High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-ß7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

Effect of radiotherapy on the DNA cargo and cellular uptake mechanisms of extracellular vesicles.

In the past decades, plenty of evidence has gathered pointing to the role of extracellular vesicles (EVs) secreted by irradiated cells in the development of radiation-induced non-targeted effects. EVs are complex natural structures composed of a phospholipid bilayer which are secreted by virtually all cells and carry bioactive molecules. They can travel certain distances in the body before being taken up by recipient cells. In this review we discuss the role and fate of EVs in tumor cells and highlight the importance of DNA specimens in EVs cargo in the context of radiotherapy. The effect of EVs depends on their cargo, which reflects physiological and pathological conditions of donor cell types, but also depends on the mode of EV uptake and mechanisms involved in the route of EV internalization. While the secretion and cargo of EVs from irradiated cells has been extensively studied in recent years, their uptake is much less understood. In this review, we will focus on recent knowledge regarding the EV uptake of cancer cells and the effect of radiation in this process.

The miRNA Content of Bone Marrow-Derived Extracellular Vesicles Contributes to Protein Pathway Alterations Involved in Ionising Radiation-Induced Bystander Responses.

Extracellular vesicles (EVs), through their cargo, are important mediators of bystander responses in the irradiated bone marrow (BM). MiRNAs carried by EVs can potentially alter cellular pathways in EV-recipient cells by regulating their protein content. Using the CBA/Ca mouse model, we characterised the miRNA content of BM-derived EVs from mice irradiated with 0.1 Gy or 3 Gy using an nCounter analysis system. We also analysed proteomic changes in BM cells either directly irradiated or treated with EVs derived from the BM of irradiated mice. Our aim was to identify key cellular processes in the EV-acceptor cells regulated by miRNAs. The irradiation of BM cells with 0.1 Gy led to protein alterations involved in oxidative stress and immune and inflammatory processes. Oxidative stress-related pathways were also present in BM cells treated with EVs isolated from 0.1 Gy-irradiated mice, indicating the propagation of oxidative stress in a bystander manner. The irradiation of BM cells with 3 Gy led to protein pathway alterations involved in the DNA damage response, metabolism, cell death and immune and inflammatory processes. The majority of these pathways were also altered in BM cells treated with EVs from mice irradiated with 3 Gy. Certain pathways (cell cycle, acute and chronic myeloid leukaemia) regulated by miRNAs differentially expressed in EVs isolated from mice irradiated with 3 Gy overlapped with protein pathway alterations in BM cells treated with 3 Gy EVs. Six miRNAs were involved in these common pathways interacting with 11 proteins, suggesting the involvement of miRNAs in the EV-mediated bystander processes. In conclusion, we characterised proteomic changes in directly irradiated and EV-treated BM cells, identified processes transmitted in a bystander manner and suggested miRNA and protein candidates potentially involved in the regulation of these bystander processes.

Extracellular Vesicles in Modifying the Effects of Ionizing Radiation.

Extracellular vesicles (EVs) are membrane-coated nanovesicles actively secreted by almost all cell types. EVs can travel long distances within the body, being finally taken up by the target cells, transferring information from one cell to another, thus influencing their behavior. The cargo of EVs comprises of nucleic acids, lipids, and proteins derived from the cell of origin, thereby it is cell-type specific; moreover, it differs between diseased and normal cells. Several studies have shown that EVs have a role in tumor formation and prognosis. It was also demonstrated that ionizing radiation can alter the cargo of EVs. EVs, in turn can modulate radiation responses and they play a role in radiation-induced bystander effects. Due to their biocompatibility and selective targeting, EVs are suitable nanocarrier candidates of drugs in various diseases, including cancer. Furthermore, the cargo of EVs can be engineered, and in this way they can be designed to carry certain genes or even drugs, similar to synthetic nanoparticles. In this review, we describe the biological characteristics of EVs, focusing on the recent efforts to use EVs as nanocarriers in oncology, the effects of EVs in radiation therapy, highlighting the possibilities to use EVs as nanocarriers to modulate radiation effects in clinical applications.

