Engineering topics

Sams, C. F.

Publications and source records attributed to Sams, C. F..

24 records · Page 2

NASA NTRS2000

T Lymphocyte Activation Threshold and Membrane Reorganization Perturbations in Unique Culture Model

Quantitative activation thresholds and cellular membrane reorganization are mechanisms by which resting T cells modulate their response to activating stimuli. Here we demonstrate perturbations of these cellular processes in a unique culture system that non-invasively inhibits T lymphocyte activation. During clinorotation, the T cell activation threshold is increased 5-fold. This increased threshold involves a mechanism independent of TCR triggering. Recruitment of lipid rafts to the activation site is impaired during clinorotation but does occur with increased stimulation. This study describes a situation in which an individual cell senses a change in its physical environment and alters its cell biological behavior.

Adams, C. L.↗

NASA NTRS1999

T cell activation responses are differentially regulated during clinorotation and in spaceflight

Studies of T lymphocyte activation with mitogenic lectins during spaceflight have shown a dramatic inhibition of activation as measured by DNA synthesis at 72 h, but the mechanism of this inhibition is unknown. We have investigated the progression of cellular events during the first 24 h of activation using both spaceflight microgravity culture and a ground-based model system that relies on the low shear culture environment of a rotating clinostat (clinorotation). Stimulation of human peripheral blood mononuclear cells (PBMCs) with soluble anti-CD3 (Leu4) in clinorotation and in microgravity culture shows a dramatic reduction in surface expression of the receptor for IL-2 (CD25) and CD69. An absence of bulk RNA synthesis in clinorotation indicates that stimulation with soluble Leu4 does not induce transition of T cells from G0 to the G1 stage of the cell cycle. However, internalization of the TCR by T cells and normal levels of IL-1 synthesis by monocytes indicate that intercellular interactions that are required for activation occur during clinorotation. Complementation of TCR-mediated signaling by phorbol ester restores the ability of PBMCs to express CD25 in clinorotation, indicating that a PKC-associated pathway may be compromised under these conditions. Bypassing the TCR by direct activation of intracellular pathways with a combination of phorbol ester and calcium ionophore in clinorotation resulted in full expression of CD25; however, only partial expression of CD25 occurred in microgravity culture. Though stimulation of purified T cells with Bead-Leu4 in microgravity culture resulted in the engagement and internalization of the TCR, the cells still failed to express CD25. When T cells were stimulated with Bead-Leu4 in microgravity culture, they were able to partially express CD69, a receptor that is constitutively stored in intracellular pools and can be expressed in the absence of new gene expression. Our results suggest that the inhibition of T cell proliferative response in microgravity culture is a result of alterations in signaling events within the first few hours of activation, which are required for the expression of important regulatory molecules.

NASA Experiment Number 9403079 1/2↗

NASA NTRS1999

The use of a spaceflight-compatible device to perform WBC surface marker staining and whole-blood mitogenic activation for cytokine detection by flow cytometry

Significant changes have recently been described regarding circulating peripheral immune cells immediately following spaceflight. Existing methods for immunophenotype staining of peripheral blood in terrestrial labs do not meet the constraints for flight on the Space Shuttle. We have recently described the development and use of the Whole Blood Staining Device (WBSD), a simple device for staining flow cytometry specimens during spaceflight. When preparing samples with the WBSD, all liquids are safely contained as the cells are moved through staining, lysis and fixation steps. Here we briefly review the use of the WBSD, and then describe another versatile adaptation, a modification to perform intracellular staining of cytokines for detection by flow cytometry. Alterations in cytokine production have been reported both in ground-based simulated microgravity culture and in astronaut samples returning from spaceflight. Data regarding microgravity effects on cytokine production for specific subpopulations of cells is lacking. Flow cytometric cytokine analysis offers the unique ability to perform simultaneous surface marker analysis and positively identity cytokine producing subsets of cells. The utilization of the WBSD provides the ability to perform rapid and routine mitogenic activation during spaceflight coupled with the ability to perform simultaneous surface marker analysis. The only external requirements for this procedure are an in-flight 37-degree incubator and the capacity for 4-degree storage.

NASA Discipline Regulatory Physiology↗

NASA NTRS1999

Leukocyte subsets and neutrophil function after short-term spaceflight

Changes in leukocyte subpopulations and function after spaceflight have been observed but the mechanisms underlying these changes are not well defined. This study investigated the effects of short-term spaceflight (8-15 days) on circulating leukocyte subsets, stress hormones, immunoglobulin levels, and neutrophil function. At landing, a 1.5-fold increase in neutrophils was observed compared with preflight values; lymphocytes were slightly decreased, whereas the results were variable for monocytes. No significant changes were observed in plasma levels of immunoglobulins, cortisol, or adrenocorticotropic hormone. In contrast, urinary epinephrine, norepinephrine, and cortisol were significantly elevated at landing. Band neutrophils were observed in 9 of 16 astronauts. Neutrophil chemotactic assays showed a 10-fold decrease in the optimal dose response after landing. Neutrophil adhesion to endothelial cells was increased both before and after spaceflight. At landing, the expression of MAC-1 was significantly decreased while L-selectin was significantly increased. These functional alterations may be of clinical significance on long-duration space missions.

short duration↗

NASA NTRS1998

Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

NASA Discipline Regulatory Physiology↗

NASA NTRS1991

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.

Endothelium, Vascular/chemistry↗