The Lymphocyte Story – an Overview of Selected Highlights on the in Vitro Activation of Human Lymphocytes in Space

Since the first flight of humans into space it is known that space flight affects the immune system; especially a weakening of the reactivity of T-lymphocytes after flight has been observed. In an in vitro experiment, proposed by Augusto Cogoli and flown in Spacelab-1 in 1983, the activation of T-lymphocytes was found to be strongly inhibited in microgravity. This surprising result triggered extended investigations in space and on the ground by us and other research teams. T-cells are that subpopulation of lymphocytes responsible for the activation of the specific immune system. The mechanism of T-cell activation is very complex; 3 different signals are required as well as an interaction between T-lymphocytes and monocytes. Cell motility based on a continuous rearrangement of the cytoskeletal network within the cell is essential for cell-cell contacts. The objective of all our experiments performed on different platforms in space as well as in simulated microgravity on ground was to understand and explain the dysfunction of the cell activation under reduced gravity conditions. On sounding rockets we have studied the influence of microgravity on the delivery of the first signal, the motility of lymphocytes as well as changes in the cytoskeletal structure and early gene expression. On long term missions we investigated many aspects of the delivery of the 2 ^nd and 3 ^rd signal, including motility and aggregate formation of lymphocytes, interaction of lymphocytes with monocytes, motility of monocytes and changes in different cytoskeletal structures.     

Influence of Fe(II) and Fe(III) on the expression of genes related to cholesterol- and fatty acid metabolism in human vascular smooth muscle cells

Iron is the major alloy component for a large variety of cardiovascular devices such as stents. In recent studies it has been shown that biodegradable iron or iron based stents exhibit good mechanical features with no pronounced neointimal proliferation. Whole genome gene profiling using DNA chip technology revealed that genes involved in cholesterol and fatty acid metabolism (low-density lipoprotein receptor, LDL-R; 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (HMGCS1) and fatty acid desaturase 1 (FADS1) are up-regulated after exposure of vascular smooth muscle cells with soluble ferrous iron. To analyze the effects of iron on these genes in detail we co-incubated human vascular smooth muscle cells for 12 and 24 h with different concentrations of ferrous (soluble iron(II)-gluconate) and ferric iron (soluble iron(III)-chloride), Ferrlecit?, a commercially available drug (ferric iron-gluconate complex) and solid iron coils. The expression of LDL-R, HMGCS1 and FADS1 was analyzed using TaqMan® Real-time PCR. After 24 h, all forms of iron led to a significant up-regulation of the examined genes. At high concentrations the expression rates declined, probably as a result of reduced metabolic activity. The most prominent effects were observed after co-incubation with Ferrlecit?, probably caused by an increased bioavailability of the iron gluconate complex. We postulate that both, bi- and trivalent forms of iron induce the expression of LDL-R, HMGCS1 and FADS1 by generation of highly reactive oxygen species. Further animal experiments using tissues from iron-stented vessels may lead to a more profound insight into iron induced expression of cholesterol- and fatty acid metabolism related genes.     

Space flight and bone formation

Major physiological changes which occur during spaceflight include bone loss, muscle atrophy, cardiovascular and immune response alterations. When trying to determine the reason why bone loss occurs during spaceflight, one must remember that all these other changes in physiology and metabolism may also have impact on the skeletal system. For bone, however, the role of normal weight bearing is a major concern and we have found no adequate substitute for weight bearing which can prevent bone loss. During the study of this problem, we have learned a great deal about bone physiology and increased our knowledge about how normal bone is formed and maintained. Presently, we do not have adequate ground based models which can mimic the tissue loss that occurs in spaceflight but this condition closely resembles the bone loss seen with osteoporosis. Although a normal bone structure will respond to application of mechanical force and weight bearing by forming new bone, a weakened osteoporotic bone may have a tendency to fracture. The study of the skeletal system during weightless conditions will eventually produce preventative measures and form a basis for protecting the crew during long term space flight. The added benefit from these studies will be methods to treat bone loss conditions which occur here on earth.     

