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NASA NTRS2004

Microgravity and Cellular Consequences in Lymphocyte Function

Mammalian cells adapt to the environment of low gravity and express a series of responses, some possibly from direct effects on cells and others based on environmental conditions created by microgravity. Human lymphocytes in microgravity culture are functionally diminished in activation and locomotion. Both processes are integral to optimal immune response to fight pathogens. The NASA Rotating-wall vessel (RWV) is a well-accepted analog for microgravity culture on the ground. Gene array experiments and immunoblotting identified upstream events in human lymphocytes adapting to microgravity analog culture. Microgravity induces selective changes, many of which are cell membrane related. Results showed that upstream of PKC in the T cell activation cascade, PLC-gamma and LAT are significantly diminished. ZAP 70 which controls LAT activation is also down regulated in modeled microgravity. Thus events governing cell shape might warrant attention in microgravity conditions. The goal of this study is to delineate response suites that are consequential, direct or indirect effects of the microgravity environment and which of these are essential to lymphocytes

Pellis, Neal R.↗

NASA NTRS2002

Neurovestibular modulation of circadian and homeostatic regulation: vestibulohypothalamic connection?

Chronic exposure to increased force environments (+G) has pronounced effects on the circadian and homeostatic regulation of body temperature (T(b)), ambulatory activity (Act), heart rate, feeding, and adiposity. By using the Brn 3.1 knockout mouse, which lacks vestibular hair cells, we recently described a major role of the vestibular system in mediating some of these adaptive responses. The present study used the C57BL6JEi-het mouse strain (het), which lacks macular otoconia, to elucidate the contribution of specific vestibular receptors. In this study, eight het and eight WT mice were exposed to 2G for 8 weeks by means of chronic centrifugation. In addition, eight het and eight WT mice were maintained as 1G controls in similar conditions. Upon 2G exposure, the WT exhibited a decrease in T(b) and an attenuated T(b) circadian rhythm. Act means and rhythms also were attenuated. Body mass and food intake were significantly lower than the 1G controls. After 8 weeks, percent body fat was significantly lower in the WT mice (P < 0.0001). In contrast, the het mice did not exhibit a decrease in mean T(b) and only a slight decrease in T(b) circadian amplitude. het Act levels were attenuated similarly to the WT mice. Body mass and food intake were only slightly attenuated in the het mice, and percent body fat, after 8 weeks, was not different in the 2G het group. These results link the vestibular macular receptors with specific alterations in homeostatic and circadian regulation.

NASA Discipline Regulatory Physiology↗

NASA NTRS1999

Lymphocyte Functions in Space - Related Conditions

Our previous studies showed that modeled (MMG) and true (STS-54 and STS-56) microgravity (MG) inhibit human lymphocyte locomotion. MMG also suppresses polyclonal and antigen-specific lymphocyte activation. Analysis of the relationship between activation deficits and the loss of locomotion in MG suggested a fundamental defect in signal transduction mechanism localized either at the PKC level or upstream at the cell membrane. FACS analysis of the expression of PKC isoforms in PBMC revealed that MMG selectively inhibits the PKC isoforms expression. The decrease was most prominent in PKC epsilon, less obvious in PKC delta and almost marginal and insignificant in PKC alpha. Western blot analysis confirmed these results (PKC epsilon protein expression was downregulated at 24, 72 and 96 hours in MG). We also found a decrease in PKC epsilon mRNA expression. MMG inhibited programmed cell death (PCD) in lymphocytes. Inhibition was observed in two types of experiments: 1) when PCD was induced by gamma-radiation of PBMC, and 2) when PCD in activated T cells was triggered by PHA-M or PMA + ionomycin restimulation. The established direct effects of MG on signal transduction mechanisms as well as on PCD in lymphocytes could contribute to the impairment of the immunity in space.

