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At least 523 records · Page 29
All sourcesNASA NTRSDOE OSTI
NASA NTRS1998

In-Vitro Induced Immunosuppression in a Rotary Cell Culture System

The function of the innate immune system is to provide a first-line of defense against infectious organisms, via control of bacterial and viral growth using antigen nonspecific means. These nonspecific immune effectors include macrophages and Natural Killing (NK) cells, and certain cytokines elicited in response to "super antigens" on the infectious agents. This innate system usually keeps most infectious agents from rapidly growing while the adaptive immune system is generating a specific response complete with immunologic memory. Compelling evidence suggests that space flight results in various immunosuppressive effects, including reduced innate and adaptive immune responses. We were particularly concerned with reduced NK activity at landing, and have asked whether the microgravity component of space flight could be responsible for the previously observed NK defect. We have conclusively demonstrated that simulated microgravity as provided by the Synthecon bioreactors does not inhibit the NK function nor the IL-2 activation of lymphokine-activated killing (LAK). Interleukin-2 is the key cytokine responsible for activation of NK cells to express LAK, as well as to support differentiation of lymphocytes during adaptive immune responses. Therefore, we have disproved our original hypothesis based on poor NK in many of the astronauts upon landing.

Grimm, Elizabeth A.↗
NASA NTRS1999

Reactions and Surface Transformations of a Bone-Bioactive Material in a Simulated Microgravity Environment

A comprehensive program to investigate the expeditious in vitro formation of three-dimensional bone-like tissue is currently underway at the University of Pennsylvania. The study reported here forms a part of that program. Three-dimensional bone-like tissue structures may be grown under the simulated microgravity conditions of NASA designed Rotating Wall Bioreactor Vessels (RWV's). Such tissue growth will have wide clinical applications. In addition, an understanding of the fundamental changes that occur to bone cells under simulated microgravity would yield important information that will help in preventing or minimizing astronaut bone loss, a major health issue with travel or stay in space over long periods of time. The growth of three-dimensional bone-like tissue structures in RWV's is facilitated by the use of microcarriers which provide structural support. If the microcarrier material additionally promotes bone cell growth, then it is particularly advantageous to employ such microcarriers. We have found that reactive, bone-bioactive glass (BBG) is an attractive candidate for use as microcarrier material. Specifically, it has been found that BBG containing Ca- and P- oxides upregulates osteoprogenitor cells to osteoblasts. This effect on cells is preceded by BBG reactions in solution which result in the formation of a Ca-P surface layer. This surface further transforms to a bone-like mineral (i.e., carbonated crystalline hydroxyapatite (c-HA)). At normal gravity, time-dependent, immersion-induced BBG reactions and transformations are greatly affected both by variations in the composition of the milieu in which the glass is immersed and on the immersion conditions. However, the nature of BBG reactions and phase transformations under the simulated microgravity conditions of RWV's are unknown, and must be understood in order to successfully use BBG as microcarrier material in RWV'S. In this paper, we report some of our recent findings in this regard using experimental and numerical methods. BBG composition 45S5, the most reactive among known bone-bioactive glasses, was chosen for the study. BBG 45S5 behavior in physiological solutions was tested in simulated microgravity and compared with that at normal gravity. On the basis of our numerical study, we have chosen the BBG granule size to be in the range 40-70 microns, and a RWV rotational speed of 10 rpm. Our numerical study has shown that these parameters enable the microcarrier to remain suspended in the medium without experiencing collisions with the wall of the vessel. Immersion-induced changes in the solution composition and the material surface were analyzed after immersion.