Changes in expression of genes involved in apoptosis in activated human T-cells in response to modeled microgravity

Space flights result in remarkable effects on various physiological systems, including a decline in cellular immune functions. Previous studies have shown that exposure to microgravity, both true and modeled, can cause significant changes in numerous lymphocyte functions. The purpose of this study was to search for microgravity-sensitive genes, and specifically for apoptotic genes influenced by the microgravity environment and other genes related to immune response. The experiments were performed on anti-CD3 and IL-2 activated human T cells. To model microgravity conditions we have utilized the NASA rotating wall vessel bioreactor. Control lymphocytes were cultured in static 1g conditions. To assess gene expression we used DNA microarray chip technology. We had shown that multiple genes (approximately 3–8% of tested genes) respond to microgravity conditions by 1.5 and more fold change in expression. There is a significant variability in the response. However, a certain reproducible pattern in gene response could be identified. Among the genes showing reproducible changes in expression in modeled microgravity, several genes involved in apoptosis as well as in immune response were identified. These are IL-7 receptor, Granzyme B, Beta-3-endonexin, Apo2 ligand and STAT1. Possible functional consequences of these changes are discussed.     

From hypergravity to microgravity: Choosing the suitable simulator

Cardiovascular system functions are impaired in altered gravity conditions. In particular endothelial cells play a major role being responsible for the integrity of the vascular wall. Due to obvious difficulties in performing continuous and exhaustive experiments in space, most of the available data have been obtained so far using various simulators of hypergravity and microgravity (µg) conditions. The consistency of the data resides on the reliability of the simulator, being a critical point in the development of the research. We exposed the cell cultures to 1) hypergravity (launch condition) using MidiCAR at Dutch Experiment Support Center (DESC, NL); 2) simulated µg using the Rotating Wall Vessel (RWV) and the Random Positioning Machine (RPM). We used two different cellular models: human umbilical vein endothelial cells (HUVEC) and human leukocytes (U937). Only few experiments on cells using RPM have been reported. To assess the RPM best operative parameters we considered data from experiments in space on U937 as reference standard. Differently, cultures in modelled µg using RWV have been extensively reported. Our data on HUVEC indicate that the two µg simulators provide analogous results in terms of proliferation and cytoskeletal organization. Finally, to investigate the effects of spaceflight on different human cells, we developed a spaceflight-like protocol consisting of an initial hypergravity phase (launch), followed by a µg simulation (orbital flight). Using this protocol, results show that hypergravity limits in our models the effects on proliferation induced by modelled µg.     

Human Reliability Analysis in Spaceflight Applications

Predicting and mitigating human error in manned spaceflight can be the difference between mission success and lost vehicle or crewmember. The National Aeronautics and Space Administration (NASA) has used the Cognitive Reliability Error Analysis Model analysis developed by the nuclear industry during the last 30 years of manned spaceflight to predict human error. Although the analysis has proven to be reliable, it does not take into account operations specific for long duration spaceflight such as crew training and ground support. This article first explains the principles of the Cognitive Reliability Error Analysis Model and how it is used at NASA. Then, the probability for error for an International Space Station ingress procedure is calculated using standard performance shaping factors developed for the nuclear power industry. Lastly, the environmental and operational constraints of space flight are used to develop new performance shaping factors specific to a NASA‐operated spacecraft. Copyright © 2012 John Wiley & Sons, Ltd.     

Preformulation Prodrug Research - Chemical and Enzymatic Hydrolysis Kinetics of the Glycerol, Glycolic Acid and Morpholino Ethyl Ester Derivatives of a Developmental Analgesic Agent (RS-82917)

The chemical and enzymatic hydrolysis of the glycerol-(2), glycolic acid-(3), and morpholino ethyl-(4) ester derivatives of 7-(4-methylthiobenzoyl) benzofuran-5-yl-acedtic acid (RS-82917,1) were evaluated for potential application as prodrugs. In aqueous solution with pH from 1-10, all three ester derivatives hydrolyzed quantitatively to 1. Rate expressions were derived from the log(rate) pH profiles for each ester, and pH-dependent intramolecular catalysis was proposed to account for the hydrolysis kinetics of all three esters. None of the ester aqueous solutions can provide two-year shelf-life at 25°C. The rate of hydrolysis was accelerated significantly by human and mouse plasma enzymes for 2 and 4 and by mouse liver homogenate for 2, 3 and 4.     

