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Introduction A functional vascular barrier controlling leukocyte recruitment into the perivascular space relies on an intact endothelial glycocalyx (EGX). Critical disease states such as sepsis or trauma can induce massive shedding of EGX components into the blood stream. Previous studies have shown that high blood levels of EGX components are correlated with bleeding in patients. The mechanism behind that observation remains to be fully elucidated. Material and methods The EGX components syndecan-1 (S1), hyaluronic acid (HA) and heparan sulfate (HS) were added to blood samples of 10 healthy male volunteers separately in three distinct concentrations to mimic three severity levels of in vitro EGX shedding. We analyzed spiked blood samples for leukocyte derived reactive oxygen species (ROS) release as a measure for innate immune activation and evaluated the impact on coagulation using clinical standard coagulation tests (SCTs) as well as rotational thrombelastometry (ROTEM®). Results Whereas ROS formation by polymorphonuclear leukocytes (PMN) was unaltered by all three substances, high concentrations of HS showed prolonged aPTT and TT compared to controls and S1 or HA. Changes in ROTEM® were discrete and mostly within normal range of values but analyses showed a significant reduction of clot firmness and formation by all EGX components compared to controls. Furthermore, alterations by HA and HS were dose dependent. Only HS showed a heparin like effect supporting the findings of SCTs. Conclusions All EGX components interfere with clot formation and strength. HS mimics heparin effects in ROTEM® that confirm detectable alterations of standard coagulation tests.

This study investigates the longitudinal course of blood-based biomarkers representing the brain parenchyma in long-duration spaceflight.

Background Antarctica challenges human explorers by its extreme environment. The effects of these unique conditions on the human physiology need to be understood to best mitigate health problems in Antarctic expedition crews. Moreover, Antarctica is an adequate Earth-bound analogue for long-term space missions. To date, its effects on human physiology have been studied mainly in male cohorts though more female expeditioners and applicants in astronaut training programs are selected. Therefore, the identification of sex differences in stress and immune reactions are becoming an even more essential aim to provide a more individualized risk management. Methods Ten female and 16 male subjects participated in three 1-year expeditions to the German Antarctic Research Station Neumayer III. Blood, saliva, and urine samples were taken 1–2 months prior to departure, subsequently every month during their expedition, and 3–4 months after return from Antarctica. Analyses included cortisol, catecholamine and endocannabinoid measurements; psychological evaluation; differential blood count; and recall antigen- and mitogen-stimulated cytokine profiles. Results Cortisol showed significantly higher concentrations in females than males during winter whereas no enhanced psychological stress was detected in both sexes. Catecholamine excretion was higher in males than females but never showed significant increases compared to baseline. Endocannabinoids and N-acylethanolamides increased significantly in both sexes and stayed consistently elevated during the confinement. Cytokine profiles after in vitro stimulation revealed no sex differences but resulted in significant time-dependent changes. Hemoglobin and hematocrit were significantly higher in males than females, and hemoglobin increased significantly in both sexes compared to baseline. Platelet counts were significantly higher in females than males. Leukocytes and granulocyte concentrations increased during confinement with a dip for both sexes in winter whereas lymphocytes were significantly elevated in both sexes during the confinement. Conclusions The extreme environment of Antarctica seems to trigger some distinct stress and immune responses but—with the exception of cortisol and blood cell counts—without any major relevant sex-specific differences. Stated sex differences were shown to be independent of enhanced psychological stress and seem to be related to the environmental conditions. However, sources and consequences of these sex differences have to be further elucidated.

Background Antarctica is a challenging environment for humans. It serves as a spaceflight ground analog, reflecting some conditions of long-duration exploration class space missions. The French-Italian Concordia station in interior Antarctica is a high-fidelity analog, located 1000 km from the coast, at an altitude of 3232 m. The aim of this field study was to characterize the extent, dynamics, and key mechanisms of the immune adaptation in humans overwintering at Concordia for 1 year. Methods This study assessed immune functions in fourteen crewmembers. Quantitative and phenotypic analyses from human blood were performed using onsite flow cytometry together with specific tests on receptor-dependent and receptor-independent functional innate and adaptive immune responses. Transcriptome analyses and quantitative identification of key response genes were assessed. Results Dynamic immune activation and a two-step escalation/activation pattern were observed. The early phase was characterized by moderately sensitized global immune responses, while after 3-4 months, immune responses were highly upregulated. The cytokine responses to an ex vivo stimulation were markedly raised above baseline levels. These functional observations were reflected at the gene transcriptional level in particular through the modulation of hypoxia-driven pathways. Conclusions This study revealed unique insights into the extent, dynamics, and genetics of immune dysfunctions in humans exposed for 1 year to the Antarctic environment at the Concordia station. The scale of immune function was imbalanced toward a sensitizing of inflammatory pathways.

