Biology: Molecular and cell biology

Cell and Molecular Biology

ISS Increment 23-24

2010

P.D. Hansen ⁽¹⁾, E. Unruh ⁽¹⁾, G. Horneck ⁽²⁾, P. Rettberg ⁽²⁾, G. Reitz ⁽²⁾, C. Panitz ⁽³⁾

BIOLAB

Aim
The aim of the experiment is to understand the mechanisms at the cellular level which underlie the following phenomena previously observed in spaceflight
- Impairment of immune functions under spaceflight conditions.
- Enhancement of responses to radiation in microgravity
- Clearly separate the effects of microgravity from other spaceflight factors by use of an onboard 1.g centrifuge.

Specific goals
1. The ability of leukocytes to phagocytose zymosan (as an analogue of bacteria) will be assessed. This process is the first line of defense against microbial infection. Phagocytosis will be quantified using luminol as a detector for reactive oxygen species produced during phagocytosis of zymosan.

General Description
Long term space missions present a number of risks for astronauts. Some effects of the space environment level appear to act at the cellular level and it is important to understand the underlying mechanisms of these effects. This project will focus on two aspects of cellular function which may have medical importance; (i) The synergy
between the effects of the space radiation environment and microgravity on cellular function & (ii) the impairment of immune functions under spaceflight conditions.
The Triplelux experiment consists of three components – Triplelux A, B & C. Three different cell types will be used; bacteria (Triplelux C), invertebrate hemocytes (Triplelux B) and a rat macrophages cell line (Triplelux A). The TRIPLELUX biosensor test will be used which will assay cellular responses by a bioluminescent or chemiluminescent reporter. This will be used for inflight measurements of gene expression (SOS lux reporter) and phagocytosis. By use of an onboard 1.g control, it will be possible to determine whether changes in these processes are caused by microgravity, radiation or a combination of these parameters.

Precursor flights
REPAIR IML-2 (STS 65)
KINETICS IML-2 (STS 65)
FUNGUS S/MM 05 (STS-81)

Related research:
Immunological and cellular reactions under influence of microgravity and cosmic radiation - The application of a biosensor with phagocytotic cells
56th ESA Parabolic Flight Campaign

The TRIPLELUX-B experiment : Immunological and cellular reactions under influence of microgravity
54th ESA Parabolic Flight Campaign

Macrophages and invertebrate hemocytes kill pathogens by phagocytosis (engulfment of pathogen) followed by a reactive oxygen burst to degrade the pathogen. This is illustrated in Fig.1.

The Triplelux experiment uses the reactive oxygen burst as measure of phagocytic activity of macrophages and hemocytes under spaceflight conditions. The reactive oxygen burst is measured by a chemiluminescent assay which where 02- radicals convert luminol to 3-Aminophthalate which results in the emission of light at 475nm,
this is illustrated below. Light emission is enhanced by the additional of exogenous hydrogen peroxide (Fig.2).

Store cells & frozen reagents until start of experiment (Fig.3)
1 – thaw stock cell cultures
2- inject stock culture into culture medium
3 – reconstitute stock culture in fresh medium, with possibility of stirring
4 – measure viability of culture
5 – transfer cultures to measurement chambers, begin cultivation
6 – Add peroxidase, zymosan & luminol to start bioluminescence
7 – measure light emission from the culture

General description of experiment protocol
Cells & selected reagents will be launched frozen and stored frozen on orbit until start of the experiment. Samples and reagents can be stored for several months on-orbit. The cells are thawed at ambient temperature (18-28°C), then experiment is run at 18°C (BIOLAB). A viability test is performed following reconstitution inside of CB (sample
taken from CB). Following the preincubation (reconstitution) period the cells are incubated with a mixture of zymosan, peroxidase& luminol. Bioluminescence associated with the phagocytosis of the zymosan particles is measured over an approximately 90 minute period. One set of samples is run under microgravity conditions & a second set on a 1.g centrifuge to permit discrimination between microgravity and radiation effects.

A frozen sample of cells is stored onboard & returned to ground for postflight experiments to assess the combined effect of cosmic radiation and exposure to microgravity. It is desirable to have the total radiation dose recorded by a passive dosimeter during the flight & an estimate of the radiation flux at the time of the experiment run.

Parameters measured
– In-flight parameters measured:
• Cell viability (downlinked video data or returned video tape)
• Data on luminescence of cultures at 485nm+/-30nm = measure of phagocytosis of Zymosan by  macrophages/granulocytes
(Downlinked)

– Post-flight parameters measured:
• Analysis of phagocytic activity of hemocytes stored frozen on ISS
• Accumulated radiation dose, recorded by dosimeter

Ground reference experiment(s)
Experiment run simultaneously if possible to maintain similar storage conditions. Protocol and experiment specific hardware identical to flight experiment, only 1.g conditions. A delay of two weeks will not cause any negative effect.

It is expected that the experiment will allow a clear discrimination between the effects of microgravity and radiation on DNA repair in bacteria and phagocytosis of zymosan by both mammalian and invertebrate leukocytes, by use of an onboard 1.g centrifuge control and measurement of accumulated radiation dose. Furthermore it will be possible to assess whether the effects of microgravity and radiation are synergistic (eg. does microgravity enhance the damaging effects of radiation by influencing the enzymatic repair reactions negatively?).

This study will provide useful information on how the spaceflight environment affects two cellular processes which are of importance for long term human space missions.

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Jason Hatton (e-mail: jason.hatton@esa.int)