Gentamicin: effect on E. coli in space

Previous investigations have shown that liquid bacterial cultures grown in space flight were not killed as effectively by antibiotic treatments as were cultures grown on Earth. However, the cause for the decreased antibiotic effectiveness remains unknown. Possible explanations include modified cell proliferation and modified antibiotic transport in the culture medium. Escherichia coli cultures were grown in space flight (STS-69 and STS-73), with and without gentamicin, on a solid agar substrate thus eliminating fluid effects and reducing the unknowns associated with space-flight bacterial cultures in suspension. This research showed that E. coli cultures grown in flight on agar for 24 to 27 hours experienced a heightened growth compared to simultaneous controls. However, addition of gentamicin to the agar killed the bacteria such that both flight and ground control E. coli samples had similar final cell concentrations. Therefore, while the reported existence of a decrease in antibiotic effectiveness in liquid cultures remains unexplained, these data suggest that gentamicin in space flight was at least as effective as, if not more effective than, on Earth, when E. coli cells were grown on agar.     

Embedding Arabidopsis Plant Cell Suspensions in Low-Melting Agarose Facilitates Altered Gravity Studies

Gravity plays a role in modulating plant growth and development and its alteration induces changes in these processes. Microgravity research has recently been extended to the use of in vitro plant cell cultures which are considered as an ideal model system to study cell proliferation and growth. In general, among the ground-based facilities available for microgravity simulation, the 2D pipette clinostat had been previously considered a suitable facility to be used for unicellular biological models although studies using single plant cell cultures raised some concerns. The incompatibility comes from the standard requirement of shaking a suspension culture for assuring its viability and active proliferation status in the control samples. Moreover, a related issue applies to the use of the random positioning machine (RPM) for cell suspension experiments. Here, we demonstrate an alternative culture method based on the immobilization of the culture before the altered gravity treatment occurs, such that it behaves as a solid object. Our immobilization procedure preserved plant cell culture viability without compromising basic cell properties as viability, morphology, cell cycle phases distribution, or chromatin organization, when compared with a standard cell suspension under shaking as a control. This approach should allow the space biology community to improve the quantity and quality of plant cell results in future simulated microgravity experiments or spaceflight opportunities.     

Biological dosimetry in the ENEIDE Mission on the International Space Station

Space radiation represents one of the major health hazards to crews of interplanetary missions. As the duration of space flight increases, according to International Space Station (ISS) and Mars mission programs, the risk associated with exposure to ionizing radiation also increases. Although physical dosimetry is routinely performed in manned space missions, it is generally accepted that direct measurement of biological endpoints (biological dosimetry) is necessary for a precise assessment of radiation risk in extraterrestrial activities. Chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) are particularly suitable to this purpose, as they can provide estimates of both equivalent radiation dose and risk. In this study, cytogenetic analysis was performed on PBL chromosomes of an Italian astronaut involved in two different 10-day missions on the ISS (Marco Polo, April 2002, and ENEIDE, May 2005). Blood samples were collected before and after flights. CAs were evaluated in either mitotic spreads or in prematurely condensed chromosomes (PCC) by Fluorescence in Situ Hybridization (FISH). In addition, blood samples were exposed to graded doses of X-rays in vitro before and after the flight and cytogenetic damage evaluated to investigate whether the space environment alters the sensitivity of human cells to ionizing radiation. The yield of baseline chromosomal aberrations was not modified following Marco Polo and ENEIDE mission. This is consistent with the low dose absorbed in these short-term space missions. Preliminary results from Marco Polo mission suggested a significant increase in intrinsic radiosensitivity of lymphocytes after landing compared to pre-flight and follow-up (6 months after landing) samples. However, this effect was not observed during the ENEIDE mission. The results suggest that intra-indi-vidual variations in radiosensitivity are significant, but they cannot be related to the space flight.     