Risin, D.↗

NASA NTRS2003

Effects of broad frequency vibration on cultured osteoblasts

Bone is subjected in vivo to both high amplitude, low frequency strain, incurred by locomotion, and to low amplitude, broad frequency strain. The biological effects of low amplitude, broad frequency strain are poorly understood. To evaluate the effects of low amplitude strains ranging in frequency from 0 to 50 Hz on osteoblastic function, we seeded MC3T3-E1 cells into collagen gels and applied the following loading protocols for 3 min per day for either 3 or 7 days: (1) sinusoidal strain at 3 Hz, with 0-3000 microstrain peak-to-peak followed by 0.33 s resting time, (2) "broad frequency vibration" of low amplitude strain (standard deviation of 300 microstrain) including frequency components from 0 to 50 Hz, and (3) sinusoidal strain combined with broad frequency vibration (S + V). The cells were harvested on day 4 or 8. We found that the S + V stimulation significantly repressed cell proliferation by day 8. Osteocalcin mRNA was up-regulated 2.6-fold after 7 days of S + V stimulation, and MMP-9 mRNA was elevated 1.3-fold after 3 days of vibration alone. Sinusoidal stimulation alone did not affect the cell responses. No differences due to loading were observed in alkaline phosphatase activity and in mRNA levels of type I collagen, osteopontin, connexin 43, MMPs-1A, -3, -13. These results suggest that osteoblasts are more sensitive to low amplitude, broad frequency strain, and this kind of strain could sensitize osteoblasts to high amplitude, low frequency strain. This suggestion implies a potential contribution of stochastic resonance to the mechanical sensitivity of osteoblasts. Copyright 2002 Elsevier Science Ltd.

Non-NASA Center↗

NASA NTRS1992

The nucleus of the optic tract. Its function in gaze stabilization and control of visual-vestibular interaction

1. Electrical stimulation of the nucleus of the optic tract (NOT) induced nystagmus and after-nystagmus with ipsilateral slow phases. The velocity characteristics of the nystagmus were similar to those of the slow component of optokinetic nystagmus (OKN) and to optokinetic after-nystagmus (OKAN), both of which are produced by velocity storage in the vestibular system. When NOT was destroyed, these components disappeared. This indicates that velocity storage is activated from the visual system through NOT. 2. Velocity storage produces compensatory eye-in-head and head-on-body movements through the vestibular system. The association of NOT with velocity storage implies that NOT helps stabilize gaze in space during both passive motion and active locomotion in light with an angular component. It has been suggested that "vestibular-only" neurons in the vestibular nuclei play an important role in generation of velocity storage. Similarities between the rise and fall times of eye velocity during OKN and OKAN to firing rates of vestibular-only neurons suggest that these cells may receive their visual input through NOT. 3. One NOT was injected with muscimol, a GABAA agonist. Ipsilateral OKN and OKAN were lost, suggesting that GABA, which is an inhibitory transmitter in NOT, acts on projection pathways to the brain stem. A striking finding was that visual suppression and habituation of contralateral slow phases of vestibular nystagmus were also abolished after muscimol injection. The latter implies that NOT plays an important role in producing visual suppression of the VOR and habituating its time constant. 4. Habituation is lost after nodulus and uvula lesions and visual suppression after lesions of the flocculus and paraflocculus. We postulate that the disappearance of vestibular habituation and of visual suppression of vestibular responses after muscimol injections was due to dysfacilitation of the prominent NOT-inferior olive pathway, inactivating climbing fibers from the dorsal cap to nodulouvular and flocculoparafloccular Purkinje cells. The prompt loss of habituation when NOT was inactivated, and its return when the GABAergic inhibition dissipated, suggests that although VOR habituation can be relatively permanent, it must be maintained continuously by activity of the vestibulocerebellum.

Review↗

NASA NTRS1999

PKC Isoform Expression in Modeled Microgravity

Our previous studies showed that modeled (MMG) and true (USA Space Shuttle Missions STS-54 and STS-56) microgravity (MG) inhibit human lymphocyte locomotion, Modeled MG also suppressed polyclonal and antigen-specific lymphocyte activation. Activation of PKC by phorbol myristate acetate (PMA) restored the microgravity-inhibited lymphocyte locomotion as well as activation by phytohaemagglutinin (PHA), whereas calcium ionophore (ionomycin) was unable to restore these functions. Based on these results we hypothesized that MG-induced changes in lymphocyte functions are caused by a fundamental defect in signal transduction mechanism. This defect may be localized either at the PKC level or upstream of PKC, most likely, at the cell membrane level. In this study we examined the expression of PKC isoforms alpha, epsilon and delta in PBMC cultured in rotating wall vessel bioreactor, developed at NASA JSC, which models microgravity by sustaining cells in continuous free fall. The assessment of the isoforms was performed by FACS analysis following cell permeabilization. A decrease in the expression of isoforms epsilon and delta, but not isoform a, was observed in PBMC cultured in microgravity conditions. These data suggest that MMG might selectively affect the expression of Ca2+ independent isoforms of PKC Molecular analysis confirm selective suppression of Ca2+ independent isoforms of PKC.