In Vivo Irradiation of Mice Induces Activation of Dendritic Cells.

It is becoming clear that ionizing radiation positively influences certain immune parameters, which opens the possibility for combining radio- and immunotherapies in cancer treatment. The presence of functionally competent dendritic cells (DCs) is crucial in mounting a successful antitumor immune response. While it has been shown that DCs are relatively radioresistant, few and contradictory data are available on how ionizing radiation alters the functional integrity of these cells. Therefore, our objective was to investigate the effect of whole-body irradiation on the function of splenic DCs. C57Bl/6 mice were irradiated with 0.1, 0.25, and 2 Gy X-rays and changes in the phenotype of splenic DCs were compared to unirradiated controls. An increase was seen in DC surface markers influencing DC-T cell interactions. In vivo cytokine production was determined by direct intracellular cytokine staining. Irradiation with 2 Gy induced a 1.6-fold increase in IL-1α production, while the combination of irradiation and lipopolysaccharide (LPS) treatment induced a 3.9-fold increase, indicating a strong synergism between irradiation and LPS stimulation. Interaction of DCs with effector and regulatory T cells was investigated in a mixed lymphocyte reaction. While DCs from control animals induced stronger proliferation of regulatory T cells, DCs from animals irradiated with 2 Gy induced stronger proliferation of effector T cells. Antigen uptake and presentation was investigated by measuring the capacity of DCs to internalize and present ovalbumine (OVA)-derived peptides on their major histocompatibility complex (MHCI) molecules. Irradiation with 2 Gy did not influence antigen uptake or presentation, while low doses stimulated antigen uptake and reduced the level of antigen presentation. In conclusion, high-dose in vivo irradiation induced increased expression of T cell costimulatory markers, enhanced production of proinflammatory cytokines and a stronger stimulation of effector T cell proliferation than that of regulatory T cells. However, it did not influence DC antigen uptake or presentation. On the other hand, low-dose irradiation increased antigen uptake and lowered antigen presentation of DCs, indicating that low- and high-dose irradiation act on different pathways in DCs.

[The effect of radiotherapy on the antitumor immune response. Possibilities to combine radiotherapy with immunotherapy]. / A sugárterápia hatása a daganatellenes immunválaszra.

The past three decades of immunology research led to a drastic increase in the knowledge of antitumor immune response mechanisms and in parallel to a rapid development in various antitumor immune therapy strategies. This will most probably result in the implementation of immunotherapeutic protocols within the standard anticancer regimens in a very near future. Though, it is obvious that combination of immunotherapy with traditional anticancer treatment modalities will only be legitimate if the combination has at least an additive, or perhaps a synergistic effect. The similarly dynamic progress in the radiobiological knowledge proved that ionizing radiation does not have a general immune suppressing effect, as it has been thought for decades, but might possess certain immune stimulatory effects, as well. It is also known by now that local irradiation due to its out-of-field effects has systemic immune modulatory capacity, too. In the light of all these novel findings the optimal combination between antitumor immunotherapy and radiotherapy has become an increasing option. The first part of the present review summarizes the main antitumor mechanisms that can be influenced by ionizing radiation, and the second part attempts to provide a comprehensive overview of those antitumor immunotherapeutic modalities that are currently being used in combination with radiotherapy in preclinical and/or clinical trials for the treatment of various tumors.

TP53inp1 Gene Is Implicated in Early Radiation Response in Human Fibroblast Cells.