International audience; Immune dysregulation is among the main adverse outcomes of spaceflight. Despite the crucial role of the antibody repertoire in host protection, the effects of spaceflight on the human antibody repertoire are unknown. Consequently, using high‐throughput sequencing, we examined the IgM repertoire of five cosmonauts 25 days before launch, after 64 ± 11 and 129 ± 20 days spent on the International Space Station (ISS), and at 1, 7, and 30 days after landing. This is the first study of this kind in humans. Our data revealed that the IgM repertoire of the cosmonauts was different from that of control subjects (n = 4) prior to launch and that two out the five analyzed cosmonauts presented significant changes in their IgM repertoire during the mission. These modifications persisted up to 30 days after landing, likely affected the specificities of IgM binding sites, correlated with changes in the V(D)J recombination process responsible for creating antibody genes, and coincided with a higher stress response. These data confirm that the immune system of approximately half of the astronauts who spent 6 months on the ISS is sensitive to spaceflight conditions, and reveal individual responses indicating that personalized approaches should be implemented during future deep‐space exploration missions that will be of unprecedented durations

Exposure to stressful environments weakens immunity evidenced by a detectable reactivation of dormant viruses. The mechanism behind this observation remains unclear. We performed next generation sequencing from RNA extracted from blood samples of 8 male subjects collected before, during and after a 12-month stay at the Antarctic station Concordia. RNA-seq data analysis was done using QIAGEN Ingenuity Pathway Analysis (IPA) software. Data revealed the inactivation of key immune functions such as chemotaxis and leukocyte recruitment which persisted after return. Next to the activation of the stress response eIF2 pathway, interferon signaling was predicted inactivated due to a downregulation of 14 downstream genes involved in antiviral immunity. Among them, the interferon stimulated genes (ISGs) IFITM2 and 3 as well as IFIT3 exhibited the strongest fold changes and IFIT3 remained downregulated even after return. Impairment of antiviral immunity in winter-over crew can be explained by the downregulation of a battery of ISGs., Highlights • Whole blood transcriptome analysis during 12-months of isolation in the Antarctic. • Data show an inactivation of key immune functions and pathways without recovery. • The IFN pathway is most affected showing a downregulation of 14 downstream genes. • The results suggest impairment of antiviral immunity and vulnerability to infection.

Horizons Autonome Assistenzsysteme mit künstlicher Intelligenz sind heute technisch möglich. Durch solche Assistenten sind Effizienzsteigerungen zu erwarten. Ein solches System mit Sprachsteuerung und Gesichtserkennung soll als Experiment auf der Internationalen Raumstation ISS während der Mission „horizons“ vom deutschen ESA-Astronauten Alexander Gerst getestet werden. Beim Alltag auf der ISS unterliegen die Astronauten 6 Monate durch Isolation und Schwerelosigkeit besonderem Stress. In medizinischer Hinsicht ist dies beim Zusammenspiel zwischen Mensch und Maschine von Interesse sowie die Akzeptanz und mögliche zukünftige Anwendungen. Das Experiment CIMON wurde von Airbus Defence and Space entwickelt und gebaut, im Auftrag des DLR-Raumfahrtmanagements.

Host defense is key to survival of every living organism. The innate immune system, the evolutionarily oldest form of defense against invading pathogens, is present in all life and the result of long years of adaptation to the environment. A fast, less-specific reply to microbial threats using cellular and humoral components without the build-up of an immunological memory are its features. As we progress exploring the universe, manned space missions will become even more frequent and longer in duration. We are striving to explore the possibilities of permanently manned space stations like the International Space Station or the planned “Deep Space Gateway” or just “Gateway” as well as further missions to Earth’s moon and Mars. Astronauts are living and working in a uniquely stressful environment. To ensure safe travel and return, it is our duty to protect them from potential hazards and health threats during their adaptation to this new living environment. The first observations gathered during the Apollo missions made it clear that astronauts become more susceptible to infection. The past years of research have identified various immune alterations. The innate system’s central mechanisms such as the generation of reactive oxygen species, phagocytosis, and regulation of the core body temperature become compromised. Yet little can we explain, and nor can we prevent these changes from happening. Nevertheless, it seems evident that maintaining a well-functioning immune system is the prerequisite for space travel. To further expand our knowledge, more research is necessary. We also need to draw information from terrestrial space analog research such as bed rest studies, parabolic flight campaigns, or Antarctic winter-over. Improving the resources aboard for inflight analyses and encouraging reporting of symptoms and disease will help to develop effective countermeasures.

