Grazia Galleri

Key gravity-sensitive signaling pathways drive T cell activation

Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity‐dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real‐time RT‐PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF‐κB, CREB, ELK, AP‐1, and STAT after crosslinking the T‐cell receptor contributes to T‐cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T‐cell activation. Since the majority of the genes were regulated by NF‐κB, CREB, and AP‐1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down‐regulated in vg. In contrast, PI3‐K, PKC, and its upstream regulator pLAT were not significantly down‐regulated by vectorless gravity. Since NF‐κB, AP‐1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T‐cell activation in altered gravity.

Detection of Mycoplasma agalactiae in sheep milk samples by polymerase chain reaction

We developed a simple and rapid method for DNA extraction from sheep milk to use for polymerase chain reaction (PCR) diagnosis of Mycoplasma agalactiae. We tested 357 samples from 21 newly infected flocks (group 1) and 87 samples from 8 flocks infected in the past (group 2). PCR results were compared with those of conventional culture. By PCR we detected 175 positives in group 1, while by culture we detected only 153. Milk samples from group 2 were negative, both by PCR assay and by culture. Our PCR is much faster than culture and reduces the time required for diagnosis from several days to 5 h. The method could be used for the routine diagnosis of contagious agalactia caused by Mycoplasma agalactiae.

The Rel/NF‐κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity

This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in μg. Immunosuppression during spaceflight is a major barrier to safe, long‐term human space habitation and travel. The goals of these experiments were to prove that μg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti‐CD28 on board the ISS. An on‐board centrifuge was used to generate a 1g simultaneous control to isolate the effects of μg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that μg‐ and 1g‐activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down‐regulated in μg. Importantly, several key immediate early genes were inhibited in μg. In particular, transactivation of Rel/NF‐κB, CREB, and SRF gene targets were down‐regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in μg and upon anti‐CD3/anti‐CD28 stimulation in simulated μg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in μg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that μg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes.

Cytoskeleton changes and impaired motility of monocytes at modelled low gravity

Summary.Investigations performed in space have shown that gravity changes affect important cellular mechanisms like proliferation, differentiation, genetic expression, cytoskeletal architecture, and motility in lymphocytes, monocytes, and other mammalian cells. In particular, a dramatic depression of the mitogenic in vitro activation of human peripheral blood lymphocytes was observed at low gravity. The hypothesis of the present work is that a reduced interaction between T lymphocytes and monocytes, essential for the second signalling pathway, might be one of the reasons for the observed depression of the in vitro activation of human lymphocytes. Cell motility and with it a continuous rearrangement of the cytoskeletal network within the cell is essential for cell-to-cell contacts. Whereas nonactivated lymphocytes in suspension are highly motile at low gravity, no data are available so far on the motility of adherent monocytes. It thus can be argued that impaired monocyte locomotion and cytoskeletal changes could be responsible for a reduced interaction of monocytes with T lymphocytes. In this study, the locomotion ability of J-111 cells, an adherent monocyte cell line, attached to colloidal gold particles on coverslips and exposed to modelled low gravity in the random positioning machine was found to be severely reduced compared with that of controls and the structures of actin, tubulin, and vinculin were affected.

Rapid and specific detection of Mycoplasma agalactiae by polymerase chain reaction.

A polymerase chain reaction (PCR)-based test was developed for the detection of Mycoplasma agalactiae in sheep milk samples. Two oligonucleotide primers were designed to amplify a 375 bp fragment of M. agalactiae chromosomal DNA. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization using a fluorescein labeled 528 bp probe. The primers allowed the amplification of fragment of M. agalactiae DNA and did not amplify any specific fragment of other mycoplasmal DNAs (M. capricolum, M. mycoides subsp. mycoides, M. mycoides subsp. capri, M. putrefaciens, M. arginini and M. bovis) or other bacterial DNAs (S. aureus, S. epidermidis, P. haemalytica, E. coli, S. agalactiae, S. dysgalactiae, S. uberis, B. cereus, P. aeruginosa, S. durans, L. lactis, L. lactis var. diacetilactis, L. mesenteroides, S. thermophilus, L. bulgaricus and L. casei). The limit of detection of PCR assay was between 2.5 and 25 fg of purified DNA and 10(2) CCU ml-1 on mycoplasma cultures. These results indicate that the PCR technique can be used as a rapid and specific diagnostic method for detection of M. agalactiae.

Creating conditions similar to those that occur during exposure of cells to microgravity induces apoptosis in human lymphocytes by 5‐lipoxygenase‐mediated mitochondrial uncoupling and cytochrome c release

Creating conditions similar to those that occur during exposure of cells to microgravity induced a sixfold increase of apoptotic bodies and DNA fragments in human lymphocytes, paralleled by an early (within 2 h) fourfold increase in 5‐lipoxygenase (5‐LOX) activity and a fivefold decrease in mitochondrial membrane potential and increase in cytochrome c release (within 4 and 8 h, respectively). Similar membrane potential and cytochrome c release were observed in isolated mitochondria treated with physiological amounts of 5‐LOX and were enhanced by creating conditions similar to those that occur during exposure of cells to microgravity. 5‐LOX inhibitors, 5,8,11,14‐eicosatetraynoic acid and caffeic acid, completely prevented apoptosis, whereas the phospholipase A2 inhibitor methyl‐arachidonoyl fluorophosphonate and the 5‐LOX activating protein inhibitor MK886 reduced it to 65–70%. The intracellular calcium chelator EGTA‐acetoxymethylester reduced 5‐LOX activity and apoptosis to 30–40% of controls, whereas the p38 mitogen‐activated protein kinase inhibitor SB203580 was ineffective. The caspase‐3 and caspase‐9 inhibitors Z‐Asp(OCH3)‐Glu(OCH3)‐Val‐Asp(OCH3)‐fluoromethylketone (FMK) and Z‐Leu‐Glu(OCH3)‐His‐Asp(OCH3)‐FMK reduced apoptotic bodies to 25–30% of the control cells. Finally, creating conditions similar to those that occur during exposure of cells to microgravity did not induce apoptosis in human lymphoma U937 cells, which did not express an active 5‐LOX.

