Engineering a mimicry of bone marrow tissue ex vivo.

Hematopoietic stem cells reside in specific niches in the bone marrow and give rise to either more stem cells or maturing hematopoietic progeny depending on the signals provided in the bone marrow microenvironment. This microenvironment is comprised of cellular components as well as soluble constituents called cytokines. The use of cytokines alone for the ex vivo expansion of stem cells in flat, two-dimensional culture flasks, dishes or bags is inadequate and, given the three-dimensionality of the in vivo bone marrow microenvironment, inappropriate. Three-dimensional culture conditions can therefore provide an ex vivo mimicry of bone marrow, recapitulate the desired niche, and provide a suitable environment for stem cell expansion and differentiation. Choice of scaffold, manipulation and reproducibility of the scaffold properties and directed structuring of the niche, by choosing pore size and porosity may inform the resident stem cells of their fate in a directed fashion. The use of bioreactors for cultivation of hematopoietic cells will allow for culture control, optimization, standardization, scale-up, and a "hands-off" operation making the end-product dependable, predictable and free of contaminants, and therefore suitable for human use and therapeutic applications.

Magnetic levitation-based Martian and Lunar gravity simulator.

Missions to Mars will subject living specimens to a range of low gravity environments. Deleterious biological effects of prolonged exposure to Martian gravity (0.38 g), Lunar gravity (0.17 g), and microgravity are expected, but the mechanisms involved and potential for remedies are unknown. We are proposing the development of a facility that provides a simulated Martian and Lunar gravity environment for experiments on biological systems in a well controlled laboratory setting. The magnetic adjustable gravity simulator will employ intense, inhomogeneous magnetic fields to exert magnetic body forces on a specimen that oppose the body force of gravity. By adjusting the magnetic field, it is possible to continuously adjust the total body force acting on a specimen. The simulator system considered consists of a superconducting solenoid with a room temperature bore sufficiently large to accommodate small whole organisms, cell cultures, and gravity sensitive bio-molecular solutions. It will have good optical access so that the organisms can be viewed in situ. This facility will be valuable for experimental observations and public demonstrations of systems in simulated reduced gravity.

Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development.

The transport of auxin controls developmental events in plants. Here, we report that in addition to maintaining vacuolar pH, the H+-pyrophosphatase, AVP1, controls auxin transport and consequently auxin-dependent development. AVP1 overexpression results in increased cell division at the onset of organ formation, hyperplasia, and increased auxin transport. In contrast, avp1-1 null mutants have severely disrupted root and shoot development and reduced auxin transport. Changes in the expression of AVP1 affect the distribution and abundance of the P-adenosine triphosphatase and Pinformed 1 auxin efflux facilitator, two proteins implicated in auxin distribution. Thus, AVP1 facilitates the auxin fluxes that regulate organogenesis.

Chronic morphine treatment promotes specific Th2 cytokine production by murine T cells in vitro via a Fas/Fas ligand-dependent mechanism.

Improper homeostasis of Th1 and Th2 cell differentiation can promote pathological immune responses such as autoimmunity and asthma. A number of factors govern the development of these cells including TCR ligation, costimulation, death effector expression, and activation-induced cell death (AICD). Although chronic morphine administration has been shown to selectively promote Th2 development in unpurified T cell populations, the direct effects of chronic morphine on Th cell skewing and cytokine production by CD4(+) T cells have not been elucidated. We previously showed that morphine enhances Fas death receptor expression in a T cell hybridoma and human PBL. In addition, we have demonstrated a role for Fas, Fas ligand (FasL), and TRAIL in promoting Th2 development via killing of Th1 cells. Therefore, we analyzed whether the ability of morphine to affect Th2 cytokine production was mediated by regulation of Fas, FasL, and TRAIL expression and AICD directly in purified Th cells. We found that morphine significantly promoted IL-4 and IL-13 production but did not alter IL-5 or IFN-gamma. Furthermore, morphine enhanced the mRNA expression of Fas, FasL and TRAIL and promoted Fas-mediated AICD of CD4(+) T cells. Additionally, blockade of Fas/FasL interaction by anti-FasL inhibited the morphine-induced production of IL-4 and IL-13 and AICD of CD4(+) T cells. These results suggest that morphine preferentially enhances Th2 cell differentiation via killing of Th1 cells in a Fas/FasL-dependent manner.

The lung in space.