We are currently entering a new era of manned space flight, which will expand human presence to the moon and Mars as outlined in the roadmaps of the International Space Exploration Coordination Group (ISECG, www.globalspaceexploration.org). This roadmap builds on the vision for “a coordinated human and robotic exploration of our solar system” within this century, and envisions permanent human colonization on the Moon and on our neighboring planet. This goal set by the established and emerging space countries and agencies, as well as by private entrepreneurs, has further fueled the pace and created impact in this new “race to space” to moon again and to Mars, following the first era in exploring the Earth orbit and the landing on the Moon in the past century. These new strategies mandate that we also need to enter a new era of holistic research to better understand human beings subjected to extreme environments for longer periods of time.

Health maintenance and disease prevention in astronauts are of high priority during space missions. To achieve this aim, basic science of molecular biology has to be joined with modern noninvasive tools allowing high-fidelity research on diagnosing and monitoring of the astronauts’ health status. Experts from biology, medicine, and engineering should merge their expertise to enable additive effects for development and improvement of stress and immune-monitoring tools under extreme environmental conditions. This will hereby ensure not only the success of space missions, especially when leaving the Earth orbit in the future, but also will provide important impetus for many other applications on Earth as well.

A prolonged stress burden is known to hamper the efficiency of both the innate and the adaptive immune systems and to attenuate the stress responses by the catecholaminergic and endocannabinoid (EC) systems. Key mechanisms of innate immunity are the eradication of pathogens through phagocytosis and the respiratory burst. We tested the concentration-dependent, spontaneous and stimulated (via TNFα and N-formylmethionine-leucyl-phenylalanine) release of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs) in vitro in response to norepinephrine (NE) and AM1241, a pharmacological ligand for the EC receptor CB2. We evaluated phagocytosis of Dectin-1 ligating zymosan particles and tested the cytokine response against Candida antigen in an in vitro cytokine release assay. Increasing concentrations of NE did not affect phagocytosis, yet stimulated ROS release was attenuated gradually reaching maximum suppression at 500 nM. Adrenergic receptor (AR) mechanisms using non-AR-selective (labetalol) as well as specific α-(prazosin) and β-(propranolol) receptor antagonists were tested. Results show that only labetalol and propranolol were able to recuperate cytotoxicity in the presence of NE, evidencing a β-receptor-mediated effect. The CB2 agonist, AM1241, inhibited phagocytosis at 10 µM and spontaneous peroxide release by PMNs. Use of the inverse CB2 receptor agonist SR144528 led to partial recuperation of ROS production, confirming the functional role of CB2. Additionally, AM1241 delayed early activation of monocytes and induced suppression of IL-2 and IL-6 levels in response to Candida via lower activity of mammalian target of rapamycin (mTOR). These findings provide new insights into key mechanisms of innate immunity under stressful conditions where ligands to the sympatho-adrenergic and EC system are released.

Although immune dysfunction by space conditions has been reported postflight, as well as during ground-based experiments, the cause(s) and nature of the immunological changes are not completely understood. Microgravity has been suggested as one of the factors responsible for the observed immune dysregulation. The goal of this study was to assess immune changes in simulated microgravity (s-μG) using an in vitro cytokine release assay. The effect of s-μG provided by the desktop random positioning machine on cell-mediated immunity was examined by analyzing interleukin 2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 10 (IL-10), in response to immune cell stimulation in whole blood samples (n = 10). Stimuli used were bacterial recall antigens, pokeweed mitogen (PWM), lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM). S-μG caused an overall inhibition of the IL-2 and IFN-γ responses to recall antigen and mitogen stimulation. More specifically, s-μG most strongly influenced the levels of all four cytokines elicited by bacterial recall antigen stimulation. In contrast, HKLM-induced TNF-α secretion was elevated. The average concentrations of TNF-α in response to PWM and LPS and IL-10 release stimulated by PWM, LPS, and HKLM were not significantly altered by s-μG. However, a variable response between individual subjects could be observed. In conclusion, our results demonstrate that the in vitro cytokine release assay can detect gravity-related immune alterations. Furthermore, the use of multiple stimuli and the associated changes in cytokine secretion has the potential to reveal information on the underlying mechanisms affected by s-μG.

Gravitational stress in general and microgravity (µg) in particular are regarded as major stress factors responsible for immune system dysfunction in space. To assess the effects of alternating µg and hypergravity (hyper-g) on immune cells, the attachment of peripheral blood mononuclear cells (PBMCs) to adhesion molecules under flow conditions and the antigen-induced immune activation in whole blood were investigated in parabolic flight (PF). In contrast to hyper-g (1.8 g) and control conditions (1 g), flow and rolling speed of PBMCs were moderately accelerated during µg-periods which were accompanied by a clear reduction in rolling rate. Whole blood analyses revealed a “primed” state of monocytes after PF with potentiated antigen-induced pro-inflammatory cytokine responses. At the same time, concentrations of anti-inflammatory cytokines were increased and monocytes displayed a surface molecule pattern that indicated immunosuppression. The results suggest an immunologic counterbalance to avoid disproportionate immune responses. Understanding the interrelation of immune system impairing and enhancing effects under different gravitational conditions may support the design of countermeasures to mitigate immune deficiencies in space.