人类骨桥蛋白(hOPN)在细胞增殖中的功能研究

为了研究人类骨桥蛋白（hOPN)与293细胞增殖。细胞周期及与细胞周期有关基因表达的关系。并对其机制进行探讨。成功地构建了hOPN真核表达载体并获得了稳定表达hOPN的细胞系，也同时获得了稳定表达EGFP的细胞系，hOPN对293细胞具有促增殖效应，hOPN蛋白激活了293细胞细胞周期蛋白A的表达。实验结果表明：hOPN通过一定的信号通路刺激了细胞周期蛋白A的表达。加快了细胞进入通过S期，从而促进了细胞的增殖。     

The effect of hyaluronan combined with KI3 complex (Hyiodine wound dressing) on keratinocytes and immune cells

Hyiodine (high molecular weight hyaluronan combined with KI₃ complex) is a new non-adhesive wound dressing which significantly improves the healing process. The aim of the study was to investigate the effects of Hyoidine on functional properties of isolated human keratinocytes and leukocytes, and on those of U937 and HL60 cell lines. While KI₃ complex inhibited the viability and proliferation of the cells tested, Hyiodine did not have any significant effect. The expression of CD11b, CD62L and CD69 on PMNL, monocytes and lymphocytes, as well as the oxidative burst of blood neutrophils, were not changed. On the contrary, Hyiodine inhibited the PMA-activated oxidative burst and significantly increased the production of IL-6 and TNF-α by lymphocytes. It was concluded that hyaluronan content of Hyiodine reduces the toxic effect of KI₃ complex on cells and speeds up the wound healing process by increasing the production of inflammatory cytokines.     

Modulation of poly (d,l-lactic acid) with amniotic fluid for improvement of the cell affinity

The objective of this study was to investigate the effects of naturally occurring amniotic fluid modified poly(d,l-lactic acid) (PDLLA) film on the culture of rat calvaria osteoblast. The characteristics of surfaces (both modified and control) were examined by contact angle measurement and electron spectroscopy for chemical analysis (XPS). Cell adhesion and proliferation were used to assess the cell behavior on modified films and control one. MTT assay was used to determine cell viability and alkaline phosphatase (ALP) activity was taken to evaluate differentiated cell function. Compared with the untreated films, cell adhesion of osteoblast was significantly higher (P < 0.05) than that found on control, and osteoblast proliferation was also greater than control one (P < 0.01) at the time interval of 4 and 7 days. Moreover, the alkaline phosphatase (ALP) activity exhibited statistic difference (P < 0.05) and cell viability demonstrated significant difference (P < 0.01) between amniotic fluid modified PDLLA films and control one. These results suggested that amniotic fluid was a suitable material when used to modify PDLLA in order to improve its biocompatibility.     

Synergistic Effects of Incubation in Rotating Bioreactors and Cumulative Low Dose 60Co γ-ray Irradiation on Human Immortal Lymphoblastoid Cells

The complex space environments can influence cell structure and function. The research results on space biology have shown that the major mutagenic factors in space are microgravity and ionizing radiation. In addition, possible synergistic effects of radiation and microgravity on human cells are not well understood. In this study, human immortal lymphoblastoid cells were established from human peripheral blood lymphocytes and the cells were treated with low dose (0.1, 0.15 and 0.2?Gy) cumulative ⁶⁰Co ??-irradiation and simulated weightlessness [obtained by culturing cells in the Rotating Cell Culture System (RCCS)]. The commonly used indexes of cell damage such as micronucleus rate, cell cycle and mitotic index were studied. Previous work has proved that Gadd45 (growth arrest and DNA-damage-inducible protein 45) gene increases with a dose-effect relationship, and will possibly be a new biological dosimeter to show irradiation damage. So Gadd45 expression is also detected in this study. The micronucleus rate and the expression of Gadd45?? gene increased with irradiation dose and were much higher after incubation in the rotating bioreactor than that in the static irradiation group, while the cell proliferation after incubation in the rotating bioreactor decreased at the same time. These results indicate synergetic effects of simulated weightlessness and low dose irradiation in human cells. The cell damage inflicted by ??-irradiation increased under simulated weightlessness. Our results suggest that during medium- and long-term flight, the human body can be damaged by cumulative low dose radiation, and the damage will even be increased by microgravity in space.     