The lung is exquisitely sensitive to gravity, which induces gradients in ventilation, blood flow, and gas exchange. Studies of lungs in microgravity provide a means of elucidating the effects of gravity. They suggest a mechanism by which gravity serves to match ventilation to perfusion, making for a more efficient lung than anticipated. Despite predictions, lungs do not become edematous, and there is no disruption to, gas exchange in microgravity. Sleep disturbances in microgravity are not a result of respiratory-related events; obstructive sleep apnea is caused principally by the gravitational effects on the upper airways. In microgravity, lungs may be at greater risk to the effects of inhaled aerosols.

Modeling intercellular interactions during carcinogenesis.

By modulating the microenvironment of malignant or premalignant cells, inhibitory or stimulatory signals from nearby cells can play a key role in carcinogenesis. However, current commonly used quantitative models for induction of cancers by ionizing radiation focus on single cells and their progeny. Intercellular interactions are neglected or assumed to be confined to unidirectional radiation bystander effect signals from cells of the same tissue type. We here formulate a parsimoniously parameterized two-stage logistic (TSL) carcinogenesis model that incorporates some effects of intercellular interactions during the growth of premalignant cells. We show that for baseline tumor rates, involving no radiation apart from background radiation, this TSL model gives acceptable fits to a number of data sets. Specifically, it gives the same baseline hazard function, using the same number of adjustable parameters, as does the commonly used two-stage clonal expansion (TSCE) model, so it is automatically applicable to the many data sets on baseline cancer that have been analyzed using the TSCE model. For perturbations of baseline rates due to radiation, the models differ. We argue from epidemiological and laboratory evidence, especially results for the atomic bomb survivors, that for radiation carcinogenesis the TSL model gives results at least as realistic as the TSCE or similar models, despite involving fewer adjustable parameters in many cases.

Removal of organic contaminants by RO and NF membranes.

Rejection characteristics of organic and inorganic compounds were examined for six reverse osmosis (RO) membranes and two nanofiltration (NF) membranes that are commercially available. A batch stirred-cell was employed to determine the membrane flux and the solute rejection for solutions at various concentrations and different pH conditions. The results show that for ionic solutes the degree of separation is influenced mainly by electrostatic exclusion, while for organic solutes the removal depends mainly upon the solute radius and molecular structure. In order to provide a better understanding of rejection mechanisms for the RO and NF membranes, the ratio of solute radius (r(i,s)) to effective membrane pore radius (r(p)) was employed to compare rejections. An empirical relation for the dependence of the rejection of organic compounds on the ratio r(i,s)/r(p) is presented. The rejection for organic compounds is over 75% when r(i,s)/r(p) is greater than 0.8. In addition, the rejection of organic compounds is examined using the extended Nernst-Planck equation coupled with a steric hindrance model. The transport of organic solutes is controlled mainly by diffusion for the compounds that have a high r(i,s)/r(p) ratio, while convection is dominant for compounds that have a small r(i,s)/r(p) ratio.

Redirection of biological heat from head to hands to support finger comfort in the cold.

INTRODUCTION: Maintaining hand comfort in the cold while sustaining optimal performance is still a challenge. There has been little research on the efficacy of transporting biological heat from the head to the hands to stabilize finger comfort, although there are notable temperature differences between these two areas in the cold. METHOD: A tubing bypass between the head and the hands was designed as an independent component in a liquid cooling/warming garment (LCWG). Seven subjects (four men, three women) were studied, comparing finger temperature (Tfing) change in two conditions: LCWG with additional bypass; and LCWG without bypass. The protocol consisted of three stages: 1) comfort stabilization, LCWG inlet water temperature 33 degrees C, water in loop in bypass condition 23 degrees C; 2) body cooling, LCWG inlet water temperature 20 degrees C; and 3) rewarming, LCWG inlet water temperature 45 degrees C. RESULTS: The time to reach the 25 degrees C Tfing discomfort criterion was significantly longer in the bypass condition (p < 0.01); Tfing was significantly higher at the same time point when Tfing of 25 degrees C was reached in the control condition (p < 0.01). CONCLUSION: The incorporation of a bypass transferring biological heat from a high to a low skin temperature area has potential to improve local finger comfort and thus increase the time personnel can work in cold environments.

Evaluation of cardiovascular risks of spaceflight does not support the NASA bioastronautics critical path roadmap.