Tumor protein 53-induced nuclear protein-1 (TP53inp1) is expressed by activation via p53 and p73. The purpose of our study was to investigate the role of TP53inp1 in response of fibroblasts to ionizing radiation. γ-Ray radiation dose-dependently induces the expression of TP53inp1 in human immortalized fibroblast (F11hT) cells. Stable silencing of TP53inp1 was done via lentiviral transfection of shRNA in F11hT cells. After irradiation the clonogenic survival of TP53inp1 knockdown (F11hT-shTP) cells was compared to cells transfected with non-targeting (NT) shRNA. Radiation-induced senescence was measured by SA-ß-Gal staining and autophagy was detected by Acridine Orange dye and microtubule-associated protein-1 light chain 3 (LC3B) immunostaining. The expression of TP53inp1, GDF-15, and CDKN1A and alterations in radiation induced mitochondrial DNA deletions were evaluated by qPCR. TP53inp1 was required for radiation (IR) induced maximal elevation of CDKN1A and GDF-15 expressions. Mitochondrial DNA deletions were increased and autophagy was deregulated following irradiation in the absence of TP53inp1. Finally, we showed that silencing of TP53inp1 enhances the radiation sensitivity of fibroblast cells. These data suggest functional roles for TP53inp1 in radiation-induced autophagy and survival. Taken together, we suppose that silencing of TP53inp1 leads radiation induced autophagy impairment and induces accumulation of damaged mitochondria in primary human fibroblasts.

Structural analysis of oval-cell-mediated liver regeneration in rats.

UNLABELLED: We have analyzed the architectural aspects of progenitor-cell-driven regenerative growth in rat liver by applying the 2-acetaminofluorene/partial hepatectomy experimental model. The regeneration is initiated by the proliferation of so-called oval cells. The oval cells at the proximal tips of the ductules have a more differentiated phenotype and higher proliferative rate. This preferential growth results in the formation of a seemingly random collection of small hepatocytes, called foci. These foci have no clonal origin, but possess a highly organized structure, which shows similarities to normal hepatic parenchyma. Therefore, they can easily remodel into the lobular structure. Eventually, the regenerated liver is constructed by enlarged hepatic lobules; no new lobules are formed during this process. The foci of the Solt-Farber experimental hepatocarcinogenesis model have identical morphological features; accordingly, they also represent only regenerative, not neoplastic, growth. CONCLUSION: Progenitor-cell-driven liver regeneration is a well-designed, highly organized tissue reaction, and better comprehension of the architectural events may help us to recognize this process and understand its role in physiological and pathological reactions.

The effect of ionizing radiation on the homeostasis and functional integrity of murine splenic regulatory T cells.

OBJECTIVE: Radiotherapy affects antitumor immune responses; therefore, it is important to study radiation effects on various compartments of the immune system. Here we report radiation effects on the homeostasis and function of regulatory T (Treg) cells, which are important in down-regulating antitumor immune responses. METHODS: C57Bl/6 mice were irradiated with 2 Gy and alterations in splenic lymphocyte fractions analyzed at different intervals. RESULTS: Total CD4+ numbers showed stronger decrease after irradiation than CD4+Foxp3+ Tregs. Tregs were less prone to radiation-induced apoptosis than CD4+Foxp3- T cells. The ratio of CD4+Foxp3- and CD4+Foxp3+ fractions within the proliferating CD4+ pool progressively changed from 74:26 in control animals to 59:41 eleven days after irradiation, demonstrating a more dynamic increase in the proliferation and regeneration of the Treg pool. The CD4+Foxp3+ fraction expressing cell-surface CTLA4, an antigen associated with Treg cell activation increased from 5.3 % in unirradiated mice to 10.5 % three days after irradiation. The expression of IL-10 mRNA was moderately upregulated, while TGF-ß expression was not affected. On the other hand, irradiation reduced Treg capacity to suppress effector T cell proliferation by 2.5-fold. CONCLUSION: Tregs are more radioresistant, less prone to radiation-induced apoptosis, and have faster repopulation kinetics than CD4+Foxp3- cells, but irradiated Tregs are functionally compromised, having a reduced suppressive capacity.

Extracellular Vesicles Derived from Bone Marrow in an Early Stage of Ionizing Radiation Damage Are Able to Induce Bystander Responses in the Bone Marrow.

