F Bk

Resting cell

Memory cell

Effector cell

Memory cell

Figure 4-18. Activation of T-lymphocytes. Resting cells in the Go phase are activated in vitro with a mitogen thus triggering the events occurring in vivo during antigenic activation, e.g., with a virus or a bacterium. Within two days the cells increase in volume and enter mitosis on the third day of activation.

T-lymphocytes from human peripheral blood may be activated in vitro by several substances of different origin, called mitogens, which are able to trigger the events occurring in vivo following exposure to an antigen (Figure 4-18). The activation of T-cells with mitogens is, therefore, a good model to simulate in vitro this key aspect of the immune response. Concanavalin A (Con A) is a widely used mitogen. Its binding to the cell membrane mimics the first signal required for T-lymphocyte activation, which usually is transmitted to the "resting" T-cells by the antigen, or a fragment of it, bound to the "antigen-presenting cell" by means of the major histocompatibility complex. Resting lymphocytes are in the G0 phase and after recognition of the first signal the complex activation process is started. The cells enter the G| phase, a number of cytokines such as interleukines and interferon-gamma are produced, cell division leads to the appearance of T-effector and T-memory cells. While antigen recognition is limited to one clone of a small number of cells, mitogen activation is polyclonal and involves a large number of T-cell clones.

The activation process consists of three phases:

a. Recognition via T-Cell Receptor, TCR (of which CD3 is the main constituent) (Figure 4-19), either of the antigen "presented" by the antigen presenting cell or of the mitogen;

b. Cell-cell interaction and exchange of signals between T-cells and "accessory" cells (in general monocytes via CD28 on the T-cell and B7 on the monocyte) respectively;

c. Expression and secretion of interleukin-2 (as autocrine signal) as well as expression of interleukin-2 receptor and recognition of interleukin-2 by T-cells.

Antigen / MHC anti-CD3 (ConA)

(accessory cell)

(accessory cell)

Antigen / MHC anti-CD3 (ConA)

IL-2

(Autocrine signal)

Figure 4-19. Three signals are required for full T-cell activation. The first signal is delivered in vivo by the antigen "presented" by the major histocompatibility complex to the T-cell receptor TCR/CD3 complex on the membrane of the cell. Such interaction can be mimicked in vitro with a mitogen (e.g., Con A) or by anti-CD3 antibody. The second signal is delivered by an "accessory" cell, usually a monocyte, canying a B7 ligand that is recognized by the CD28 receptor on the T-cell. During the activation process, T-cells produce a and fS subunits of the IL-2 receptor that combine with the y subunit present on the membrane and secrete IL-2 as autocrine third signal.

IL-2

(Autocrine signal)

Figure 4-19. Three signals are required for full T-cell activation. The first signal is delivered in vivo by the antigen "presented" by the major histocompatibility complex to the T-cell receptor TCR/CD3 complex on the membrane of the cell. Such interaction can be mimicked in vitro with a mitogen (e.g., Con A) or by anti-CD3 antibody. The second signal is delivered by an "accessory" cell, usually a monocyte, canying a B7 ligand that is recognized by the CD28 receptor on the T-cell. During the activation process, T-cells produce a and fS subunits of the IL-2 receptor that combine with the y subunit present on the membrane and secrete IL-2 as autocrine third signal.

The endpoint of the activation is mitosis of the T-cell, which is maximal 72 h after addition of the mitogen. The mitotic index, an indicator of the proliferation rate triggered by the mitogen, is determined by treating the cells either with a pulse of a radioactive marker (e.g., trititated thymidine) or of a marker that can be detected via enzyme-linked immunosorbent assay (e.g., bromodeoxyuridine).

Ex Vivo and In Vivo

Ex vivo experiments are based on blood samples drawn from space crewmembers prior to, during, and after flight, which are diluted with culture medium and incubated in the presence of concanavalin A. In vivo studies consist of the application of antigens to the skin of space crewmembers in order to determine the delayed hypersensitivity, i.e., the specific response of T-lymphocytes, prior to, during, and after flight to a number of antigens (delayed-type hypersensitivity test or skin test). In vitro experiments are based on immune cells isolated from the peripheral blood of healthy donors (not necessarily astronauts) a few hours before the experiment is started, either in the space or in the ground laboratory, and cultured in a standard culture medium in the presence of a mitogen.

Figure 4-20. Mitotic index of T-cells from three space crewmembers exposed in vitro to Con A. Whole-blood samples taken 9 and 2 days before launch (L-), during flight on the 3rd mission day (L+), and 0, 7, and 13 days after landing (R+) were cultured for three days in the presence of the mitogen. The inflight samples were incubated in a centrifuge at 1 g. The data are expressed as counts per minute (cpm) of tritiated thymidine incorporated into DNA.

