Cellular immunity in cosmonauts during long duration spaceflight on board the orbital MIR station.

To investigate the effect of spaceflight on cell mediated immunity we tested delayed-type hypersensitivity (DTH) in 5 cosmonauts on three missions in the orbital space station MIR. DTH was determined by the intradermal application of seven antigens and a control using the standardized Multitest Mérieux. This multiple prick puncture test was applied prior to, during, and following missions, which lasted for up to 177 d. In four of the five cosmonauts, reaction scores of DTH-responses below the warning level were noted during flight (two subjects) or following landing (two subjects). In-flight reductions of DTH-responses were possibly induced by a series of stressful extravehicular activities and recovered to normal levels after landing. The results confirm earlier observations of a decreased lymphocyte function following spaceflights determined by means of mitogenic responsiveness of lymphocytes. Thus, the notion of a possibly impaired cell-mediated immunity under stress in association with spaceflight gains further support.

Cultivation of hamster kidney cells in a dynamic cell culture system in space (Spacelab IML-1 Mission).

Cell proliferation, tissue plasminogen activator (t-PA) production and metabolic changes of Hamster Kidney cells (HaK) grown on microcarriers in an automatic Dynamic Cell Culture System (DCCS) were determined on the first International Microgravity Mission (IML-1) Spacelab (22-30 January 1992). The DCCS was designed for two cell culture chambers (volume: 200 microliters each), one operating as a hatch system, the other as a perfusion system. Medium exchange was achieved with an osmotic pump (flow rate 1 microliter h-1). Two major items were investigated: the biological performance of the DCCS in space and the effect of microgravity on HaK cells. The results obtained demonstrated that (1) the DCCS can be used for biological experiments on long term Spacelab missions. In fact, higher cell densities and higher concentration of glucose but lower concentration of lactate in the perfusion chambers than in the batch chambers were measured. The concentration of t-PA, glutamine and ammonia was similar in all chambers. (2) Microgravity had no effect on cell growth and metabolism of HaK cells.

Effect of head-down tilt bedrest (10 days) on lymphocyte reactivity.

Immunological responses of six healthy males to 10 days of head-down tilt bedrest (HDT) were assessed. Lymphocyte responsiveness was severely reduced immediately before, during, and immediately after the HDT, even though the lymphocyte numbers did not change. By contrast, delayed-type hypersensitivity was not affected. No dramatic changes were found in WBC counts and lymphocyte subpopulations, with the only exception of natural killer (NK) cells which transiently decreased immediately after HDT. Plasma cortisol levels were elevated above normal immediately before and during the HDT. The data suggest that the mitogenic response of lymphocytes was affected by psychological and fluid shift stress. These results are compared with data obtained during and after spaceflight. We conclude that the stress of HDT induces changes in immunological responsiveness that are strikingly similar to those arising from the stress of spaceflight.

Reduced lymphocyte activation in space: role of cell-substratum interactions.

We investigated the effect of substratum adhesiveness on stimulated lymphocyte blastogenesis by reducing and blocking cell adhesion with poly (2-hydroxyethyl methacrylate) (poly-HEMA) in a simple on-ground system. Cells grown on medium-thick and thick poly-HEMA films were rounded in shape and displayed no signs of spreading. By contrast, on tissue culture plastic and very thin poly-HEMA films, they showed clear signs of spreading. The mitogenic response of lymphocytes grown on thick poly-HEMA films was reduced by up to 68% of the control (tissue culture plastic). Interferon-gamma production was near zero when the cells were grown on the least adhesive substratum. On uncoated plastic, activated lymphocytes subjected to high gravity (20g) exhibited an increased proliferation rate (40%) compared with 1g. By contrast, on poly-HEMA, high gravity did not improve lymphocyte responsiveness. These results show that activated lymphocytes need to anchor and spread prior to achieving an optimal proliferation response. We conclude that decreased lymphocyte adhesion could contribute to the depressed in vitro lymphocyte responsiveness found in the microgravity conditions of space flight.

Effect of a herbal yeast food supplement and long-distance running on immunological parameters.