Ionizing radiation (IR)-induced bystander effects contribute to biological responses to radiation, and extracellular vesicles (EVs) play important roles in mediating these effects. In this study we investigated the role of bone marrow (BM)-derived EVs in the bystander transfer of radiation damage. Mice were irradiated with 0.1Gy, 0.25Gy and 2Gy, EVs were extracted from the BM supernatant 24 h or 3 months after irradiation and injected into bystander mice. Acute effects on directly irradiated or EV-treated mice were investigated after 4 and 24 h, while late effects were investigated 3 months after treatment. The acute effects of EVs on the hematopoietic stem and progenitor cell pools were similar to direct irradiation effects and persisted for up to 3 months, with the hematopoietic stem cells showing the strongest bystander responses. EVs isolated 3 months after irradiation elicited no bystander responses. The level of seven microRNAs (miR-33a-3p, miR-140-3p, miR-152-3p, miR-199a-5p, miR-200c-5p, miR-375-3p and miR-669o-5p) was altered in the EVs isolated 24 hour but not 3 months after irradiation. They regulated pathways highly relevant for the cellular response to IR, indicating their role in EV-mediated bystander responses. In conclusion, we showed that only EVs from an early stage of radiation damage could transmit IR-induced bystander effects.

Prostate Cancer Survivors Present Long-Term, Residual Systemic Immune Alterations.

BACKGROUND: The development of cancer and anti-tumor therapies can lead to systemic immune alterations but little is known about how long immune dysfunction persists in cancer survivors. METHODS: We followed changes in the cellular immune parameters of prostate cancer patients with good prognostic criteria treated with low dose rate brachytherapy before and up to 3 years after the initiation of therapy. RESULTS: Patients before therapy had a reduced CD4+ T cell pool and increased regulatory T cell fraction and these alterations persisted or got amplified during the 36-month follow-up. A significant decrease in the total NK cell number and a redistribution of the circulating NK cells in favor of a less functional anergic subpopulation was seen in patients before therapy but tumor regression led to the regeneration of the NK cell pool and functional integrity. The fraction of lymphoid DCs was increased in patients both before therapy and throughout the whole follow-up. Increased PDGF-AA, BB, CCL5 and CXCL5 levels were measured in patients before treatment but protein levels rapidly normalized. CONCLUSIONS: while NK cell dysfunction recovered, long-term, residual alterations persisted in the adaptive and partly in the innate immune system.

Analysis of the common deletions in the mitochondrial DNA is a sensitive biomarker detecting direct and non-targeted cellular effects of low dose ionizing radiation.

One of the key issues of current radiation research is the biological effect of low doses. Unfortunately, low dose science is hampered by the unavailability of easily performable, reliable and sensitive quantitative biomarkers suitable detecting low frequency alterations in irradiated cells. We applied a quantitative real time polymerase chain reaction (qRT-PCR) based protocol detecting common deletions (CD) in the mitochondrial genome to assess direct and non-targeted effects of radiation in human fibroblasts. In directly irradiated (IR) cells CD increased with dose and was higher in radiosensitive cells. Investigating conditioned medium-mediated bystander effects we demonstrated that low and high (0.1 and 2Gy) doses induced similar levels of bystander responses and found individual differences in human fibroblasts. The bystander response was not related to the radiosensitivity of the cells. The importance of signal sending donor and signal receiving target cells was investigated by placing conditioned medium from a bystander response positive cell line (F11-hTERT) to bystander negative cells (S1-hTERT) and vice versa. The data indicated that signal sending cells are more important in the medium-mediated bystander effect than recipients. Finally, we followed long term effects in immortalized radiation sensitive (S1-hTERT) and normal (F11-hTERT) fibroblasts up to 63 days after IR. In F11-hTERT cells CD level was increased until 35 days after IR then reduced back to control level by day 49. In S1-hTERT cells the increased CD level was also normalized by day 42, however a second wave of increased CD incidence appeared by day 49 which was maintained up to day 63 after IR. This second CD wave might be the indication of radiation-induced instability in the mitochondrial genome of S1-hTERT cells. The data demonstrated that measuring CD in mtDNA by qRT-PCR is a reliable and sensitive biomarker to estimate radiation-induced direct and non-targeted effects.