Subject 1

Subject 2

Subject 3

Figure 4-20. Mitotic index of T-cells from three space crewmembers exposed in vitro to Con A. Whole-blood samples taken 9 and 2 days before launch (L-), during flight on the 3rd mission day (L+), and 0, 7, and 13 days after landing (R+) were cultured for three days in the presence of the mitogen. The inflight samples were incubated in a centrifuge at 1 g. The data are expressed as counts per minute (cpm) of tritiated thymidine incorporated into DNA.

The objective of in vitro studies during spaceflight is to investigate the biological mechanism of T-cell activation under the influence of gravitational changes. The experiments in vitro in microgravity have contributed to understand certain aspects of signal transduction in T-cells. Studies ex vivo and in vivo on the immune cells and on the delayed hypersensitivity of astronauts on board Spacelab and the Mir space station, respectively, have helped to distinguish between the effects of gravity and those of physical and psychological stress. Briefly, the in vivo and the ex vivo studies permitted to establish that the depression of the T-cell-dependent immune response is due to the psychological and physical stress of spaceflight on the neuroendocrine system of the astronaut rather than to weightlessness per se (Figure 4-20).

In an experiment performed with blood samples from four astronauts in a multi-g centrifuge on Spacelab SLS-1 we were able to see that the threshold of sensitivity in T-cells ranges between 0 g and 0.6 g (Figure 4-21). An experiment designed to narrow the sensitivity gap by using a 0.2 g centrifuge was lost with Columbia STS-107. A new attempt will be undertaken on board the ISS.

Figure 4-21. Mitotic index of T-cells from four space crewmembers exposed in vitro to Con A. Whole-blood samples taken during flight from four crewmembers and incubated for three days in the presence of the mitogen at 0 g and in a centrifuge providing 0.6, 1.0, 1.36, or 1.75 g. The data are expressed as counts per minute of tritiated thymidine incorporated into DNA.

Subject I Subject 2 Subject 3 Subject 4

Figure 4-21. Mitotic index of T-cells from four space crewmembers exposed in vitro to Con A. Whole-blood samples taken during flight from four crewmembers and incubated for three days in the presence of the mitogen at 0 g and in a centrifuge providing 0.6, 1.0, 1.36, or 1.75 g. The data are expressed as counts per minute of tritiated thymidine incorporated into DNA.

Table 4-02 gives an overview on the most important findings on the effect of spaceflight on mammalian cell proliferation. Besides lymphocytes, hybridoma cells and macrophages also showed remarkable proliferation rate changes, whereas no changes were observed with embryonic lung cells, hamster kidney cells, rat myoblasts, or rat osteoblasts. However, in embryonic lung cells, the glucose consumption from the medium was 20% higher in the flight cultures than in the ground control, thus pointing to important metabolic changes that were not further investigated.

Cell type

Effect

Remarks

T lymphocytes with monocytes as accessory cells, human

60-90% reduction of mitotic index upon activation by Con A of resuspended cells (6 independent experiments); 100% increase of activation of cells attached to microcarrier beads

Experiments in Spacelab 1, D-l, SLS-1, IML-2-all, except the first one, with a onboard 1-g control

7E3 hybridoma cells

40% increase of cell number after 4 d in space

Spacelab IML-l-onboard 1-g control

Bone marrow derived macrophages, mice femora and tibiae

Up to 60% increase in cell number after 6 d in space

Space shuttle STS-57, -60, and-62-no onboard 1-g control; incubation temperature between 23 and27°C

WI38 embryonic lung cells, human

No effect of growth rate during 28 d in space

Skylab-automatic medium supply; no onboard 1-g control

Kidney cells, hamster

No alteration of cell number in cells attached to microcarrier beads after 7 d in space

Spacelab IML-l-onboard 1-g control

L8 myoblast cells, rat

No change of proliferation rate in cells attached to collagen-coated microcarriers beads

Space shuttle-no onboard 1-g control

Osteoblasts, rat

No change in cell growth rate

Spacelab IML-2-no onboard 1-g control

Table 4-02. Effects of space.flight on cell proliferation in mammalian cells.

Table 4-02. Effects of space.flight on cell proliferation in mammalian cells.

As said above, proliferation is the end-point of a biological process. To understand the mechanism of such effects, it is necessary to analyze intermediate signal transduction pathways. Such studies are outlined below.