Radin, S.↗
NASA NTRS1999

A Study of Aberrant Glycosylation in Simulated Microgravity Using Laser Induced AutoFluorescence and Flow Cytometry

A number of pathologies and cellular dysfunctions including neoplasms have been correlated with autofluorescence. The complications of aging and diabetes have been associated with the accumulation of non-enzymatic glycosylations of tissue macromolecules. These products are known as the Advanced Glycosylated End Products (AGEs). A physical property associated with AGEs is the emission of 570 mn or 630 nm light energy (autofluorescence) following the absorption of 448 mm energy associated with the argon laser. This investigation sought to assess the induction of argon-laser induced autofluorescence in a variety of in vitro culture systems. Different fluorescence intensities distinguished tumor lines from normal cell populations. Laser-stimulated autofluorescence discriminated primary cultures of lymphocytes grown in the presence of excess glucose as opposed to normal glucose concentrations. The effects of deglycosylating agents upon laser-induced autofluorescence were also assessed. The studies included studies of cell cycle analysis using Propidium Iodide stained DNA of cells grown in simulated microgravity using NASA Bioreactor Vessels in media of normal and elevated glucose concentrations.

Lawless, B. DeSales↗
NASA NTRS1998

Develop Recovery Systems for Separations of Salts from Process Streams for use in Advanced Life Support System

The main objectives of this project were the development of a four-compartment electrolytic cell using high selective membranes to remove nitrate from crop residue leachate and convert it to nitric acid, and the development of an six compartment electrodialysis cell to remove selectively sodium from urine wastes. The recovery of both plant inedible biomass and human wastes nutrients to sustain a biomass production system are important aspects in the development of a controlled ecological life support system (CELSS) to provide the basic human needs required for life support during long term space missions. A four-compartment electrolytic cell has been proposed to remove selectively nitrate from crop residue and to convert it to nitric acid, which is actually used in the NASA-KSC Controlled Ecological Life Support System to control the pH of the aerobic bioreactors and biomass production chamber. Human activities in a closed system require large amount of air, water and minerals to sustain life and also generate wastes. Before using human wastes as nutrients, these must be treated to reduce organic content and to remove some minerals which have adverse effects on plant growth. Of all the minerals present in human urine, sodium chloride (NACl) is the only one that can not be used as nutrient for most plants. Human activities also requires sodium chloride as part of the diet. Therefore, technology to remove and recover sodium chloride from wastes is highly desirable. A six-compartment electrodialysis cell using high selective membranes has been proposed to remove and recover NaCl from human urine.

Colon, Guillermo↗
NASA NTRS1999

Training Under-Represented Students in Biological Research at Fisk University

The objectives of our training and research project in biology at Fisk are to motivate and train our African-American undergraduate and graduate students by (a) teaching the basic principles and applications of different biological, biochemical and biophysical research techniques; (b) providing a "hands on experience" with laboratory instrumentation (c) requiring the students to participate in the proposed research project entitled "Cyanobacterial Bioreactors for Oxygen and Ammonia Production under "CELSS" Conditions" to gain confidence in independently conducting experiments and (d) providing training in scientific data collection and presentation to peers in scientific conferences or meetings.

Gunasekaran, Muthukumaran↗
NASA NTRS1998

Analysis of proteins involved in biodegradation of crop biomass

The biodegradation of crop biomass for re-use in crop production is part of the bioregenerative life support concept proposed by the National Aeronautics and Space Administration (NASA) for long duration, manned space exploration. The current research was conducted in the laboratory to evaluate the use of electrophoretic analysis as a means of rapidly assaying for constitutive and induced proteins associated with the bacterial degradation of crop residue. The proteins involved in crop biomass biodegradation are either constitutive or induced. As a result, effluent and cultures were examined to investigate the potential of using electrophoretic techniques as a means of monitoring the biodegradation process. Protein concentration for optimum banding patterns was determined using the Bio-Rad Protein Assay kit. Four bacterial soil isolates were obtained from the G.W. Carver research Farm at Tuskegee University and used in the decomposition of components of plant biomass. The culture, WDSt3A was inoculated into 500 mL of either Tryptic Soy Broth or Nutrient Broth. Incubation, with shaking of each flask was for 96 hours at 30 C. The cultures consistently gave unique banding patterns under denaturing protein electrophoresis conditions, The associated extracellular enzymes also yielded characteristic banding patterns over a 14-day period, when native electrophoresis techniques were used to examine effluent from batch culture bioreactors. The current study evaluated sample preparation and staining protocols to determine the ease of use, reproducibility and reliability, as well as the potential for automation.