New Discoveries in Radiation Science.

This series of 16 articles (8 original articles and 8 reviews) was written by internationally recognized scientists attending the 44th Congress of the European Radiation Research Society (Pécs, Hungary) [...].

Blood-Derived Biomarkers of Diagnosis, Prognosis and Therapy Response in Prostate Cancer Patients.

Prostate cancer is among the most frequent cancers in men worldwide. Despite the fact that multiple therapeutic alternatives are available for its treatment, it is often discovered in an advanced stage as a metastatic disease. Prostate cancer screening is based on physical examination of prostate size and prostate-specific antigen (PSA) level in the blood as well as biopsy in suspect cases. However, these markers often fail to correctly identify the presence of cancer, or their positivity might lead to overdiagnosis and consequent overtreatment of an otherwise silent non-progressing disease. Moreover, these markers have very limited if any predictive value regarding therapy response or individual risk for therapy-related toxicities. Therefore, novel, optimally liquid biopsy-based (blood-derived) markers or marker panels are needed, which have better prognostic and predictive value than the ones currently used in the everyday routine. In this review the role of circulating tumour cells, extracellular vesicles and their microRNA content, as well as cellular and soluble immunological and inflammation- related blood markers for prostate cancer diagnosis, prognosis and prediction of therapy response is discussed. A special emphasis is placed on markers predicting response to radiotherapy and radiotherapy-related late side effects.

Oxidative Stress and Gene Expression Modifications Mediated by Extracellular Vesicles: An In Vivo Study of the Radiation-Induced Bystander Effect.

Radiation-induced bystander effect is a biological response in nonirradiated cells receiving signals from cells exposed to ionising radiation. The aim of this in vivo study was to analyse whether extracellular vesicles (EVs) originating from irradiated mice could induce modifications in the redox status and expression of radiation-response genes in bystander mice. C57BL/6 mice were whole-body irradiated with 0.1-Gy and 2-Gy X-rays, and EVs originating from mice irradiated with the same doses were injected into naïve, bystander mice. Lipid peroxidation in the spleen and plasma reactive oxygen metabolite (ROM) levels increased 24 h after irradiation with 2 Gy. The expression of antioxidant enzyme genes and inducible nitric oxide synthase 2 (iNOS2) decreased, while cell cycle arrest-, senescence- and apoptosis-related genes were upregulated after irradiation with 2 Gy. In bystander mice, no significant alterations were observed in lipid peroxidation or in the expression of genes connected to cell cycle arrest, senescence and apoptosis. However, there was a systemic increase in the circulating ROM level after an intravenous EV injection, and EVs originating from 2-Gy-irradiated mice caused a reduced expression of antioxidant enzyme genes and iNOS2 in bystander mice. In conclusion, we showed that ionising radiation-induced alterations in the cellular antioxidant system can be transmitted in vivo in a bystander manner through EVs originating from directly irradiated animals.

Influence of sample preparation optimization on the accuracy of dose assessment of an automatic non-fluorescent MN scoring system.