Interestingly enough, the mitotic index is increased significantly in a number of cell types cultured at 10 g in a centrifuge. In a study conducted on HeLa cells11 it could be seen that proliferation rate is increased at 10 g while motility tracked on colloidal gold was strongly reduced compared to 1-g controls. In analogy with the considerations made with Paramecium, it was

'1 HeLa cells are an established line of human epithelial cells derived from a cervical cancer.

speculated that at 10 g the cells switch their energy turnover from motion towards mitosis.

Figure 4-22. Aggregates of lymphocytes incubated for 78 h in the presence of Con A in the NIZEMI facility. The arrow indicates a cell moving out of the aggregate. Courtesy of M. Cogoli-Greuter, Zero-gLife Tec GmbH, Zurich, Switzerland.

T+12 min T+16min

Figure 4-22. Aggregates of lymphocytes incubated for 78 h in the presence of Con A in the NIZEMI facility. The arrow indicates a cell moving out of the aggregate. Courtesy of M. Cogoli-Greuter, Zero-gLife Tec GmbH, Zurich, Switzerland.

3.2.2 Morphology and Motility

Important cellular functions are regulated by cell-cell interactions. This is particularly important in the activation of T-lymphocytes. It was possible to show that white blood cells are capable of autonomous movements and interactions in microgravity (Figure 4-22). Again, this is a surprising and unpredictable finding. It was thought that mammalian cells can move only on a substratum, and that gravity is somehow driving the motion. Moreover, it was also seen that the cytoskeleton undergoes structural changes few seconds after exposure to 0 g (Figure 4-23). The cytoskeleton plays an important role during signal transduction, in particular, in the interaction of the cytoskeleton with G-proteins. Alteration of microtubules and increased apoptosis in space were detected in Jurlcat cells, a T-cell derived cell line. As shown in Table 403, leukocytes are again the cells showing the most remarkable effects of microgravity on the cytoskeleton.

The attachment to a substratum of adhesion-dependent cells was tested in microgravity with human embryonic kidney cells in an experiment carried out in an incubator installed in the flight deck of Space Shuttle mission

STS-8. Microcarriers were added inflight to the cells in culture at 37°C. Scanning electron microscopy showed that attachment took place qualitatively and quantitatively as in the ground controls, thus confirming that the related membrane functions are not altered at 0 g. Similar conclusions have been arrived at from clinostat experiments with human colorectal carcinoma cells.

Cell type

Effect

Remarks

T lymphocytes with monocytes as accessory cells, human

Normal attachment of Con A to the cell membrane; slightly retarded patching and capping; cell motion in the presence of Con A is higher at 0 g than at 1 g; elongated cell shape and contraction waves. In the presence of Con A: cell motion as above; formation of cell aggregates smaller than at 1 g; cells move out/in of aggregates; Apoptotic cells in suspension at Oxg, normal morphology at 0 g in microcarrier-attached cells

Sounding rockets

NIZEMI rotating microscope, on board Spacelab IML-2; Spacelab D-l, SLS-1-onboard 1-g control

A431 epidermoid cells, human

No change in clustering of the receptors of epidermal growth factor

Sounding rocket

Embryonic kidney cells, hamster

Normal attachment to microcarrier beads

Space Shuttle-onboard 1-g control

WI38 embryonic lung cells, human

No changes of ultrastructure, no effect on cell migration

28d culture in Skylab; onboard time-lapse cine cameras-no onboard 1-g control

Erythrocytes, human

Dramatic decrease of cell aggregation

2 experiments in Space Shuttle, no 1 g control

L8 myoblasts, rat

Cells fail to fuse and differentiate into myoblasts and show atypical morphology in culture after exposure to 0 g

Space Shuttle-no onboard 1-g control

Jurkat cells, human T-cell line

Significant changes of the cytoskeleton; large bundles of vimentin are formed after 30 sec in

Alteration of the microtubules and increased apoptosis

Sounding rockets

Shuttle flight-onboard 1-g control

Friend leukemia-virus transformed cells, murine

No changes in the ultrastructure of the cell

Spacelab-onboard 1-g control

Cerebellum cells, murine

At Oxg Cells form aggregates that are larger in number but smaller in size than in the inflight 1-g controls

New instrument in Spacelab IML-2— onboard 1-g control

Osteoblast cell line MC2T3-E1

Changes in cell shape and extracellular matrix

Space Shuttle-onboard 1-g control

Tubulin/microtubules

Almost no self organization of tubulin into microtubuli

Sounding rocket-onboard 1-g control

Table 4-03. Effect of space.flight on morphology and motility of mammalian cells.

Table 4-03. Effect of space.flight on morphology and motility of mammalian cells.