The effect of a food supplement on immunological parameters of 16 long-distance runners was tested in a randomized, double-blind and placebo-controlled trial. The supplement comprised plasmolysed herbal yeast, malt, honey, and orange juice. No statistically significant differences between the two groups regarding the following variables were detected at three sessions at rest and immediately after a 21 km run: total and differential white blood cell counts, numbers of B- and T-cells and T-subpopulations, concanavalin-A-induced lymphocyte proliferation, serum levels of immunoglobulins, neopterin, IL-2 receptors, beta 2-microglobulin, complement factor b, c4 and c3c, and c1-inactivator. These findings suggest that the effects of the tested food supplement on these parameters are negligible with respect to improvements in the immunological status of long-distance runners. The changes observed immediately after the run had a transient character. In both groups, however, low lymphocyte counts, IgG subclass 2 levels and c1-inactivator levels were noted at rest, which indicate that the immune status of endurance athletes may be affected by training.

A ground-based model to study the effects of weightlessness on lymphocytes.

The mitogenic response of human lymphocytes was found to be markedly reduced in weightlessness conditions as compared to normal gravity. One possible explanation is that due to the non-existent sedimentation in space the lymphocytes could not adhere and spread on a substratum. Thus, we investigated the effect of substratum adhesiveness on lymphocyte responsiveness by reducing and blocking cell adhesion with poly-HEMA in a simple on-ground system. Lymphocyte adhesiveness was assessed by measuring the proportion of non-adhesive, slightly, and strongly adhesive 51Cr-radiolabelled cells on uncoated and poly-HEMA coated plastic. The amount of cell spreading on surfaces with varying adhesiveness was determined by measuring the area of cells. Cells grown on medium and thick poly-HEMA films were rounded in shape. By contrast, on tissue culture plastic, they showed clear signs of spreading. The mitogenic response of lymphocytes grown on thick poly-HEMA films was reduced by up to 68% of the control (tissue culture plastic). Interferon-gamma production was virtually nil when the cells were grown on the least adhesive substratum. These results show that activated lymphocytes need to anchor and spread prior to achieving an optimal proliferation response. We conclude that decreased lymphocyte adhesion could contribute to the depressed in vitro lymphocyte responsiveness found in the microgravity conditions of space flight.

Metabolic control of glucose degradation in yeast and tumor cells.

Regulation of glucose degradation in both yeasts and tumor cells is very similar in many respects. In both cases it leads to excretion of intermediary metabolites (e.g., ethanol, lactate) in those cell types where uptake of glucose is unrestricted (Saccharomyces cerevisiae, Bowes melanoma cells). The similarities between glucose metabolism observed in yeast and tumor cells is explained by the fact that cell transformation of animal cells leads to inadequate expression of (proto-)oncogenes, which force the cell to enter the cell cycle. These events are accompanied by alterations at the signal transduction level, a marked increase of glucose transporter synthesis, enhancement of glycolytic key enzyme activities, and slightly reduced respiration of the tumor cell. In relation to homologous glucose degradation found in yeast and tumor cells there exist strong similarities on the level of cell division cycle genes, signal transduction and regulation of glycolytic key enzymes. It has been demonstrated that ethanol and lactate excretion in yeast and tumor cells, respectively, result from an overflow reaction at the point of pyruvate that is due to a carbon flux exceeding the capacity of oxidative breakdown. Therefore, the respiratory capacity of a cell determines the amount of glycolytic breakdown products if ample glucose is available. This restricted flux is also referred to as the respiratory bottleneck. The expression "catabolite repression", which is often used in textbooks to explain ethanol and acid excretion, should be abandoned, unless specific mechanisms can be demonstrated. Furthermore, it was shown that maximum respiration and growth rates are only obtained under optimum culture conditions, where the carbon source is limiting.

Mammalian cell cultivation in space.

Equipment used in space for the cultivation of mammalian cells does not meet the usual standard of earth bound bioreactors. Thus, the development of a space worthy bioreactor is mandatory for two reasons: First, to investigate the effect on single cells of the space environment in general and microgravity conditions in particular, and second, to provide researchers on long term missions and the Space Station with cell material. However, expertise for this venture is not at hand. A small and simple device for animal cell culture experiments aboard Spacelab (Dynamic Cell Culture System; DCCS) was developed. It provides 2 cell culture chambers, one is operated as a batch system, the other one as a perfusion system. The cell chambers have a volume of 200 microliters. Medium exchange is achieved with an automatic osmotic pump. The system is neither mechanically stirred nor equipped with sensors. Oxygen for cell growth is provided by a gas chamber that is adjacent to the cell chambers. The oxygen gradient produced by the growing cells serves to maintain the oxygen influx by diffusion. Hamster kidney cells growing on microcarriers were used to test the biological performance of the DCCS. On ground tests suggest that this system is feasible.

