Lost in Space? Unmasking the T Cell Reaction to Simulated Space Stressors.

The space environment will expose astronauts to stressors like ionizing radiation, altered gravity fields and elevated cortisol levels, which pose a health risk. Understanding how the interplay between these stressors changes T cells' response is important to better characterize space-related immune dysfunction. We have exposed stimulated Jurkat cells to simulated space stressors (1 Gy, carbon ions/1 Gy photons, 1 µM hydrocortisone (HC), Mars, moon, and microgravity) in a single or combined manner. Pro-inflammatory cytokine IL-2 was measured in the supernatant of Jurkat cells and at the mRNA level. Results show that alone, HC, Mars gravity and microgravity significantly decrease IL-2 presence in the supernatant. 1 Gy carbon ion irradiation showed a smaller impact on IL-2 levels than photon irradiation. Combining exposure to different simulated space stressors seems to have less immunosuppressive effects. Gene expression was less impacted at the time-point collected. These findings showcase a complex T cell response to different conditions and suggest the importance of elevated cortisol levels in the context of space flight, also highlighting the need to use simulated partial gravity technologies to better understand the immune system's response to the space environment.

Correction: Sallam et al. DNA Methylation Alterations in Fractionally Irradiated Rats and Breast Cancer Patients Receiving Radiotherapy. Int. J. Mol. Sci. 2022, 23, 16214.

Radia Tamarat and Susana Constantino Rosa Santos were not included as authors in the original publication [...].

The role of connexin proteins and their channels in radiation-induced atherosclerosis.

Radiotherapy is an effective treatment for breast cancer and other thoracic tumors. However, while high-energy radiotherapy treatment successfully kills cancer cells, radiation exposure of the heart and large arteries cannot always be avoided, resulting in secondary cardiovascular disease in cancer survivors. Radiation-induced changes in the cardiac vasculature may thereby lead to coronary artery atherosclerosis, which is a major cardiovascular complication nowadays in thoracic radiotherapy-treated patients. The underlying biological and molecular mechanisms of radiation-induced atherosclerosis are complex and still not fully understood, resulting in potentially improper radiation protection. Ionizing radiation (IR) exposure may damage the vascular endothelium by inducing DNA damage, oxidative stress, premature cellular senescence, cell death and inflammation, which act to promote the atherosclerotic process. Intercellular communication mediated by connexin (Cx)-based gap junctions and hemichannels may modulate IR-induced responses and thereby the atherosclerotic process. However, the role of endothelial Cxs and their channels in atherosclerotic development after IR exposure is still poorly defined. A better understanding of the underlying biological pathways involved in secondary cardiovascular toxicity after radiotherapy would facilitate the development of effective strategies that prevent or mitigate these adverse effects. Here, we review the possible roles of intercellular Cx driven signaling and communication in radiation-induced atherosclerosis.

DNA Methylation Alterations in Fractionally Irradiated Rats and Breast Cancer Patients Receiving Radiotherapy.

Radiation-Induced CardioVascular Disease (RICVD) is an important concern in thoracic radiotherapy with complex underlying pathophysiology. Recently, we proposed DNA methylation as a possible mechanism contributing to RICVD. The current study investigates DNA methylation in heart-irradiated rats and radiotherapy-treated breast cancer (BC) patients. Rats received fractionated whole heart X-irradiation (0, 0.92, 6.9 and 27.6 Gy total doses) and blood was collected after 1.5, 3, 7 and 12 months. Global and gene-specific methylation of the samples were evaluated; and gene expression of selected differentially methylated regions (DMRs) was validated in rat and BC patient blood. In rats receiving an absorbed dose of 27.6 Gy, DNA methylation alterations were detected up to 7 months with differential expression of cardiac-relevant DMRs. Of those, SLMAP showed increased expression at 1.5 months, which correlated with hypomethylation. Furthermore, E2F6 inversely correlated with a decreased global longitudinal strain. In BC patients, E2F6 and SLMAP exhibited differential expression directly and 6 months after radiotherapy, respectively. This study describes a systemic radiation fingerprint at the DNA methylation level, elucidating a possible association of DNA methylation to RICVD pathophysiology, to be validated in future mechanistic studies.