In an experiment with Friend cells* conducted in Biorack during IML-1, extensive analysis (scanning, transmission, volume measurements) of the ultrastructure of cells cultured for six days in the presence of DMSO did not reveal differences between the cell cultures at 0 g and in the onboard 1-g centrifuge. As mentioned above, in an experiment with WI38 human embryonic lung cells carried out in Skylab, cinematographic recording, phase, electron and scanning microscopy produced no observable differences in ultrastructure and in cell migration between flight and ground controls.

Several permanent phenotypic alterations were recorded in cell cultures of rat myoblasts, which were recultured on Earth after return from a 10-day Space Shuttle flight (STS-45). The differences included altered morphology and failure to fuse and differentiate into myotubes. Unfortunately, the spaceflight cultures were accommodated in an automated cell culture apparatus in a middeck locker of the Space Shuttle, and there was no onboard 1-g control. Consequently, the cause of the altered phenotype is unknown.

Changes in bone extracellular matrix and osteoblast shape were detected in cell cultures in real microgravity by Hughes-Fulford et al. (2002). Such changes were not caused by an alteration of the transcription determined with the Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) technology, translation of fibronectin, nor by altered matrix formation.

Figure 4-23. Vimentin filaments in Jurkat cells (a derived T-cell Une) flown on the sounding rocket Maxus detected with fluorescent antibodies. Courtesy of G. Sciola.

* In 1956, Charlotte Friend described a new "virus-like" agent that caused a malignant disease of the hematopoietic system in mice. These cell lines came to be known as FriendEtythroleukemia Cells (FELC).

3.2.3 Signal Transduction and Gene Expression

In this section, I discuss in some detail how exposure to microgravity may change the production and secretion of specific cell products. Such changes usually reflect important alterations of the signal transduction pathway that may be located at different steps such as the recognition of an activator or the genetic expression of a cytokine. Several techniques have been used in space experiments like the simple determination of proteins in the cell culture medium, the measurement of gene expression with RT-PCR technology, or with the modern and sophisticated microarray technology. Signal transduction is an extremely complicated process involving membrane receptors, G-proteins, the cytoskeleton, several protein kinases, transcription factors, and oncogenes. Many aspects of such process are still obscure and extensively studied worldwide. Microgravity may offer a new tool to study this subject from a new perspective.

Table 4-04 presents an overview of the most important data on genetic expression and metabolism of mammalian cells in space. Thereby it is important to distinguish between genetic expression determined as the amount of a protein (generally a cytokine) newly produced and secreted in response to a specific signal and, more properly, as the specific mRNA determined quanti- and qualitatively, with either the RT-PCR or the microarray technology.

It has been possible to identify the failure of the expression of the interleukin-2 receptor in T-lymphocytes as one of the possible causes of the loss of activation in microgravity. In a ground-based study conducted in the random positioning machine in our laboratory Walther could show, with the PCR-RT technology, that genetic expression of IL-2 is depressed at early time of mitogenic activation of T cells. Moreover, the genetic expression of the alpha subunit of the IL-2 receptor (IL-2Ra) is depressed in the random positioning machine while that of the beta subunit remains unchanged. A differential effect of simulated microgravity on the genetic expression of two strictly related components of the IL-2 receptor points to a direct effect of gravity on the activation mechanisms of T cells.

Investigations conducted by Hashemi et al. (1999) on human peripheral mononuclear cells in conditions of simulated microgravity (by means of a fast rotating clinostat) as well as in space have confirmed that the expression of IL-2Ra is inhibited at 0 g in T-cells exposed to anti-CD3 (a component of the TCR). However, such inhibition disappears in the clinostat and is partially restored in actual microgravity when activation is carried out with phorbol ester and calcium ionophore thus bypassing the TCR and Protein Kinase C (PKC). These data point to PKC as a possible key element of the sensitivity to gravity of T-cells and are in agreement with the findings on Jurlcat cells described below.

Cell type

Effect

Remarks

T lymphocytes with monocytes as accessory cells, human

500% increase of interferon-a secretion induced by various agents

Con A activation of cells attached to microcarrier beads: 2.5 fold increase in interferon-yproduction and 2 fold increase in production of IL-2.