INTRODUCTION: Occurrence of serious cardiac dysrhythmias and diminished cardiac and vascular function are the primary cardiovascular risks of spaceflight identified in the 2005 NASA Bioastronautics Critical Path Roadmap. METHODS: A review of the literature was conducted on experimental results and observational data obtained from spaceflight and relevant ground simulation studies that addressed occurrence of cardiac dysrhythmias, cardiac contractile and vascular function, manifestation of asymptomatic cardiovascular disease, orthostatic intolerance, and response to exercise stress. RESULTS: Based on data from astronauts who have flown in space, there is no compelling experimental evidence to support significant occurrence of cardiac dysrhythmias, manifestation of asymptomatic cardiovascular disease, or reduction in myocardial contractile function. Although there are post-spaceflight data that demonstrate lower peripheral resistance in astronauts who become presyncopal compared with non-presyncopal astronauts, it is not clear that these differences are the result of decreased vascular function. However, the evidence of postflight orthostatic intolerance and reduced exercise capacity is well substantiated by both spaceflight and ground experiments. Although attenuation of baroreflex function(s) may contribute to postflight orthostatic instability, a primary mechanism of orthostatic intolerance and reduced exercise capacity is reduced end-diastolic and stroke volume associated with lower blood volumes and consequent cardiac remodeling. CONCLUSION: Data from the literature on the current population of astronauts support the notion that the primary cardiovascular risks of spaceflight are compromised hemodynamic responses to central hypovolemia resulting in reduced orthostatic tolerance and exercise capacity rather than occurrence of cardiac dysrhythmias, reduced cardiac contractile and vascular function, or manifestation of asymptomatic cardiovascular disease. These observations warrant a critical review and revision of the 2005 Bioastronautics Critical Path Roadmap.

An information maximization model of eye movements.

We propose a sequential information maximization model as a general strategy for programming eye movements. The model reconstructs high-resolution visual information from a sequence of fixations, taking into account the fall-off in resolution from the fovea to the periphery. From this framework we get a simple rule for predicting fixation sequences: after each fixation, fixate next at the location that minimizes uncertainty (maximizes information) about the stimulus. By comparing our model performance to human eye movement data and to predictions from a saliency and random model, we demonstrate that our model is best at predicting fixation locations. Modeling additional biological constraints will improve the prediction of fixation sequences. Our results suggest that information maximization is a useful principle for programming eye movements.

Proton-induced genetic damage in lacZ transgenic mice.

The plasmid-based lacZ transgenic mouse model system was used to evaluate the mutagenic and genotoxic potential of 250 MeV/nucleon proton radiation by evaluating the frequency of micronucleated polychromatic reticulocytes in peripheral blood and bone marrow and the mutant frequencies of the lacZ reporter transgene in spleen and brain, respectively. Doses of 0.1-2 Gy produced dose- and time-dependent changes in the frequency of micronucleated polychromatic reticulocytes within 48 h, with peak induction up to sixfold above control levels. The frequency of micronucleated polychromatic reticulocytes returned to control levels within 1 week after exposure. With doses of 4 Gy, the elevation in the frequency of micronucleated polychromatic reticulocytes was delayed up to 1 week after exposure, but complete recovery to control levels was observed at 16 weeks postirradiation. Significant increase in mutant frequencies in brain tissue was observed at 8 week after proton exposure at doses as low of 0.1 Gy. Mutant frequencies in spleen increased up to twofold above spontaneous mutant frequencies at 8 weeks after exposure to 0.5-1 Gy. These effects appeared saturated at doses >1 Gy for both tissues, possibly due to elimination of damaged cells from the tissue systems. These in vivo results highlight the importance of considering tissue specificity, dose and temporal dependence when assessing radiation effects.

Radiation responses of perfused tracheal tissue.

We are using a novel perfusion system to examine the effects of radiation on a model respiratory tissue. Tracheas taken from young adult male Fischer 344 rats are embedded in a growth factor-enriched agarose matrix that is mounted in a special apparatus designed to allow growth medium to periodically wash the epithelial surface of the lumen. A comparison of the microarray expression profiles of freshly harvested tracheas and tracheas maintained in perfusion culture for 24 h shows no significant difference except for an increase in expression of a few metabolism- and surfactant-related genes. Perfusion culture samples exposed to 4 Gy of X rays show a lower than expected increase in expression for some cell cycle- and repair-related genes.

Induction of anchorage-independent growth in primary human cells exposed to protons or HZE ions separately or in dual exposures.