Crawford, Kamau↗
NASA NTRS1999

Loss of Signal Transduction and Inhibition of Lymphocyte Locomotion in a Ground-Based Model of Microgravity

Inflammatory adherence to, and locomotion through the interstitium is an important component of the immune response. Conditions such as true gravity (TG) and modeled microgravity (MMG) severely inhibit lymphocyte locomotion in vitro through gelled Type I collagen (Pellis et al., 1994, 1997). We used the rotating-wall vessel bioreactor (RWV) as a prototype for modeled microgravity. After observing that lymphocyte locomotion was severely affected in modeled microgravity, we found that polyclonal activation of lymphocytes before exposure to modeled microgravity reversed the locomotion inhibition. Phorbol myristate acetate (PMA) treatment of normal peripheral blood lymphocytes, after exposure to modeled microgravity, restored lymphocyte locomotion by 84%. Calcium ionophore had no effect on modeled microgravity-exposed lymphocytes. Therefore, the signal pathways involving calcium may not be affected by modeled microgravity. However, direct activation of Protein Kinase C (PKC) with PMA was effective in restoring locomotion in modeled microgravity almost comparable to normal levels in lymphocytes cultured in static T flasks. Thus, events either at the level of PKC or upstream are affected by modeled microgravity. Treatment of lymphocytes with mitomycin C prior to exposure to modeled microgravity, followed by PMA, restored locomotion to the same extent as nonmitomycin C-treated lymphocytes exposed to modeled microgravity (80-85%). Therefore 1) new DNA synthesis is not necessary to restore locomotion and 2) traditional activation and locomotion share common pathways up to PKC. Thereafter the signals diverge. Furthermore PMA added immediately before or after initiation of modeled microgravity prevents the loss of lymphocyte locomotion.

Sundaresan, Alamelu↗
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 NTRS1999

Gravitational Effects on Signal Transduction

An understanding of the mechanisms by which individual cells perceive gravity and how these cells transduce and respond to gravitational stimuli is critical for the development of long-term manned space flight experiments. We now propose to use a well-characterized model erythroid cell system and to investigate gravitational perturbations of its erythropoietin (Epo) signaling pathway and gene regulation. Cells will be grown at 1-G and in simulated microgravity in the NASA Rotating Wall Vessel bioreactor (RWV). Cell growth and differentiation, the Epo-receptor, the protein kinase C pathway to the c-myc gene, and the protein phosphatase pathway to the c-myb gene will be studied and evaluated as reporters of gravitational stimuli. The results of these experiments will have impact on the problems of 1) gravitational sensing by individual cells, and 2) the anemia of space flight. This ground-based study also will serve as a Space Station Development Study in gravitational effects on intracellular signal transduction.

Sytkowski, Arthur J.↗
NASA NTRS1997

Endothelial Cell Morphology and Migration are Altered by Changes in Gravitational Fields

Many of the physiological changes of the cardiovascular system during space flight may originate from the dysfunction of basic biological mechanisms caused by microgravity. The weightlessness affects the system when blood and other fluids move to the upper body causing the heart to enlarge to handle the increased blood flow to the upper extremities and decrease circulating volume. Increase arterial pressure triggers baroreceptors which signal the brain to adjust heart rate. Hemodynarnic studies indicate that the microgravity-induced headward fluid redistribution results in various cardiovascular changes such as; alteration of vascular permeability resulting in lipid accumulation in the lumen of the vasculature and degeneration of the the vascular wall, capillary alteration with extensive endothelial invagination. Achieving a true microgravity environment in ground based studies for prolonged periods is virtually impossible. The application of vector-averaged gravity to mammalian cells using horizontal clinostat produces alterations of cellular behavior similar to those observed in microgravity. Similarly, the low shear, horizontally rotating bioreactor (originally designed by NASA) also duplicates several properties of microgravity. Additionally, increasing gravity, i.e., hypcrgravity is easily achieved. Hypergravity has been found to increase the proliferation of several different cell lines (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. The effect of altered gravity on cells maybe similar to those of other physical forces, i.e. shear stress. Previous studies examining laminar flow and shear stress on endothelial cells found that the cells elongate, orient with the direction of flow, and reorganize their F-actin structure, with concomitant increase in cell stiffness. These studies suggest that alterations in the gravity environment will change the behavior of most cells, including vascular cells. However, few studies have been directed at assessing the effect of altered gravitational field on vascular cell fiction and metabolism, Using image analysis we examined how bovine aortic endothelial cells altered their morphological characteristics and their response to a denudation injury when cells were subjected to simulated microgravity and hypergravity.