Depression of genetic expression of IL-2 and IL-2 receptor a-unit, but not of /3-unit. Depression of genetic expression of IL-2 receptor a-unit and of CD69; depression restored bypassing TCR activation by phorbol ester and calcium ionophore

Salyut 6-incubator switched off during crew-sleep period: Spacelab-onboard 1- g control

Random positioning machine

Shuttle flight-onboard 1-g control

Monocytes as accessory cells in T-lymphocyte culture, human

Contradictory results:

Nearly total inhibition ofIL-1 production in resuspended cells: normal IL-1 secretion

Spacelab SLS-1 and IML-2, respectively—onboard 1-g control

Jurkat cells, human T cell line

Normal production of IL-2 after induction with anti-CD3 monoclonal antibodies in the presence of THP-1 cells: 100% inhibition of IL-2 production induced by calcium ionophor andphorbolester. The distribution ofPKC is altered in 0 g

Russian biosatellite

Spacelab IML-2-onboard 1-g control

THP-1, myelomonocytic cell line

Normal production of IL-1 ¡3 after induction with anti-CD3 monoclonal antibodies in the presence of Jurkat cells; 85% inhibition of IL-1 P production induced by phorbolester

Russian biosatellite

7E3 hybridoma cells

Production of monoclonal antibodies, consumption of glucose and glutamin as well as secretion of lactate and ammonia decreased

Spacelab IML-l-onboard 1-g control

Spleen cells, murine

Increased secretion of interferon-a upon stimulation with polyinosini-polycytidylic acid

Space Shuttle middeck, ambient temperature

B6MP102 macrophage cell line

Increased secretion of IL-1 and interferon-y induced by lipolysaccharide

Space Shuttle middeck, ambient temperature-no onboard 1-g control

Bone-marrow-derived macrophages, mice femora and tibiae

150% increase ofIL-6 secretion, up to 100% decrease of phenotypic marker expression of MHC-II and MAC-2

Space shuttle STS-57, -60, and-62-no onboard 1-g control-incubation between 23 and27°C

Friend leukemia-virus transformed cells, murine

No changes in metabolic behavior: glucose and glutamin consumption, production of haemoglobin, lactate and ammonia in dimethylsulfoxide-inducedproduction of hemoglobin.

Spacelab-onboard 1-g control

Table 4-04. Effect of spaceflight on signal transduction, genetic expression, and metabolism in mammalian cells.

Table 4-04. Effect of spaceflight on signal transduction, genetic expression, and metabolism in mammalian cells.

A number of interesting effects were observed by Chapes et al. (1992) in space cultures of three types of immune cells. However, the cultures were kept in the middeck of the Space Shuttle at ambient temperature throughout the incubation time instead of in an incubator at 37°C (an obvious requirement in work with mammalian cells), so the results must be interpreted with caution. The anchorage-dependent bone marrow-derived macrophage cell line B6MP102 secreted, upon activation with lipopolysaccharide, significantly more IL-1 and TNF-a in space than on the ground. Murine spleen cells, stimulated with poly I:C released significantly more IFN-a in space than on Earth. Also, human lymphocytes as well as murine lymph node T-cells activated with Con A produced significantly more IFN-g in space than on Earth. Experiments on Shuttle flight STS-50 found that cellular cytotoxicity caused by TNF-a was inhibited. This was confirmed in experiments on later flights (STS-54 and STS-57) and it was found that TNF mediated cytotoxicity was restored to levels observed in the ground controls in the presence of inhibitors of PKC. The authors conclude that spaceflight ameliorates the action of TNF by affecting PKC in target cells, but none of these experiments were accompanied by onboard 1-g controls (only ground controls were available) so what aspect of spaceflight is effective has not been established.

The metabolic data of an experiment with Hybridoma 7E3-N cells'2 in Spacelab IML-1 revealed another interesting behavior pattern: the production per cell of monoclonal antibodies, the glucose and glutamine consumption per cell, as well as the secretion per cell of waste products like lactate and ammonia were lower at 0 g than at 1 g. In fact, the lack of significant differences of metabolite concentrations in the supernatants at 0 g and 1 g is only apparent since approximately 40% more cells were present in the cultures at 0 g than in those at 1 g. Although there is not yet an explanation, the data show that gravitational unloading had significant effect on hybridoma cell metabolism. It appears that the transition from a two-dimensional configuration, as in the case of cells sedimented to the flat bottom of the culture flask at 1 g, to a three-dimensional configuration, as for free-floating cells at 0 g, increased cell proliferation despite a lower metabolic turnover. It appears also that the biosynthesis of a specific cell product was coupled to the glucose/glutamine consumption and to the lactate/ammonia secretion rather than to the proliferation rate.

12 Hybridoma is a type of hybrid cell produced by fusing a normal cell with a tumor cell. When lymphocytes (antibody-producing cells) are fused to the tumor cells, the resulting hybridomas produce antibodies and maintain rapid, sustained growth, producing large amounts of an antibody. Hybridomas are the source of monoclonal antibodies.