Effect of long-term physical exercise on lymphocyte reactivity: similarity to spaceflight reactions.

The response of critical immunological parameters in seven athletes to the sustained physical stress of marathon running was assessed. Variables analysed were the responsiveness of lymphocytes (measured as mitogenic response to concanavalin A), the numbers of lymphocytes, their subsets, and leukocyte numbers. In addition, blood levels of cortisol, epinephrine, and norepinephrine were determined. After the run, lymphocyte responsiveness was severely depressed to 1-70% of the resting values, even though the lymphocyte counts did not change. Leukocyte counts were elevated 2.8-fold. No dramatic changes were found within the lymphocyte subsets, although an increase in pan T-cells and the helper/inducer subset 2 d after the run was significant. In addition, the numbers of B-cells decreased significantly. No change was observed within the suppressor/cytotoxic subset. Cortisol increased 2.1-fold, epinephrine 3.2-fold and norepinephrine 2.7-fold. All these parameters returned to baseline values within 2 d. These data were compared with data obtained during and after spaceflight. We conclude that prolonged physical stress of marathon running induces changes in immunological responsiveness that are strikingly similar to those arising from the stress of spaceflight.

Cultivation of single cells in space.

The purpose of this review is to present an updated and comprehensive analysis of the experiments with single cells performed in space. Especially the results of the investigations performed in Biorack on the D-1 mission clearly show that important cellular functions are changing in microgravity. Cell proliferation, differentiation, metabolism, membrane properties, and cytoplasmic streaming underwent significant alteration during exposure to space flight conditions in a variety of single cells cultures spanning from bacteria to mammalian cells. These findings open new and interesting perspectives to basic and applied research in microgravity. The focus of this paper is on the cultivation of mammalian cells in space laboratories and on the related instrumentation. While Biorack is a useful and efficient instrument for simple studies in Spacelab, the development of new facilities like incubators with automated fixation devices as well as of more complex bioreactors is strongly recommended.

Dynamic cell culture system: a new cell cultivation instrument for biological experiments in space.

The prototype of a miniaturized cell cultivation instrument for animal cell culture experiments aboard Spacelab is presented (Dynamic cell culture system: DCCS). The cell chamber is completely filled and has a working volume of 200 microliters. Medium exchange is achieved with a self-powered osmotic pump (flowrate 1 microliter h-1). The reservoir volume of culture medium is 230 microliters. The system is neither mechanically stirred nor equipped with sensors. Hamster kidney (Hak) cells growing on Cytodex 3 microcarriers were used to test the biological performance of the DCCS. Growth characteristics in the DCCS, as judged by maximal cell density, glucose consumption, lactic acid secretion and pH, were similar to those in cell culture tubes.

Chronic granulomatous disease: mode of action of sulfamethoxazole/trimethoprim.

Four possible modes of action for the clinically observed effectiveness of sulfamethoxazole/trimethoprim in chronic granulomatous disease were evaluated: (1) inhibition of bacterial catalase, (2) improvement of granulocyte oxygen metabolism, (3) synergism of the antibiotic with nonoxygen-dependent granulocyte killing mechanisms, and (4) a purely antibiotic effect based on uptake and concentration of the antibiotic by and within granulocytes. While the first three mechanisms were excluded, the fourth mechanism is highly probable; sulfamethoxazole was found to reach granulocyte associated concentrations 1.7-fold and trimethoprim 4.1-fold of extracellular levels. Penicillin G, a known nonpenetrating antibiotic, reached 0.3-fold, and tetracycline, a known penetrating agent, 7.1-fold the extracellular level. These findings indicate that sulfamethoxazole/trimethoprim is an antibiotic combination uniquely suited for the long-term prophylaxis of infections in patients with defects of intracellular phagocyte killing.

Chronic granulomatous disease: effect of sulfamethoxazole/trimethoprim on neutrophil microbicidal function.

Normal and chronic granulomatous disease (CGD) neutrophils accumulated sulfamethoxazole (SMX) 3-fold and trimethoprim (TMP) 14-fold, possibly through a non-ionic diffusion and pH-partition mechanism. CGD neutrophils incubated with SMX/TMP after phagocytosis of S. aureus killed the bacteria. These findings explain the clinically observed beneficial effect of SMX/TMP in the treatment of infections in CGD and in other conditions characterized by impaired phagocyte microbicidal capacity.