Space flight affects motility and cytoskeletal structures in human monocyte cell line J-111.

Certain functions of immune cells in returning astronauts are known to be altered. A dramatic depression of the mitogenic in vitro activation of human lymphocytes was observed in low gravity. T‐cell activation requires the interaction of different type of immune cells as T‐lymphocytes and monocytes. Cell motility based on a continuous rearrangement of the cytoskeletal network within the cell is essential for cell–cell contacts. In this investigation on the International Space Station we studied the influence of low gravity on different cytoskeletal structures in adherent monocytes and their ability to migrate. J‐111 monocytes were incubated on a colloid gold substrate attached to a cover slide. Migrating cells removed the colloid gold, leaving a track recording cell motility. A severe reduction of the motility of J‐111 cells was found in low gravity compared to 1g in‐flight and ground controls. Cell shape appeared more contracted, whereas the control cells showed the typical morphology of migrating monocytes, i.e., elongated and with pseudopodia. A qualitative and quantitative analysis of the structures of F‐actin, β‐tubulin and vinculin revealed that exposure of J‐111 cells to low gravity affected the distribution of the different filaments and significantly reduced the fluorescence intensity of F‐actin fibers. Cell motility relies on an intact structure of different cytoskeletal elements. The highly reduced motility of monocytes in low gravity must be attributed to the observed severe disruption of the cytoskeletal structures and may be one of the reasons for the dramatic depression of the in vitro activation of human lymphocytes.

Enzyme Biosensors for Biomedical Applications: Strategies for Safeguarding Analytical Performances in Biological Fluids.

Enzyme-based chemical biosensors are based on biological recognition. In order to operate, the enzymes must be available to catalyze a specific biochemical reaction and be stable under the normal operating conditions of the biosensor. Design of biosensors is based on knowledge about the target analyte, as well as the complexity of the matrix in which the analyte has to be quantified. This article reviews the problems resulting from the interaction of enzyme-based amperometric biosensors with complex biological matrices containing the target analyte(s). One of the most challenging disadvantages of amperometric enzyme-based biosensor detection is signal reduction from fouling agents and interference from chemicals present in the sample matrix. This article, therefore, investigates the principles of functioning of enzymatic biosensors, their analytical performance over time and the strategies used to optimize their performance. Moreover, the composition of biological fluids as a function of their interaction with biosensing will be presented.

5-Lipoxygenase-dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment

The functional adaptation of the immune system to the surrounding environment is also a fundamental issue in space. It has been suggested that a decreased number of lymphocytes might be a cause of immunosuppression, possibly due to the induction of apoptosis. Early activation of 5‐lipoxygenase (5‐LOX) might play a central role in the initiation of the apoptotic program. The goal of the role of apoptosis in lymphocyte depression (ROALD) experiment, flown on the International Space Station as part of the BIO‐4 mission of the European Space Agency, was to ascertain the induction of apoptosis in human lymphocytes under authentic microgravity, and to elucidate the possible involvement of 5‐LOX. Our results demonstrate that exposure of human lymphocytes to microgravity for 48 h onboard the ISS remarkably increased apoptotic hallmarks such as DNA fragmentation (~3‐fold compared to ground‐based controls) and cleaved‐poly (ADP‐ribose) polymerase (PARP) protein expression (~3‐fold), as well as mRNA levels of apoptosis‐related markers such as p53 (~3‐fold) and calpain (~4‐fold); these changes were paralleled by an early increase of 5‐LOX activity (~2‐fold). Our findings provide a molecular background for the immune dysfunction observed in astronauts during space missions, and reveal potential new markers to monitor health status of ISS crew members.—Battista, N., Meloni, M. A., Bari, M., Mastrangelo, N., Galleri, G., Rapino, C., Dainese, E., Finazzi Agrò, A., Pippia, P., Maccarrone, M. 5‐Lipoxygenase‐dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment. FASEB J. 26, 1791‐1798 (2012). www.fasebj.org

Exposure of human lymphocytes and lymphoblastoid cells to simulated microgravity strongly affects energy metabolism and DNA repair

Exposure of freshly drawn lymphocytes and lymphoblastoid cells (LB and COR3) to simulated microgravity decreased the intracellular ATP concentration to 50%–40% of the value found in normal growth conditions. The decrease was reversible although recovery to normal values occurred only slowly both in lymphocytes and in lymphoblastoid cells. Poly(ADP‐ribose) polymerase (PARP ) activity was increased indicating that cells exposed to conditions of reduced gravitation experience stress. Exposure to microgravity forces cells into a condition of metabolic quiescence in which they appear to be particularly sensitive to subsequent exposures to a genotoxic agent. Thus, treatment of cells with the strong redox agent potassium bromate under microgravity conditions, indicated an impairment in repair of DNA 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), an oxidized derivative of deoxyguanosine. We conclude that gravitational modulation of the kind routinely obtained under laboratory conditions and during spaceflights is a stressful process to which cells appear to be extremely sensitive. These effects may reflect the physiological alterations observed in astronauts and in animals following spaceflights or exposure to conditions of simulated microgravity.