The CellBox-2 Mission to the International Space Station: Thyroid Cancer Cells in Space.

A spaceflight to the International Space Station (ISS) is a dream of many researchers. We had the chance to investigate the effect of real microgravity (CellBox-2 Space mission) on the transcriptome and proteome of FTC-133 human follicular thyroid cancer cells (TCC). The cells had been sent to the ISS by a Falcon 9 rocket of SpaceX CRS-13 from Cape Canaveral (United States) and cultured in six automated hardware units on the ISS before they were fixed and returned to Earth. Multicellular spheroids (MCS) were detectable in all spaceflight hardware units. The VCL, PXN, ITGB1, RELA, ERK1 and ERK2 mRNA levels were significantly downregulated after 5 days in space in adherently growing cells (AD) and MCS compared with ground controls (1g), whereas the MIK67 and SRC mRNA levels were both suppressed in MCS. By contrast, the ICAM1, COL1A1 and IL6 mRNA levels were significantly upregulated in AD cells compared with 1g and MCS. The protein secretion measured by multianalyte profiling technology and enzyme-linked immunosorbent assay (AngiogenesisMAP®, extracellular matrix proteins) was not significantly altered, with the exception of elevated angiopoietin 2. TCC in space formed MCS, and the response to microgravity was mainly anti-proliferative. We identified ERK/RELA as a major microgravity regulatory pathway.

Unloading-Induced Cortical Bone Loss is Exacerbated by Low-Dose Irradiation During a Simulated Deep Space Exploration Mission.

Spaceflight-induced bone losses have been reliably reproduced in Hind-Limb-Unloading (HLU) rodent models. However, a considerable knowledge gap exists regarding the effects of low-dose radiation and microgravity together. Ten-week-old male C57BL/6J mice, randomly allocated to Control (CONT), Hind-Limb Unloading (HLU), and Hind-Limb Unloading plus Irradiation (HLUIR), were acclimatized at 28 °C, close to thermoneutral temperature, for 28 days prior to the 14-day HLU protocol. HLUIR mice received a 25 mGy dose of X-ray irradiation, simulating 14 days of exposure to the deep space radiation environment, on day 7 of the HLU protocol. Trabecular bone mass was similarly reduced in HLU and HLUIR mice when compared to CONT, with losses driven by osteoclastic bone resorption rather than changes to osteoblastic bone formation. Femoral cortical thickness was reduced only in the HLUIR mice (102 µm, 97.5-107) as compared to CONT (108.5 µm, 102.5-120.5). Bone surface area was also reduced only in the HLUIR group, with no difference between HLU and CONT. Cortical losses were driven by osteoclastic resorption on the posterior endosteal surface of the distal femoral diaphysis, with no increase in the numbers of dead osteocytes. In conclusion, we show that low-dose radiation exposure negatively influences bone physiology beyond that induced by microgravity alone.

An appraisal of folates as key factors in cognition and ageing-related diseases.

Folic acid (FA) is often consumed as a food supplement and can be found in fortified staple foods in various western countries. Even though FA supplementation during pregnancy is known to prevent severe congenital anomalies in the developing child (e.g., neural tube defects), much less is known about its influence on cognition and neurological functioning. In this review, we address the advances in this field and situate how folate intake during pregnancy, postnatal life, adulthood and in the elderly affects cognition. In addition, an association between folate status and ageing, dementia and other neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis is discussed. While its role in the incidence and severity of these diseases is becoming apparent, the underlying action of folates and related metabolites remains elusive. Finally, the potential of FA as a nutraceutical has been proposed, although the efficacy will highly depend on the interplay with other micronutrients, the disease stage and the duration of supplementation. Hence, the lack of consistent data urges for more animal studies and (pre)clinical trials in humans to ascertain a potential beneficial role for folates in the treatment or amelioration of cognitive decline and ageing-related disorders.

Pathological effects of ionizing radiation: endothelial activation and dysfunction.