我国航天测控系统的现状与发展

随着我国航天任务的日益复杂化、多样化，对测控的需求也向“高、精、尖”方向发展，航天测控系统再一次面临新的机遇与挑战。在综合分析国内外航天测控发展历程与现状的基础上，总结了航天测控的发展特点与发展趋势，提出了符合我国国情的“优化地基测控网、建设和发展天基测控网、构建深空探测网，循序渐进构建天地空一体化测控通信系统”的整体发展思路，并对我国地基、天基、深空探测测控通信系统发展及重点技术进行了分析探讨。     

Fish Inner Ear Otolith Growth Under Real Microgravity (Spaceflight) and Clinorotation

Using late larval stages of cichlid fish (Oreochromis mossambicus) we have shown earlier that the biomineralization of otoliths is adjusted towards gravity by means of a neurally guided feedback loop. Centrifuge experiments, e.g., revealed that increased gravity slows down otolith growth. Microgravity thus should yield an opposite effect, i.e., larger than normal otoliths. Consequently, late larval cichlids (stage 14, vestibular system operational) were subjected to real microgravity during the 12 days FOTON-M3 spaceflight mission (OMEGAHAB-hardware). Controls were kept at 1g on ground within an identical hardware. Animals of another batch were subsequently clinorotated within a submersed fast-rotating clinostat with one axis of rotation (2d-clinostat), a device regarded to simulate microgravity. Temperature and light conditions were provided in analogy to the spaceflight experiment. Controls were maintained at 1g within the same aquarium. After all experiments, animals had reached late stage 21 (fish can swim freely). Maintenance under real microgravity during spaceflight resulted in significantly larger than normal otoliths (both lapilli and sagittae, involved in sensing gravity and the hearing process, respectively). This result is fully in line with an earlier spaceflight study in the course of which otoliths from late-staged swordtails Xiphophorus helleri were analyzed. Clinorotation resulted in larger than 1g sagittae. However, no effect on lapilli was obtained. Possibly, an effect was present but too light to be measurable. Overall, spaceflight obviously induces an adaptation of otolith growth, whereas clinorotation does not fully mimic conditions of microgravity regarding late larval cichlids.     

Molecular cocrystals of 2-amino-5-chlorobenzooxazole

Three adduct structures of 2-amino-5-chlorobenzooxazole (2-ABOX) have been prepared and studied using single-crystal X-ray techniques. These adduct structures are indole-2-carboxylic acid [(2-ABOX)(ICA)] 1, N-methylpyrrole-2-carboxylic acid [(2-ABOX)(MPCA)] 2 and 3,5-dinitrobenzoic acid [(2-ABOX)(DNBA)] 3. Having previously studied a series of carboxylic acid adducts of 2-aminothiazole derivatives, these current structures were examined to determine the spatial differences between the constituent non-hydrogen atoms of the dominate R₂²(8) graph set hydrogen-bonding association. The average distances between the carboxylate oxygen/heterocyclic nitrogen and carboxylate oxygen/2-amino nitrogen are 2.687(5) Å and 2.796(5) Å, respectively for thirteen 2-aminothiazole adduct structures, and 2.604(8) Å and 2.817(8) Å, respectively for the three 2-ABOX complexes reported in this paper. These distances show that 2-aminooxazole derivatives are dissimilar to their 2-aminothiazole counterparts, in terms of intermolecular associations with carboxylic acids.     

The effects of types of degradable polymers on porcine chondrocyte adhesion, proliferation and gene expression

Understanding how a specific biomaterial may influence chondrocyte adhesion, proliferation and gene expression is important in cartilage tissue engineering. In this study several biodegradable polymers that are commonly used in tissue engineering were evaluated with respect to their influence on chondrocyte attachment, proliferation and gene expression. Primary cultures of porcine chondrocytes were performed in films made of poly-L-lactic acid (PLLA), poly-D,L-lactic acid (PDLLA), poly-(lactide-co-glycolide) (PLGA), or polycaprolactone (PCL). Chondrocytes adhered to PDLLA or PLGA after 1-day incubation better than to PLLA or PCL. After 7 or 14 day culture, the cell numbers on PDLLA or PLGA was still higher than PLLA or PCL. The results suggested that cell attachment and growth might depend on degradation rate of biodegradable polymers. Along with the fact that PDLLA or PLGA supported expression of chondrocyte specific genes more than PLLA or PCL, the former two materials seemed to be more suitable for cartilage tissue engineering than the latter ones. Besides, we found that chondrocyte phenotype prior to seeding was important in the expression of ECM proteins.