In an experiment with Friend cells during the IML-1 mission, the amount of hemoglobin produced upon induction with Dimethyl Sulfoxide (DMSO) was the same in the flight 0-g and ground 1-g samples. The counts of haemoglobin-positive cells show that 60 to 70% of the cells were induced to express haemoglobin upon exposure to DMSO. Again, there were no significant differences between cultures at 1 g and 0 g. The metabolic analyses on glucose and glutamine consumption, as well as on lactate and ammonia production, clearly reflected the fact that Friend cells do not change their behavior in microgravity.

Production of tissue plasminogen activator (t-PA) by hamster kidney cells was determined during the IML-1 mission. Tissue plasminogen activator is a substance of high pharmaceutical value since it is used to prevent the formation of blood clots where there is risk of thrombosis. There was no difference in metabolic data on t-PA production, data on the consumption of glucose and glutamine from the medium, nor on the secretion of waste products like ammonium and lactate between the cultures kept at 0 g and those kept at 1 g in the onboard centrifuge or in the ground laboratory.

Limouse et al. (1991) and deGroot et al. (1990, 1991) were the first researchers to investigate intermediate steps of signal transduction in space. The former suggested that the function of Protein Kinase C (PKC) is altered in Jurlcat cells exposed to 0 g. Hatton and Schmitt (1999) continued these studies, and showed that the intracellular distribution of PKC was changed in microgravity. The use of the RT-PCR technology was introduced for the first time in space experiments to study the activation of epidermoid cells by epidermal growth factor. A significant depression of the expression of the early oncogenes c-fos, c-jun was detected (deGroot et al. 1990, 1991). With the same technology, Kumei et al. (1996) were able to show that the amount of mRNA of the enzyme prostaglandin G/H synthase-2 is remarkably enhanced in rat osteaoblasts cultured in space. Akiyama et al. (1996) have developed a RT-PCR procedure tailored to the peculiar constraints of spaceflight, in particular to very low amounts of biological material. Thanks to this progress, the RT-PCR technology will certainly contribute to important investigations on future space missions.

Hammond et al. (1999) grew primary human renal cell cultures in a steady state 0-g environment onboard the STS-90 Neurolab mission for six days. Gene expression analysis using microarray technology was used to monitor gene expression. More than 1,632 genes changed at steady state.

The identification of clock genes in mammalian cells raises a new and exciting question: Does gravity and spaceflight in general interfere with the expression of clock genes? It is conceivable that altered gravitational conditions may have an influence on the mechanisms regulating the circadian rhythms. Experiments in such direction are planned on future missions.

3.3 Conclusions

Based on the experimental data outlined in the previous sections, we can answer a number of questions and draw some conclusions on the sensitivity to gravity at the cellular level:

a. The function of receptors (like the TCR) seems not to be influenced as shown by the normal binding of Con A to the T-cell membrane;

b. The membrane function is not affected as well as shown by the normal patching and capping of the membrane proteins interacting with Con A;

c. Cell-cell interactions and autonomous movements are occurring under 0 g conditions as shown by the experiments conducted with NIZEMI;

d. There are changes in the cytoskeleton and in cell shape as shown in several experiments. This may have an important impact on signal transduction as G-proteins, a pivotal element in the signal transduction pathway between receptor and protein lipase C are interacting with the cytoskeleton;

e. PKC is probably one of the key elements affected by altered gravity;

f. The consequence of all this is the depression of the genetic expression of IL-2 and IL-2Ra in T-cells and probably also of the oncogenes c-fos and c-myc in epidermoid cells (depression of oncogenes expression may also occur in T-cells, such experiments will be conducted soon in space);

g. The differential genetic expression under simulated microgravity in the random positioning machine of IL-2Ra (depressed) and of IL-2Ra (unchanged) is a strong argument in favor of direct effects of gravity at the cellular level;

h. It seems that cells undergoing differentiation processes are more sensitive to gravitational changes than cells, like cancer cells, that have reached the endpoint of their development.

4 SPACE RESEARCH IN CELL BIOLOGY: ISSUES

The results and impact of the findings in space biology are not well known to the majority of the scientific community. Main reasons are the limited access to space laboratories and the difficulty to repeat the experiments to confirm their results and to increase their statistical significance. Nevertheless, the data collected so far confirm the scientific, technological, and biomedical relevance of space biology. Some of the problems preventing a large community of scientists from conducting experiments in space are outlined here.

First, the access to space is limited. Only a small number of projects can be accommodated on board a Spacelab or SpaceHab mission. The consequence is that the statistical significance of the data is sometimes questionable and the reproducibility of important results is difficult to verify by independent team. For instance, less than 20 experiments were hosted in each of the seven Biorak flights. In addition, the number of flight opportunities in Spacelab, Mir, ISS, automated satellites, and sounding rockets is very low compared to the number of investigations proposed.