Melhado, Caroline↗
NASA NTRS2001

Differentiation and Genomic Instability in a Human Mammary Cell Model

Harvest of prophylactic mastectomy specimens from an obligate heterozygote for ataxia-telangiectasia provided autologous fibroblasts as well epithelial cells (HMEC). The routine availability of these autologous cells has provided an opportunity to study cell-cell interactions in coculture and monoculture, and in 3-dimensional cultures grown in the NASA rotating bioreactor. HMEC and stromal fibroblasts grown in 2-dimensional monoculture were both observed to produce extracellular matrix. Similar matrix was encountered in 3-dimensional cultures containing HMEC. Metaphases were analyzed. For stromal fibroblasts, genomic aberrations were found in 18% of metaphase spreads. For HMEC, aberrations were greater such that a majority were found to be abnormal. The level of genomic instability determined for these noncancerous cells in 2-dimensional monoculture should be useful for generating a human cell model that can correlate the effects of differentiation in 3-dimensional coculture on the level of genomic instability.

Richmond, R.↗
NASA NTRS2000

Controlling Microbial Byproducts using Model-Based Substrate Monitoring and Control Strategies

We have developed a computer-controlled bioreactor system to study various aspects of microbially-mediated nitrogen cycling. The system has been used to investigate methods for controlling microbial denitrification (the dissimilatory reduction of nitrate to N2O and N2) in hydroponic plant growth chambers. Such chambers are key elements of advanced life support systems being designed for use on long duration space missions, but nitrogen use efficiency in them is reduced by denitrification. Control software architecture was designed which permits the heterogeneous control of system hardware using traditional feedback control, and quantitative and qualitative models of various system features. Model-based feed forward control entails prediction of future systems in states and automated regulation of system parameters to achieve desired and avoid undesirable system states. A bacterial growth rate model based on the classic Monod model of saturation kinetics was used to evaluate the response of several individual denitrifying species to varying environmental conditions. The system and models are now being applied to mixed microbial communities harvested from the root zone of a hydroponic growth chamber. The use of a modified Monod organism interaction model was evaluated as a means of achieving more accurate description of the dynamic behavior of the communities. A minimum variance parameter estimation routine was also' used to calibrate the constant parameters in the model by iterative evaluation of substrate (nitrate) uptake and growth kinetics. This representation of processes and interactions aids in the formulation of control laws. The feed forward control strategy being developed will increase system autonomy, reduce crew intervention and limit the accumulation of undesirable waste products (NOx).

Smernoff, David T.↗
NASA NTRS2001

ISS Biotechnology Facility - Overview of Analytical Tools for Cellular Biotechnology Investigations

The ISS Biotechnology Facility (BTF) platform provides scientists with a unique opportunity to carry out diverse experiments in a microgravity environment for an extended period of time. Although considerable progress has been made in preserving cells on the ISS for long periods of time for later return to Earth, future biotechnology experiments would desirably monitor, process, and analyze cells in a timely way on-orbit. One aspect of our work has been directed towards developing biochemical sensors for pH, glucose, oxygen, and carbon dioxide for perfused bioreactor system developed at Johnson Space Center. Another aspect is the examination and identification of new and advanced commercial biotechnologies that may have applications to on-orbit experiments.