The endothelium, a tissue that forms a single layer of cells lining various organs and cavities of the body, especially the heart and blood as well as lymphatic vessels, plays a complex role in vascular biology. It contributes to key aspects of vascular homeostasis and is also involved in pathophysiological processes, such as thrombosis, inflammation, and hypertension. Epidemiological data show that high doses of ionizing radiation lead to cardiovascular disease over time. The aim of this review is to summarize the current knowledge on endothelial cell activation and dysfunction after ionizing radiation exposure as a central feature preceding the development of cardiovascular diseases.

Regional vulnerability and spreading of hyperphosphorylated tau in seeded mouse brain.

We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool.

Growth of Endothelial Cells in Space and in Simulated Microgravity - a Comparison on the Secretory Level.

BACKGROUND/AIMS: Endothelial cells exposed to the Random Positioning Machine (RPM) reveal three different phenotypes. They grow as a two-dimensional monolayer and form three-dimensional (3D) structures such as spheroids and tubular constructs. As part of the ESA-SPHEROIDS project we want to understand how endothelial cells (ECs) react and adapt to long-term microgravity. METHODS: During a spaceflight to the International Space Station (ISS) and a subsequent stay onboard, human ECs (EA.hy926 cell line) were cultured for 12 days in real microgravity inside an automatic flight hardware, specially designed for use in space. ECs were cultivated in the absence or presence of vascular endothelial growth factor, which had demonstrated a cell-protective effect on ECs exposed to an RPM simulating microgravity. After cell fixation in space and return of the samples, we examined cell morphology and analyzed supernatants by Multianalyte Profiling technology. RESULTS: The fixed samples comprised 3D multicellular spheroids and tube-like structures in addition to monolayer cells, which are exclusively observed during growth under Earth gravity (1g). Within the 3D aggregates we detected enhanced collagen and laminin. The supernatant analysis unveiled alterations in secretion of several growth factors, cytokines, and extracellular matrix components as compared to cells cultivated at 1g or on the RPM. This confirmed an influence of gravity on interacting key proteins and genes and demonstrated a flight hardware impact on the endothelial secretome. CONCLUSION: Since formation of tube-like aggregates was observed only on the RPM and during spaceflight, we conclude that microgravity may be the major cause for ECs' 3D aggregation.

Immune sensitization during 1 year in the Antarctic high-altitude Concordia Environment.

BACKGROUND: Antarctica is a challenging environment for humans. It serves as a spaceflight ground analog, reflecting some conditions of long-duration exploration class space missions. The French-Italian Concordia station in interior Antarctica is a high-fidelity analog, located 1000 km from the coast, at an altitude of 3232 m. The aim of this field study was to characterize the extent, dynamics, and key mechanisms of the immune adaptation in humans overwintering at Concordia for 1 year. METHODS: This study assessed immune functions in fourteen crewmembers. Quantitative and phenotypic analyses from human blood were performed using onsite flow cytometry together with specific tests on receptor-dependent and receptor-independent functional innate and adaptive immune responses. Transcriptome analyses and quantitative identification of key response genes were assessed. RESULTS: Dynamic immune activation and a two-step escalation/activation pattern were observed. The early phase was characterized by moderately sensitized global immune responses, while after 3-4 months, immune responses were highly upregulated. The cytokine responses to an ex vivo stimulation were markedly raised above baseline levels. These functional observations were reflected at the gene transcriptional level in particular through the modulation of hypoxia-driven pathways. CONCLUSIONS: This study revealed unique insights into the extent, dynamics, and genetics of immune dysfunctions in humans exposed for 1 year to the Antarctic environment at the Concordia station. The scale of immune function was imbalanced toward a sensitizing of inflammatory pathways.

Modulation of Iberian Ribbed Newt Complement Component C3 by Stressors Similar to those Encountered during a Stay Onboard the International Space Station.

The complement system plays an important role in inflammation, innate and acquired immunity, as well as homeostasis. Despite these functions, the effects of spaceflight conditions on the complement system have not yet been intensively studied. Consequently, we investigated the effects of five types of chronic stressors, similar to those encountered during a stay onboard the International Space Station, on C3 expression in larvae of the urodele amphibian Pleurodeles waltl. We focused on C3 because it is a critical component of this system. These studies were completed by the analysis of adult mice exposed to two models of inflight stressors. Our data show that simulating space radiation, or combining a modification of the circadian rhythm with simulated microgravity, affects the amount of C3 proteins. These results suggest that C3 expression could be modified under real spaceflight conditions, potentially increasing the risk of inflammation and associated tissue damage.