Travelers on space missions will be exposed to a complex radiation environment that includes protons and heavy charged particles. Since protons are present at much higher levels than are heavy ions, the most likely scenario for cellular radiation exposure will be proton exposure followed by a hit by a heavy ion. Although the effects of individual ion species on human cells are being investigated extensively, little is known about the effects of exposure to both radiation types. One useful measure of mammalian cell damage is induction of the ability to grow in a semi-solid agar medium highly inhibitory to the growth of normal human cells, termed neoplastic transformation. Using primary human cells, we evaluated induction of soft-agar growth and survival of cells exposed to protons only or to heavy charged particles (600 MeV/nucleon silicon) only as well as of cells exposed to protons followed after a 4-day interval by silicon ions. Both ions alone efficiently transformed the human cells to anchorage-independent growth. Initial experiments indicate that the dose responses for neoplastic transformation of cells exposed to protons and then after 4 days to silicon ions appear similar to that of cells exposed to silicon ions alone.

Suppression of DNA-PK by RNAi has different quantitative effects on telomere dysfunction and mutagenesis in human lymphoblasts treated with gamma rays or HZE particles.

Basic to virtually all relevant biological effects of ionizing radiation is the underlying damage produced in DNA and the subsequent cellular processing of such damage. The damage can be qualitatively different for different kinds of radiations, and the genetics of the biological systems exposed can greatly affect damage processing and ultimate outcome--the biological effect of concern. The accurate repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and function. Incorrect repair of such lesions results in chromosomal rearrangements and mutations that can lead to cancer and heritable defects in the progeny of irradiated parents. We have focused on the consequent phenotypic effects of faulty repair by examining connections between cellular radiosensitivity phenotypes relevant for carcinogenesis after exposure to ionizing radiation, and deficiencies in various components of the non-homologous end-joining (NHEJ) system. Here we produced deficiencies of individual components of the DNA-dependent protein kinase (DNA-PK) holoenzyme (Ku86 and the catalytic subunit, DNA-PKcs), both singly and in combination, using RNA interference (RNAi) in human lymphoblastoid cell lines. Exposure of cells exhibiting reduced protein expression to either gamma rays or 1 GeV/nucleon iron particles demonstrated differential effects on telomere dysfunction and mutation frequency as well as differential effects between radiation qualities.

Gene expression changes in normal human skin fibroblasts induced by HZE-particle radiation.

Studies have shown that radiation exposure affects global gene expression in mammalian cells. However, little is known about the effects of HZE particles on gene expression. To study these effects, human skin fibroblasts were irradiated with HZE particles of different energies and LETs. The data obtained from these experiments indicate that changes in gene expression are dependent on the energy of the radiation source. Particles with the highest energy, i.e. iron, induced the biggest expression changes in terms of numbers of genes and magnitudes of changes. Many genes were found to undergo significant expression changes after HZE-particle irradiation, including CDKN1A/p21, MDM2, TNFRSF6/fas, PCNA and RAD52. Unlike X rays, HZE particles expose cells to two types of radiation: primary ions and delta rays. We hypothesized that the biological effects of delta rays, which are secondary electron emissions, should resemble the effects of X rays. To explore this idea, gene expression changes between cells that had been irradiated with HZE particles and X rays were compared. The results support our hypothesis since the number of genes that commonly changed after exposure to both radiations increased as a function of particle energy.

Effects of iron ions, protons and X rays on human lens cell differentiation.

We have investigated molecular changes in cultured differentiating human lens epithelial cells exposed to high-energy accelerated iron-ion beams as well as to protons and X rays. In this paper, we present results on the effects of radiation on gene families that include or are related to DNA damage, cell cycle regulators, cell adhesion molecules, and cell cytoskeletal function. A limited microarray survey with a panel of cell cycle-regulated genes illustrates that irradiation with protons altered the gene expression pattern of human lens epithelial cells. A focus of our work is CDKN1A (p21(CIP1/WAF1)), a protein that we demonstrate here has a role in several pathways functionally related to LET-responsive radiation damage. We quantitatively assessed RNA and protein expression in a time course before and after single 4-Gy radiation doses and demonstrated that transcription and translation of CDKN1A are both temporally regulated after exposure. Furthermore, we show qualitative differences in the distribution of CDKN1A immunofluorescence signals after exposure to X rays, protons or iron ions, suggesting that LET effects likely play a role in the misregulation of gene function in these cells. A model of molecular and cellular events is proposed to account for precataractous changes in the human lens after exposure to low- or high-LET radiations.