Risin, Diana↗

NASA NTRS2006

Microgravity and Immunity: Changes in Lymphocyte Gene Expression

Earlier studies had shown that modeled and true microgravity (MG) cause multiple direct effects on human lymphocytes. MG inhibits lymphocyte locomotion, suppresses polyclonal and antigen-specific activation, affects signal transduction mechanisms, as well as activation-induced apoptosis. In this study we assessed changes in gene expression associated with lymphocyte exposure to microgravity in an attempt to identify microgravity-sensitive genes (MGSG) in general and specifically those genes that might be responsible for the functional and structural changes observed earlier. Two sets of experiments targeting different goals were conducted. In the first set, T-lymphocytes from normal donors were activated with antiCD3 and IL2 and then cultured in 1g (static) and modeled MG (MMG) conditions (Rotating Wall Vessel bioreactor) for 24 hours. This setting allowed searching for MGSG by comparison of gene expression patterns in zero and 1 g gravity. In the second set - activated T-cells after culturing for 24 hours in 1g and MMG were exposed three hours before harvesting to a secondary activation stimulus (PHA) thus triggering the apoptotic pathway. Total RNA was extracted using the RNeasy isolation kit (Qiagen, Valencia, CA). Affymetrix Gene Chips (U133A), allowing testing for 18,400 human genes, were used for microarray analysis. In the first set of experiments MMG exposure resulted in altered expression of 89 genes, 10 of them were up-regulated and 79 down-regulated. In the second set, changes in expression were revealed in 85 genes, 20 were up-regulated and 65 were down-regulated. The analysis revealed that significant numbers of MGS genes are associated with signal transduction and apoptotic pathways. Interestingly, the majority of genes that responded by up- or down-regulation in the alternative sets of experiments were not the same, possibly reflecting different functional states of the examined T-lymphocyte populations. The responder genes (MGSG) might play an essential role in adaptation to MG and/or be responsible for pathologic changes encountered in Space and thus represent potential targets for molecular-based countermeasures

Risin, D.↗

NASA NTRS2020

Robotic Specialization in Autonomous Robotic Structural Assembly

Robotic in-space assembly of large space structures is a long-term NASA goal to reduce launch costs and enable larger scale missions. Recently, researchers have proposed using discrete lattice building blocks and co-designed robots to build high-performance, scalable primary structure for various on-orbit and surface applications. These robots would locomote on the lattice and work in teams to build and reconfigure building-blocks into functional structure. However, the most reliable and efficient robotic system architecture, characterized by the number of different robotic 'species' and the allocation of functionality between species, is an open question. To address this problem, we decompose the robotic building-block assembly task into functional primitives and, in simulation, study the performance of the the variety of possible resulting architectures. For a set consisting of five process types (move self, move block, move friend, align bock, fasten block), we describe a method of feature space exploration and ranking based on energy and reliability cost functions. The solution space is enumerated, filtered for unique solutions, and evaluated against energy and reliability cost functions for various simulated build sizes. We find that a 2 species system, dividing the five mentioned process types between one unit cell transport robot and one fastening robot, results in the lowest energy cost system, at some cost to reliability. This system enables fastening functionality to occupy the build front while reducing the need for that functional mass to travel back and forth from a feed station. Because the details of a robot design affect the weighting and final allocation of functionality, a sensitivity analysis was conducted to evaluate the effect of changing mass allocations on architecture performance. Future systems with additional functionalities such as repair, inspection, and others may use this process to analyze and determine alternative robot architectures.

Bernus, Borbala↗

NASA NTRS2002

Tensile Properties of Hydrogels and of Snake Skin

Stimulus-responsive or 'smart' gels are of potential interest as sensors and actuators, in industrial separations, and as permeable delivery systems. In most applications, a certain degree of mechanical strength and toughness will be required, yet the large-strain behavior of gels has not been widely reported. Some exceptions include work on gelatin and other food gels, some characterization of soft gels applicable for in-vitro cell growth studies, and toughness determinations on commercial contact lens materials. In general, it can be anticipated that the gel stiffness will increase with increasing degree of crosslinking, but the tensile strength may go through a maximum. Gel properties can be tailored by varying not only the degree of crosslinking, but also the polymer concentration and the nature of the polymer backbone (e.g. its stiffness or solubility). Polypeptides provide an especially interesting case, where secondary structure affects trends in moduli and conformational transitions may accompany phase changes. A few papers on the tensile properties of responsive gels have begun to appear. The responsive hydrogel chosen for the present study, crosslinked hydroxypropylcellulose, shrinks over a rather narrow temperature range near 44 C. Some vertebrate skin is also subject to substantial strain. Among reptiles, the morphologies of the skin and scales show wide variations. Bauer et al. described the mechanical properties and histology of gecko skin; longitudinal tensile properties of snake skin were examined by Jayne with reference to locomotion. The present measurements focus on adaptations related to feeding, including the response of the skin to circumferential tension. Tensile properties will be related to interspecific and regional variation in skin structure and folding.

Hinkley, Jeffrey A.↗