Jeevarajan, A. S.↗
NASA NTRS1996

Significance of Plant Root Microorganisms in Reclaiming Water in CELSS

Since many microorganisms demonstrate the ability to quickly break down complex mixtures of waste and environmental contaminants, examining their potential use for water recycling in a closed environment is appealing. Water contributes approximately 90 percent of the life sustaining provisions in a human space habitat. Nearly half of the daily water requirements will be used for personal hygiene and dish washing. The primary contaminants of the used "gray" water will be the cleansing agents or soaps used to carry out these functions. Reclaiming water from the gray water waste streams is one goal of the NASA program, Controlled Ecological Life Support Systems (CELSS). The microorganisms of plane roots are well documented to be of a beneficial effect to promote plant growth. Most plants exhibit a range of bacteria and fungi which can be highly plant-specific. In our investigations with lettuce grown in hydroponic culture, we identified a microflora of normal rhizosphere. When the roots were exposed to an anionic surfactant, the species diversity changed, based on morphological characteristics, with the numbers of species being reduced from 7 to 2 after 48 hours of exposure. In addition, the species that became dominant in the presence of the anionic surfactant also demonstrated a dramatic increase in population density which corresponded to the degradation of the surfactant in the root zone. The potential for using these or other rhizosphere bacteria as a primary or secondary waste processor is promising, but a number of issues still warrant investigation; these include but are not limited to: (1) the full identification of the microbes, (2) the classes of surfactants the microbes will degrade, (3) the environmental conditions required for optimal processing efficiency and (4) the ability of transferring the microbes to a non-living solid matrix such as a bioreactor.

Bubenheim, David L.↗
NASA NTRS1998

The Dynamics of Cascaded Monod System Models Through Five Levels

A Monod system model is a set of ordinary differential equations where the terms resemble those which Monod described in his 1949 paper. We focus on the multiple trophic level case in which each trophic level uses only one of the trophic levels for its perpetuation, and no two trophic entities use the same trophic cascaded level. The treatment derives from a primary producer progressively through five trophic levels. Stability types are identified and are related to persistence, and the consequences of some intuitive scaling structures are developed. These considerations are useful to some theoretical questions in ecology and to applications such as bioreactor operation.

Blackwell, Charles C.↗
NASA NTRS1998

The Dynamics of Cascaded Monod System Models Through Five Levels

In the context of this paper, a Monod system model is a set of ordinary differential equations in which the terms resemble those which Motion presented in his 1949 paper. Attention is directed to the multiple trophic level case in which each trophic level exploits only one of the trophic levels for its perpetuation, and no two trophic entities exploit the same trophic level (cascaded). The treatment expands from a primary producer progressively through five trophic levels. Types of stability are identified and are related to persistence, and the consequences of some intuitive scaling structures are developed. These considerations are relevant to some theoretical questions in ecology and to applications such as bioreactor operation.

Blackwell, Charles↗
NASA NTRS1998

The Impact of Deviation from Michaelis-Menten Saturation on Mathematical Model Stability Properties

Based on purely abstract ecological theory, it has been argued that a system composed of two or more consumers competing for the same resource cannot persist. By analysis on a Monod format mathematical model, Hubble and others demonstrated that this assertion is true for all but very special cases of such competing organisms which are determined by an index formed by a grouping of. the parameters which characterize the biological processes of the competing organisms. In the laboratory, using a bioreactor, Hansen and Hubble obtained confirmatory results for several cases of two competing species, and they characterized it as "qualitative confirmation" of the assertion. This result is amazing, since the analysis required the exact equality of the hey index, and it seems certain that no pair of organism species could have exactly equal values. It is quite plausible, however, that pairs of organism species could have approximately equal indices, and the question of how different they could be and still have coexistence of the two (or more) presents itself. In this paper, the pursuit of this question and a compatible resolution is presented.