PURPOSE: Automatizing the scoring of the cytokinesis-blocked micronucleus assay spares a lot of valuable time. The dose-effect relationship can be applied reliably for dose estimation if the quality of the slides is the same from the perspective of the used image processing algorithm. This aspect brings in additional requirements against the quality of the slides compared to the conventional visual scoring. MATERIALS AND METHODS: An add-in software was created to the non-fluorescent RS-MN automatic MN scoring system which is capable of measuring quantitatively the degree of typical anomalies. The image processing is less reliable when the presence of these anomalies is more frequent. The behavior of the designed sample quality parameters (SQPs) was tested on in vitro irradiated peripheral blood samples (0, 1, and 2 Gy) obtained from a healthy donor and also on samples from patients undergoing low dose-rate brachytherapy. RESULTS: We examined 20 different SQPs and identified two that are independent and correlate significantly with the error of the fully automatic MN frequency. One is related to the size of the cells and the other reflects the homogeneity of the environment. An equation was established which presents a connection between the error of the auto MN frequency and the SQPs. By adding a fourth cleaning step to the conventional sample preparation and changing the pre-dripping temperature of the slide, the SQP can be modified, and consequently, the sample quality can be improved. The gain in accuracy is 54 ± 10 MN per 1000 binucleated cells, which corresponds to the effects of 0.5 Gy. Around the lowest limit of detection (<0.5 Gy), it means a 50-100% drop in the error of dose, which is significant. With sample quality harmonization, the positive predictive value was raised to 80-93% depending on the dose. CONCLUSIONS: With the technique described in this paper, the suitability for automated scoring of a micronucleus slide can be tested quantitatively and objectively. A method is presented with which in some cases the uncertainty of the assessed doses due to variance in sample quality can be decreased or if it is not possible its bias can be predicted. The proposed protocol leads to more reliable estimation of dose. The SQPs are designed in a way that they have the potential to be adapted to similar systems.

A scanning and image processing system with integrated design for automated micronucleus scoring.

Purpose: Our aim was to design a compact and cost-effective optical microscopic system for automated non-fluorescent micronucleus (MN) scoring whose performance can reach the accuracy of visual scoring with the help of minimal user interaction and also gives an option for fully automatic scoring with an accuracy suitable for triage purposes.Materials and methods: The concept of Radometer MN-Series (RS-MN) microscopic system designed by Radosys was to develop hardware and software layers in parallel in order to optimize the performance in automated MN scoring. A MN assay slide is automatically scanned by the RS-MN then the binucleated cells and micronuclei are automatically identified. Processing 1000 cells takes 10-60 minutes by automatic scoring (scanning plus image processing) depending on sample quality and required accuracy. The manual revision of the cell gallery takes an extra 10 minutes per sample. Dose response curves are determined for manual, automatic and semi-automatic scoring methods.Results: The combination of object-sensitive autofocusing method and the multi-layer image acquisition is able to reduce the minimum resolvable dose by 14%. The MN yields obtained from the manual, semi-automatic and automatic scoring methods are well correlating (Pearson's correlation coefficients are between 0.977 and 0.998). In order to compare the reliability of the results of visual and automatic scoring, an extended analysis on uncertainty contributors was performed. For a dose of 1 Gy, the estimated relative uncertainty from the Poisson characteristics of MN yield is 17-19% for the manual and 20-38% for the automated scoring. Other uncertainty factors (differences in donor radiosensitivity, scorer performance, and sample preparations) can contribute to this error fall within a similar range: 3-16%. Taking into account all of the possible uncertainties, the minimum resolvable dose for the manual (0.48 Gy) is the two-thirds of that of the automatic scoring (0.61 Gy).Conclusions: The results verify that the fully automatic mode of RS-MN is suitable for triage purposes. The performance of the user interacted semi-automatic mode is comparable with the reference manual scoring. Its performance reaches up to other non-fluorescent automatic systems and offers a compact and cost-effective alternative.

The effect of ionising radiation on the phenotype of bone marrow-derived extracellular vesicles.