Second, the resources available in a space laboratory are very limited. Energy, weight and volume of the payload, as well as crew time have to be shared among several users from different disciplines, such as material and fluid sciences, medicine and biology. The incubation temperatures usually available are 22°C and 37°C. While the last value is adequate for all mammalian cells, 22°C is often offered as a compromise for "ambient temperature". Freezing conditions are limited to -10°C or -20°C, a large difference from the standard preservation conditions on ground, which usually include -80°C and -180°C. This means significant restriction of the manipulations, analytical procedures such as microscopic and biochemical determination, and controlled storage and stowage of biological samples in orbit. Another disturbing limitation is the so-called late access time, i.e., the latest time at which biological samples can be delivered for installation on board. This time ranges between 15 and 25 h before launch (See Chapter 3, Section 2.2). Also, several living probes must undergo special treatment in order to be viable for the processing in orbit. The consequence is that the flight experiment protocols are less sophisticated and comprehensive than those of equivalent investigations on Earth. ESA has supported programs of investigations to assess condition for optimum preservations of biological samples before and after experimentation in space. Another important issue is that of the proper controls. There is a consensus in the scientific community today that centrifuges providing 1-g in flight are necessary to control for all the other spaceflight environment factors, such as vibrations, accelerations, temperature fluctuations, and cosmic radiation typical. While Biorack was fitted with an onboard 1-g centrifuge, most of the experiments performed in other Shuttle flights lacked such control. Newer facilities like Biopack, Kubik, Biolab, and the Modular Cultivation System are equipped with centrifuges providing centrifugal accelerations ranging from 0 g to 1 g.

Third, the safety of the astronauts requires severe acceptance criteria for instruments and biological materials on board. For example, the tolerance limits for out-gassing of toxic or bad-smelling gases, and for electromagnetic contamination are extremely low; sharp edges must be avoided; and biological/chemical contamination's derived from viruses and bacteria or biological fluids must be prevented by independent triple containment

(Figure 4-24). Moreover, instruments shall not interfere, both electrically and acoustically, with each other.

Figure 4-24. ISS Expedition-6 Astronaut Kenneth D. Bowersox works with an experiment in a portable glovebox facility in the Destiny laboratory on the International Space Station. Photo courtesy of NASA.

Figure 4-24. ISS Expedition-6 Astronaut Kenneth D. Bowersox works with an experiment in a portable glovebox facility in the Destiny laboratory on the International Space Station. Photo courtesy of NASA.

Fourth, the period between the acceptance of a proposal and the flight of the experiment flight can span over several years. This is without counting the delay due to technical problems with the flight vehicles. For example, the first flight of the Space Shuttle took place in 1981 instead of 1978, as originally planned when the call for experiment proposals was issued. Another long delay followed the loss of Challenger in 1996, and the same holds true now after the loss of Columbia. The consequence is that many science proposals are obsolete at the time of flight. Requests of updates of flight protocols or new requirements during the preparation of the experiments are very difficult to have approved by the space agencies.

Fifth, failures due to instrument malfunctions, break-down of resources, and crew errors may even cause the total loss of an investigation prepared for years, often without an opportunity for a reflight.

5 CELL BIOLOGY IN SPACE: OUTLOOK

The Mir space station and Spacelab eras delivered invaluable lessons on how to carry out life and physical science research in space and on the management of emergency situations during spaceflight. Such know-how will be very useful during ISS operation in the next 20 years. ISS will be dedicated mainly to microgravity experimentation and technology. Cell and plant biology will play a prominent role. The European Modular Cultivation System (mainly for plants) and Biolab (mainly for cells) will host dozens of experiments. Whole experiment cycles will be repeated. Cells and plants will be cultivated over generations. Bioprocesses with interesting commercial return may develop. One biotechnological application will certainly consist of closed ecological life support systems aimed at the recycling of anthropogenetic water, carbon anhydride and other biological waste, and the production of fresh food (vegetables as well as animal) in space. A new ESA facility for cell biology experiments, called Biopack and designed to bridge the gap between Spacelab and Biolab on ISS, was lost with Columbia STS-107. Another instrument, called Kubik, will be used on ISS (see Figure 3-10), while the experiments will be transported to orbit by the Russian Soyuz spaceships.

In the meantime, several investigations are being carried out on the ground with the random positioning machine, or three-dimensional clinostat, invented by Hoson. Such a machine shall be used first to select biological systems suitable for basic investigations or for profitable bioprocesses in space and, second, to optimize experimental protocols of investigations selected for spaceflight.

