletal Dynamics

Glial scar formation through astrocytes impairs neural regeneration following spinal cord injury, head trauma, or stroke. Astrocyte migration towards the lesion and induction of a reactive astroglial phenotype require dynamic cytoskeletal protein rearrangements. In this glial cell model, hypergravity was used to alter cytoskeletal dynamics, i.e. stabilizing microtubules while destabilizing actin filaments. We hypothesize that increased gravitational (mechanical) loading by means of centrifugation (hypergravity) modulates in vitro astrocyte function in a way that could reduce their potential for scar formation. We exposed primary murine cortical astrocytes to 2g using two types of custom-designed hypergravity platforms at DLR (Cologne, Germany) and assessed a variety of parameters important for glial scarring in vitro. The platforms unlike commercial laboratory centrifuges model physiological hypergravity and allow for cell cultivation and live-cell imaging. Primary astrocytes were isolated from wildtype (C57BL/6J) as well as transgenic LifeAct-GFP mice and subjected to increased gravitational load. We measured cell proliferation and survival, after 7 days of exposure to 2g, as well as spreading and migration rate online for 24h. We visualized morphological features, cytoskeletal actin filament dynamics, reactivity markers and investigated expression levels of focal adhesion-related proteins. The exposure to 2g hypergravity induced a decrease in cell spreading (20%) coincidental with an inhibited migratory behavior (40%) and altered cytoskeletal dynamics. Astrocytic proliferation and survival were not affected. The expression of the focal adhesion marker vinculin was increased by 70–80%. We conclude that hypergravity attenuates astrocyte spreading and migration. These parameters are crucial for glial scar formation, while basic cellular processes, such as proliferation and apoptosis were unchanged. The response appears to be mediated through altered cytoskeletal dynamics and may provide targets for therapies promoting neuronal regeneration

Human neural network activity reacts to gravity changes in vitro

During spaceflight, humans experience a variety of physiological changes due to deviations from familiar earth conditions. Specifically, the lack of gravity is responsible for many effects observed in returning astronauts. These impairments can include structural as well as functional changes of the brain and a decline in cognitive performance. However, the underlying physiological mechanisms remain elusive. Alterations in neuronal activity play a central role in mental disorders and altered neuronal transmission may also lead to diminished human performance in space. Thus, understanding the influence of altered gravity at the cellular and network level is of high importance. Previous electrophysiological experiments using patch clamp techniques and calcium indicators have shown that neuronal activity is influenced by altered gravity. By using multi-electrode array (MEA) technology, we advanced the electrophysiological investigation covering single-cell to network level responses during exposure to decreased (micro-) or increased (hyper-) gravity conditions. We continuously recorded in real-time the spontaneous activity of human induced pluripotent stem cell (hiPSC)-derived neural networks in vitro. The MEA device was integrated into a custom-built environmental chamber to expose the system with neuronal cultures to up to 6 g of hypergravity on the Short-Arm Human Centrifuge at the DLR Cologne, Germany. The flexibility of the experimental hardware set-up facilitated additional MEA electrophysiology experiments under 4.7 s of high-quality microgravity (10–6 to 10–5 g) in the Bremen drop tower, Germany. Hypergravity led to significant changes in activity. During the microgravity phase, the mean action potential frequency across the neural networks was significantly enhanced, whereas different subgroups of neurons showed distinct behaviors, such as increased or decreased firing activity. Our data clearly demonstrate that gravity as an environmental stimulus triggers changes in neuronal activity. Neuronal networks especially reacted to acute changes in mechanical loading (hypergravity) or de-loading (microgravity). The current study clearly shows the gravity-dependent response of neuronal networks endorsing the importance of further investigations of neuronal activity and its adaptive responses to micro- and hypergravity. Our approach provided the basis for the identification of responsible mechanisms and the development of countermeasures with potential implications on manned space missions