Calcium, oxidative stress and connexin channels, a harmonious orchestra directing the response to radiotherapy treatment?

Although radiotherapy is commonly used to treat cancer, its beneficial outcome is frequently hampered by the radiation resistance of tumor cells and adverse reactions in normal tissues. Mechanisms of cell-to-cell communication and how intercellular signals are translated into cellular responses, have become topics of intense investigation, particularly within the field of radiobiology. A substantial amount of evidence is available demonstrating that both gap junctional and paracrine communication pathways can propagate radiation-induced biological effects at the intercellular level, commonly referred to as radiation-induced bystander effects (RIBE). Multiple molecular signaling mechanisms involving oxidative stress, kinases, inflammatory molecules, and Ca2+ are postulated to contribute to RIBE. Ca2+ is a highly versatile and ubiquitous second messenger that regulates diverse cellular processes via the interaction with various signaling cascades. It furthermore provides a fast system for the dissemination of information at the intercellular level. Channels formed by transmembrane connexin (Cx) proteins, i.e. hemichannels and gap junction channels, can mediate the cell-to-cell propagation of increases in intracellular Ca2+ by ministering paracrine and direct cell-cell communication, respectively. We here review current knowledge on radiation-induced signaling mechanisms in irradiated and bystander cells, particularly focusing on the contribution of oxidative stress, Ca2+ and Cx channels. By illustrating the tight interplay between these different partners, we provide a conceptual framework for intercellular Ca2+ signaling as a key player in modulating the RIBE and the overall response to radiation.

Current Evidence for Developmental, Structural, and Functional Brain Defects following Prenatal Radiation Exposure.

Ionizing radiation is omnipresent. We are continuously exposed to natural (e.g., radon and cosmic) and man-made radiation sources, including those from industry but especially from the medical sector. The increasing use of medical radiation modalities, in particular those employing low-dose radiation such as CT scans, raises concerns regarding the effects of cumulative exposure doses and the inappropriate utilization of these imaging techniques. One of the major goals in the radioprotection field is to better understand the potential health risk posed to the unborn child after radiation exposure to the pregnant mother, of which the first convincing evidence came from epidemiological studies on in utero exposed atomic bomb survivors. In the following years, animal models have proven to be an essential tool to further characterize brain developmental defects and consequent functional deficits. However, the identification of a possible dose threshold is far from complete and a sound link between early defects and persistent anomalies has not yet been established. This review provides an overview of the current knowledge on brain developmental and persistent defects resulting from in utero radiation exposure and addresses the many questions that still remain to be answered.

Gallium-68-labelled NOTA-oligonucleotides: an optimized method for their preparation.

One of the most essential aspects to the success of radiopharmaceuticals is an easy and reliable radiolabelling protocol to obtain pure and stable products. In this study, we optimized the bioconjugation and gallium-68 ((68) Ga) radiolabelling conditions for a single-stranded 40-mer DNA oligonucleotide, in order to obtain highly pure and stable radiolabelled oligonucleotides. Quantitative bioconjugation was obtained for a disulfide-functionalized oligonucleotide conjugated to the macrocylic bifunctional chelator MMA-NOTA (maleimido-mono-amide (1,4,7-triazanonane-1,4,7-triyl)triacetic acid). Next, this NOTA-oligonucleotide bioconjugate was radiolabelled at room temperature with purified and pre-concentrated (68) Ga with quantitative levels of radioactive incorporation and high radiochemical and chemical purity. In addition, high chelate stability was observed in physiological-like conditions (37 °C, PBS and serum), in the presence of a transchelator (EDTA) and transferrin. A specific activity of 51.1 MBq/nmol was reached using a 1470-fold molar excess bioconjugate over (68) Ga. This study presents a fast, straightforward and reliable protocol for the preparation of (68) Ga-radiolabelled DNA oligonucleotides under mild reaction conditions and without the use of organic solvents. The methodology herein developed will be applied to the preparation of oligonucleotidic sequences (aptamers) targeting the human epidermal growth factor receptor 2 (HER2) for cancer imaging.