The problems encountered in the preparation and execution of experiments in space shall not, however, discourage those scientists who might be interested to carry out experiments during spaceflight. The question these scientists should ask is the following: Am I ready to accept all the kind of hurdles to perform a space investigation that has great chances to fail, when the same resources used in a ground laboratories would allow the conducting of other interesting studies on the most challenging questions of today's biology?

Nevertheless, there are at least four good reasons motivating the efforts and the patience of space biologists. One is the scientific curiosity to expose living systems to conditions that have never been experienced before throughout evolution, such as microgravity and cosmic radiation. The unexpected and important results of several experiments show that even very simple organisms display drastic changes in microgravity. In this context, microgravity can be considered as a new tool to study complex biological mechanisms from a new perspective. For example, in cell cultures the transition from 1 g to 0 g changes the geometry of the system from a two-dimension to a three-dimension environment. Most living systems are thermodynamically very complicated non-equilibrium systems. Therefore, they may follow interesting bifurcations. Microgravity may favor a new path that is not even suspected in normal gravity. The study of such a path contributes to the clarification of unknown biological processes. As microgravity and cosmic radiation are not reproducible on Earth, the only way to perform this research is to go to space. Simulations in devices like clinostats are a useful and necessary complement, but not a replacement for space. In general, the data from ground-based simulations are qualitatively but not quantitatively similar to those obtained in space.

Another reason is to study specific physiological functions at the cellular level, either in vitro, i.e., in a test tube, or ex vivo, i.e., in cells drawn from tests subjects exposed to the conditions of spaceflight. Examples are the studies of the immune system with peripheral blood lymphocytes, or the bone system with condrocytes and osteoblasts. Such studies have shown evidence, for instance, of the effect of physical and psychological stress on the human immune system. This is a very interesting topic of neuroimmunology, a young discipline of growing importance in today's hectic life. Another intriguing problem is to determine the impact of gravity on the cell. Such question is obviously clearly answered in the case of the plants with their geotropism. It is also true that all major discoveries in cellular biology never took gravity into account.

A third reason is the technological return of space cell biology. The constraints of spaceflight result in high-tech challenges in the application of analytical techniques and in the development of flight instrumentation. Examples are the adaptation of the RT-PCR technology to the very small amounts of biological material available. In fact, the limits of weight and volume in orbit do not permit the use of the same samples or aliquots that biologists are accustomed to working with on ground. An example of sophisticated instrumentation is the development of the space bioreactor installed in Biorack. The introduction of microsensors, a new pH control system based on the electrolysis of water instead of the traditional neutralisation of acidity with NaOH, and piezoelectric micropump for fresh medium supply, opened new ways to the bioreactor technology.

In addition, basic research with single cells in space may show new perspectives in biotechnology and biochemical bioengineering. The fact that mammalian single cells undergo profound alterations in microgravity has nourished hypotheses and speculations on their possible commercial and medical applications. Bioprocessing in space is one promising theme for the commercial exploitation of the ISS. Several pharmaceutical companies have manifested their interest in joint application research programs with national and international space agencies. In Europe, ESA has started a Microgravity Application Program (MAP) to support application-oriented projects with participation of non-aerospace industries. An example of such activity is the first MAP project that started in May 2000 and that is aimed at the development of instruments, such as bioreactors, and technologies for tissue engineering. The objectives of the project are: to develop procedures of in vitro organogenesis of pancreatic islets, thyroid tissue, liver, vessels and cartilage; to study the mechanism of organogenesis in low-g; to define the requirements of a modular space bioreactor for medically relevant organ-like structures; and to set up procedures for the production of implants for medical applications. Experiments will begin in the random positioning machine on the ground and continue on board the ISS. There is also a strong support by NASA in the U.S. An example is the tissue-engineering project dedicated to cartilage conducted by Freed and Vunjak-Novalcovic (2002) at MIT in Boston (Figure 4-25).

Finally, the last but not least reason is the exploration of space. This includes trips to an Earth orbit as well to the planets of the Solar System and, in a far future, to other planetary systems. It is important that the adaptation of the physiological functions of humans and other mammals, as well as other organisms like plants, invertebrates, and microbes are investigated and clarified. It was and it will be an irresistible drive of our mankind to explore first all continents of the planet Earth and, later, any accessible site of the Universe as soon as the required technology becomes available. Space exploration includes also the search for extraterrestrial life. The study of terrestrial life out of the terrestrial environment will contribute to the identification and understanding of alien forms of life.

Figure 4-25. On board the SpaceHab module in the Space Shuttle Atlantis, astronauts Carl Walz and Jerome Apt analyze a bovine cartilage for its pH, C02, and ()-> content. Photo courtesy of NASA.
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