Efficient production of reactive oxygen species in neural precursor cells after exposure to 250 MeV protons.

The space radiation environment is composed of highly energetic ions, dominated by protons, that pose a range of potential health risks to astronauts. Traversals of these particles through certain tissues may compromise the viability and/or function of sensitive cells, including neural precursors found within the dentate subgranular zone of the hippocampus. Irradiation has been shown to deplete these cells in vivo, and reductions of these critical cells are believed to impair neurogenesis and cognition. To more fully understand the mechanisms underlying the behavior of these precursor cells after irradiation, we have developed an in vitro neural precursor cell system and used it to assess acute (0-48 h) changes in ROS and mitochondrial end points after exposure to Bragg-peak protons of 250 MeV. Relative ROS levels were increased at nearly all doses (1-10 Gy) and postirradiation times (6-24 h) compared to unirradiated controls. The increase in ROS after proton irradiation was more rapid than that observed with X rays and showed a well-defined dose response at 6 and 24 h, increasing approximately 10% and 3% per gray, respectively. However, by 48 h postirradiation, ROS levels fell below controls and coincided with minor reductions in mitochondrial content. Use of the antioxidant alpha-lipoic acid (before or after irradiation) was shown to eliminate the radiation-induced rise in ROS levels. Our results corroborate earlier studies using X rays and provide further evidence that elevated ROS are integral to the radioresponse of neural precursor cells.

Cytotoxic effects of low- and high-LET radiation on human neuronal progenitor cells: induction of apoptosis and TP53 gene expression.

The induction of apoptosis, TP53 expression, caspase activation and cell toxicity were investigated after exposure of cells of the human neuronal progenitor cell line Ntera2 (NT2) to low-LET radiation (gamma and X rays). The data indicates that irradiation of NT2 cells quickly induced TP53 expression, which was followed in time by an increase in caspase activity, and ultimately resulted in the induction of apoptosis. Induction of apoptosis was dependent on dose, and the highest levels were measured 48 h after exposure. For comparison, the level of apoptosis induced by high-LET particle radiation (1 GeV/ nucleon iron ions) was also determined and was found to be dependent on dose. The relative biological effectiveness (RBE) was estimated from the slopes of the dose-response curves for the induction of apoptosis. The RBE(max) for apoptosis 48 h after exposure was at least 3.4. In short, exposure to high-LET radiation results in a more efficient and greater induction of apoptosis in human neuronal progenitor cells than low-LET radiation.

A longitudinal study of operant responding in rats irradiated when 2 months old.

Two-month-old rats were exposed to 56Fe particles (1, 1.5, 2 Gy; 1 GeV/nucleon). They were tested on the performance of an ascending fixed-ratio operant task (bar pressing for food reward) at 7, 11 and 15 months after irradiation. Previous research had shown that for the same rats tested at 3 months after exposure, only the rats exposed to 2 Gy of 56Fe particles showed a significant disruption of performance compared to control (0 Gy) rats. When these rats were tested 7, 11 and 15 months after exposure, all irradiated groups showed significantly poorer performance than the controls. These results suggest that there is an interaction between irradiation and age such that 56Fe-particle-induced performance deficits can develop several months after exposure.

High-LET radiation induces inflammation and persistent changes in markers of hippocampal neurogenesis.

Exposure to heavy-ion radiation is considered a potential health risk in long-term space travel. It may result in the loss of critical cellular components in complex systems like the central nervous system (CNS), which could lead to performance decrements that ultimately could compromise mission goals and long-term quality of life. Specific hippocampal-dependent cognitive impairment occurs after whole-body 56Fe-particle irradiation, and while the pathogenesis of this effect is not yet clear, it may involve damage to neural precursor cells in the hippocampal dentate gyrus. We irradiated mice with 1-3 Gy of 12C or 56Fe ions and 9 months later quantified proliferating cells and immature neurons in the dentate subgranular zone (SGZ). Our results showed that reductions in these cells were dependent on the dose and LET. When compared with data for mice that were studied 3 months after 56Fe-particle irradiation, our current data suggest that these changes are not only persistent but may worsen with time. Loss of precursor cells was also associated with altered neurogenesis and a robust inflammatory response. These results indicate that high-LET radiation has a significant and long-lasting effect on the neurogenic population in the hippocampus that involves cell loss and changes in the microenvironment.