Concerted Uranium Research in Europe (CURE): toward a collaborative project integrating dosimetry, epidemiology and radiobiology to study the effects of occupational uranium exposure.

The potential health impacts of chronic exposures to uranium, as they occur in occupational settings, are not well characterized. Most epidemiological studies have been limited by small sample sizes, and a lack of harmonization of methods used to quantify radiation doses resulting from uranium exposure. Experimental studies have shown that uranium has biological effects, but their implications for human health are not clear. New studies that would combine the strengths of large, well-designed epidemiological datasets with those of state-of-the-art biological methods would help improve the characterization of the biological and health effects of occupational uranium exposure. The aim of the European Commission concerted action CURE (Concerted Uranium Research in Europe) was to develop protocols for such a future collaborative research project, in which dosimetry, epidemiology and biology would be integrated to better characterize the effects of occupational uranium exposure. These protocols were developed from existing European cohorts of workers exposed to uranium together with expertise in epidemiology, biology and dosimetry of CURE partner institutions. The preparatory work of CURE should allow a large scale collaborative project to be launched, in order to better characterize the effects of uranium exposure and more generally of alpha particles and low doses of ionizing radiation.

EPI-CT: design, challenges and epidemiological methods of an international study on cancer risk after paediatric and young adult CT.

Computed tomography (CT) has great clinical utility and its usage has increased dramatically over the years. Concerns have been raised, however, about health impacts of ionising radiation exposure from CTs, particularly in children, who have a higher risk for some radiation induced diseases. Direct estimation of the health impact of these exposures is needed, but the conduct of epidemiological studies of paediatric CT populations poses a number of challenges which, if not addressed, could invalidate the results. The aim of the present paper is to review the main challenges of a study on the health impact of paediatric CTs and how the protocol of the European collaborative study EPI-CT, coordinated by the International Agency for Research on Cancer (IARC), is designed to address them. The study, based on a common protocol, is being conducted in Belgium, Denmark, France, Germany, the Netherlands, Norway, Spain, Sweden and the United Kingdom and it has recruited over one million patients suitable for long-term prospective follow-up. Cohort accrual relies on records of participating hospital radiology departments. Basic demographic information and technical data on the CT procedure needed to estimate organ doses are being abstracted and passive follow-up is being conducted by linkage to population-based cancer and mortality registries. The main issues which may affect the validity of study results include missing doses from other radiological procedures, missing CTs, confounding by CT indication and socioeconomic status and dose reconstruction. Sub-studies are underway to evaluate their potential impact. By focusing on the issues which challenge the validity of risk estimates from CT exposures, EPI-CT will be able to address limitations of previous CT studies, thus providing reliable estimates of risk of solid tumours and leukaemia from paediatric CT exposures and scientific bases for the optimisation of paediatric CT protocols and patient protection.

MorphoNeuroNet: an automated method for dense neurite network analysis.

High content cell-based screens are rapidly gaining popularity in the context of neuronal regeneration studies. To analyze neuronal morphology, automatic image analysis pipelines have been conceived, which accurately quantify the shape changes of neurons in cell cultures with non-dense neurite networks. However, most existing methods show poor performance for well-connected and differentiated neuronal networks, which may serve as valuable models for inter alia synaptogenesis. Here, we present a fully automated method for quantifying the morphology of neurons and the density of neurite networks, in dense neuronal cultures, which are grown for more than 10 days. MorphoNeuroNet, written as a script for ImageJ, Java based freeware, automatically determines various morphological parameters of the soma and the neurites (size, shape, starting points, and fractional occupation). The image analysis pipeline consists of a multi-tier approach in which the somas are segmented by adaptive region growing using nuclei as seeds, and the neurites are delineated by a combination of various intensity and edge detection algorithms. Quantitative comparison showed a superior performance of MorphoNeuroNet to existing analysis tools, especially for revealing subtle changes in thin neurites, which have weak fluorescence intensity compared to the rest of the network. The proposed method will help determining the effects of compounds on cultures with dense neurite networks, thereby boosting physiological relevance of cell-based assays in the context of neuronal diseases.