OBJECTIVES: Ionising radiation-induced alterations affecting intercellular communication in the bone marrow (BM) contribute to the development of haematological pathologies. Extracellular vesicles (EVs), which are membrane-coated particles released by cells, have important roles in intercellular signalling in the BM. Our objective was to investigate the effects of ionising radiation on the phenotype of BM-derived EVs of total-body irradiated mice. METHODS: CBA mice were irradiated with 0.1 Gy or 3 Gy X-rays. BM was isolated from the femur and tibia 24 h after irradiation. EVs were isolated from the BM supernatant. The phenotype of BM cells and EVs was analysed by flow cytometry. RESULTS: The mean size of BM-derived EVs was below 300 nm and was not altered by ionising radiation. Their phenotype was very heterogeneous with EVs carrying either CD29 or CD44 integrins representing the major fraction. High-dose ionising radiation induced a strong rearrangement in the pool of BM-derived EVs which were markedly different from BM cell pool changes. The proportion of CD29 and CD44 integrin-harbouring EVs significantly decreased and the relative proportion of EVs with haematopoietic stem cell or lymphoid progenitor markers increased. Low-dose irradiation had limited effect on EV secretion. CONCLUSIONS: Ionising radiation induced selective changes in the secretion of EVs by the different BM cell subpopulations. ADVANCES IN KNOWLEDGE: The novelty of the paper consists of performing a detailed phenotyping of BM-derived EVs after in vivo irradiation of mice.

Mitochondrial DNA damage in the hair bulb: can it be used as a noninvasive biomarker of local exposure to low LET ionizing radiation?

Purpose: Our aim was to evaluate whether mitochondrial DNA (mtDNA) damage in hair bulbs could be a suitable biomarker for the detection of local exposure to ionizing radiation.Materials and methods: Mouse hair was collected 4 and 24 hours, 3 and 10 days after single whole-body exposure to 0, 0.1, and 2 Gy radiation. Pubic hair (treated area) and scalp hair (control area) were collected from 13 prostate cancer patients before and after fractioned radiotherapy with an average total dose of 2.7 Gy to follicles after five fractions. Unspecified lesion frequency of mtDNA was analyzed with long PCR, large mtDNA deletion levels were tested with real-time PCR.Results: Unspecified lesion frequency of mtDNA significantly increased in mouse hair 24 hours after irradiation with 2 Gy, but variance among samples was high. No increase in lesion frequency could be detected after 0.1 Gy irradiation. In prostate cancer patients, there was no significant change in either the unspecified lesion frequency or in the proportion of 4934-bp deleted mtDNA in pubic hair after radiotherapy. The proportions of murine 3860-bp common deletion, human 4977-bp common deletion and 7455-bp deleted mtDNA were too low to be analyzed reliably.Conclusions: Our results suggest that the unspecified lesion frequency and proportion of large deletions of mtDNA in hair bulbs are not suitable biomarkers of exposure to ionizing radiation.

Extracellular vesicles mediate low dose ionizing radiation-induced immune and inflammatory responses in the blood.

PURPOSE: Radiation-induced bystander effects (RIBE) imply the involvement of complex signaling mechanisms, which can be mediated by extracellular vesicles (EVs). Using an in vivo model, we investigated EV-transmitted RIBE in blood plasma and radiation effects on plasma EV miRNA profiles. MATERIALS AND METHODS: C57Bl/6 mice were total-body irradiated with 0.1 and 2 Gy, bone marrow-derived EVs were isolated, and injected systemically into naive, 'bystander' animals. Proteome profiler antibody array membranes were used to detect alterations in plasma, both in directly irradiated and bystander mice. MiRNA profile of plasma EVs was determined by PCR array. RESULTS: M-CSF and pentraxin-3 levels were increased in the blood of directly irradiated and bystander mice both after low and high dose irradiations, CXCL16 and lipocalin-2 increased after 2 Gy in directly irradiated and bystander mice, CCL5 and CCL11 changed in bystander mice only. Substantial overlap was found in the cellular pathways regulated by those miRNAs whose level were altered in EVs isolated from the plasma of mice irradiated with 0.1 and 2 Gy. Several of these pathways have already been associated with bystander responses. CONCLUSION: Low and high dose effects overlapped both in EV-mediated alterations in signaling pathways leading to RIBE and in their systemic manifestations.