Blackwell, Charles↗
NASA NTRS2002

Development of a New Membrane Casting Apparatus for Studying Macrovoid Defects in Low-G

A new membrane-casting apparatus is developed for studying macrovoid defects in polymeric membranes made by the wet- and dry-casting process in low-gravity. Macrovoids are large (10-50 micron), open cavities interspersed among the smaller pores in the substructure under the gelled skin surface layer of the cast membrane. Although their occurrence is considered endemic to the wet- and dry-casting process since they can lead to compaction or skin rupture in the membrane process, recent studies suggest several useful applications such as transdermal and osmotic drug delivery systems, miniature bioreactors, etc. However, lack of knowledge about the macrovoid formation mechanism is an obstacle to further development of applications using them. An on-going debate is the role of the surface-tension-driven solutocapillary convection during macrovoid formation. The rapid growth of macrovoids within 1-5 seconds and the high polymer concentration in and near macrovoids make it difficult to explain the mechanism of macrovoid growth by diffusion alone, which is the widely accepted hypothesis proposed by Reuvers et al. The hypothesis advanced by our research group can explain this rapid growth via a mechanism that involves diffusion from the casting solution in the meta-stable region to the macrovoid enhanced by solutocapillary convection induced by the steep nonsolvent concentration gradient in the vicinity of the macrovoid. Since macrovoid growth is hypothesized to be the interplay of a solutocapillary-induced driving force counteracted by viscous drag and buoyancy, eliminate the latter provides a means for testing this hypothesis. Moreover, free convection mass transfer in the nonsolvent immersion bath used to cause phase-separation in membrane casting complicates developing a model for both the wet-casting process and macrovoid growth. The low-g environment minimizes gravitationally induced free convection thereby permitting a tractable solution to the ternary diffusion equations that characterize membrane formation. NASA's Parabolic Flight Research Aircraft provides a small window of low-g (approximately 25 s) that can be used to study macrovoid development in both wet- and dry-cast membranes if an appropriate casting apparatus is used. This casting apparatus should be able to cast the membrane in both low- and high-g in a manner so that essential one-dimensional mass transfer conditions are achieved to insure lateral uniformity in the membrane. The apparatus used in previous research on membrane casting in low-gravity was operated with the plunger driven mechanism. The spring-loaded plunger pushes the bottom block containing the polymer casting solution well directly under the absorbent chamber located in the upper stationary block. However, membranes made via this casting apparatus often displayed lateral nonuniformities that precluded obtaining quantitative information on the macrovoid growth process. Thus, it was necessary to determine the reason for these structural irregularities observed in the low-g casting apparatus. Both experimental as well as computer simulation studies of the low-g casting apparatus established that the impulsive action of the plunger caused the undesired structural nonuniformities. The simulation results showed that the width-to-depth aspect ratio of the shallow well that contains the casting solution in this apparatus was not an important factor in minimizing this problem. Even for a 40:1 (width : depth) aspect ratio, any convection induced by the horizontal motion of the interface of the casting solution will be damped out within 6.25x10(exp 4) seconds. However, the experimental studies revealed that the impulsive motion of the plunger caused a 'sloshing' of the casting solution that had to be eliminated. Therefore, the plungerdriven mechanism was changed to a cam-driven mechanism that did not cause any impulsive motion of the casting solution. Other refinements to this new membrane-casting apparatus include provision for removing the membranes from the casting wells in a less destructive manner. This was accomplished by using a slit geometry for the casting well that permitted disassembly for removal of the cast membrane. The materials used in the construction of this casting apparatus were chosen to insure wetting at the side walls and to maintain precise control of the thickness of the polymer solution in the casting well. An additional provision in this new casting apparatus is the ability to carry out both wet- as well as dry-casting. As such, this apparatus permitted the first studies of the wet-casting of polymeric membranes in low-g. Both wet- and dry-casting experiments on NASA's KC-135 research aircraft employing this new membrane-casting apparatus are scheduled in July 2002. The morphology of the resulting membranes will be characterized using an environmental scanning electron microscope (ESEM). The results of these low-g studies will be reported later.

Lee, Hanyong↗