Fluid shear stress in the vasculature is the driving force for natural bypass growth, a fundamental endogenous mechanism to counteract the detrimental consequences of vascular occlusive disease, such as stroke or myocardial infarction. This process, referred to as "arteriogenesis," relies on local recruitment of leukocytes, which supply growth factors to preexisting collateral arterioles enabling them to grow. Although several mechanosensing proteins have been identified, the series of mechanotransduction events resulting in local leukocyte recruitment is not understood. In a mouse model of arteriogenesis (femoral artery ligation), we found that endothelial cells release RNA in response to increased fluid shear stress and that administration of RNase inhibitor blocking plasma RNases improved perfusion recovery. In contrast, treatment with bovine pancreatic RNase A or human recombinant RNase1 interfered with leukocyte recruitment and collateral artery growth. Our results indicated that extracellular RNA (eRNA) regulated leukocyte recruitment by engaging vascular endothelial growth factor receptor 2 (VEGFR2), which was confirmed by intravital microscopic studies in a murine cremaster model of inflammation. Moreover, we found that release of von Willebrand factor (VWF) as a result of shear stress is dependent on VEGFR2. Blocking VEGFR2, RNase application, or VWF deficiency interfered with platelet-neutrophil aggregate formation, which is essential for initiating the inflammatory process in arteriogenesis. Taken together, the results show that eRNA is released from endothelial cells in response to shear stress. We demonstrate this extracellular nucleic acid as a critical mediator of mechanotransduction by inducing the liberation of VWF, thereby initiating the multistep inflammatory process responsible for arteriogenesis.

Space flight exerts a specific conglomerate of stressors on humans that can modulate the immune system. The mechanism remains to be elucidated and the consequences for cosmonauts in the long term are unclear. Most of the current research stems from short-term spaceflights as well as pre- and post-flight analyses due to operational limitations. Immune function of 12 cosmonauts participating in a long-duration (>140 days) spaceflight mission was monitored pre-, post-, and on two time-points in-flight. While the classical markers for stress such as cortisol in saliva where not significantly altered, blood concentrations of the endocannabinoid system (ECS) were found to be highly increased in-flight indicating a biological stress response. Moreover, subjects showed a significant rise in white blood cell counts. Neutrophils, monocytes and B cells increased by 50% whereas NK cells dropped by nearly 60% shortly after landing. Analysis of blood smears showed that lymphocyte percentages, though unchanged pre- and post-flight were elevated in-flight. Functional tests on the ground revealed stable cellular glutathione levels, unaltered baseline and stimulated ROS release in neutrophils but an increased shedding of L-selectin post-flight. In vitro stimulation of whole blood samples with fungal antigen showed a highly amplified TNF and IL-1β response. Furthermore, a significant reduction in CD4+CD25+CD27low regulatory T cells was observed post-flight but returned to normal levels after one month. Concomitantly, high in-flight levels of regulatory cytokines TGF-β, IL-10 and IL-1ra dropped rapidly after return to Earth. Finally, we observed a shift in the CD8+ T cell repertoire toward CD8+ memory cells that lasted even one month after return to Earth. Conclusion: Long-duration spaceflight triggered a sustained stress dependent release of endocannabinoids combined with an aberrant immune activation mimicking features of people at risk for inflammation related diseases. These effects persisted in part 30 days after return to Earth. The currently available repertoire of in-flight testing as well as the post-flight observation periods need to be expanded to tackle the underlying mechanism for and consequences of these immune changes in order to develop corresponding mitigation strategies based on a personalized approach for future interplanetary space explorations.

Changes in wall shear stress of blood vessels are assumed to be an important component of many physiological and pathophysiological processes. However, due to technical limitations experimental in vivo data are rarely available. Here, we investigated two-photon excitation fluorescence microscopy as an option to measure vessel diameter as well as blood flow velocities in a murine hindlimb model of arteriogenesis (collateral artery growth). Using line scanning at high frequencies, we measured the movement of blood cells along the vessel axis. We found that peak systolic blood flow velocity averaged 9 mm/s and vessel diameter 42 µm in resting collaterals. Induction of arteriogenesis by femoral artery ligation resulted in a significant increase in centerline peak systolic velocity after 1 day with an average of 51 mm/s, whereas the averaged luminal diameter of collaterals (52 µm) changed much less. Thereof calculations revealed a significant fourfold increase in hemodynamic wall shear rate. Our results indicate that two-photon line scanning is a suitable tool to estimate wall shear stress e.g., in experimental animal models, such as of arteriogenesis, which may not only help to understand the relevance of mechanical forces in vivo, but also to adjust wall shear stress in ex vivo investigations on isolated vessels as well as cell culture experiments.