CXCL10 upregulation in radiation-exposed human peripheral blood mononuclear cells as a candidate biomarker for rapid triage after radiation exposure.

PURPOSE: In case of a nuclear accident, individuals with high-dose radiation exposure (>1-2 Gy) should be rapidly identified. While ferredoxin reductase (FDXR) was recently suggested as a radiation-responsive gene, the use of a single gene biomarker limits radiation dose assessment. To overcome this limitation, we sought to identify reliable radiation-responsive gene biomarkers. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from mice after total body irradiation, and gene expression was analyzed using a microarray approach to identify radiation-responsive genes. RESULTS: In light of the essential role of the immune response following radiation exposure, we selected several immune-related candidate genes upregulated by radiation exposure in both mouse and human PBMCs. In particular, the expression of ACOD1 and CXCL10 increased in a radiation dose-dependent manner, while remaining unchanged following lipopolysaccharide (LPS) stimulation in human PBMCs. The expression of both genes was further evaluated in the blood of cancer patients before and after radiotherapy. CXCL10 expression exhibited a distinct increase after radiotherapy and was positively correlated with FDXR expression. CONCLUSIONS: CXCL10 expression in irradiated PBMCs represents a potential biomarker for radiation exposure.

Evaluation of natural chronic low dose radiation exposure on telomere length and transcriptional response of shelterin complex in individuals residing in Kerala coast, India.

The high level natural radiation areas (HLNRA) of Kerala coast provide unique opportunity to study the biological effect of chronic low dose ionizing radiation (LDIR) on human population below 100 mSv. The radiation level in this area varies from < 1.0-45 mGy /year due to patchy distribution of monazite in the sand, which contains 232Th (8-10%), 238U (0.3%), and their decay products. Telomere length attrition has been correlated to DNA damage due to genotoxic agents. The objective of the present study is to evaluate the effect of natural chronic LDIR exposure on telomere length and transcriptional response of telomere specific and DNA damage repair genes in peripheral blood mononuclear cells (PBMCs) of individuals from normal level natural radiation areas (NLNRA) and HLNRA of Kerala coast, southwest India. Blood samples were collected from 71 random male donors (24-80 years) from NLNRA (≤1.50 mGy/year; N = 19) and two HLNRA dose groups [1.51-10 mGy/year (N = 17); > 10 mGy/year, (N = 35)]. Genomic DNA was isolated from PBMCs and relative telomere length (RTL) was determined using real time q-PCR. Radio-adaptive response (RAR) study was carried out in PBMCs of 40 random males from NLNRA (N = 20) and HLNRA (>10 mGy/year; N = 20), where PBMCs were given a challenged dose of 2.0 Gy gamma radiation at 4 h. Transcriptional profile of telomere specific (TRF1, TRF2, POT1, TIN2, TPP1, RAP1), DNA damage response (RAD17, ATM, CHEK1) and base excision repair pathway (BER) (OGG1, XRCC1, NTH1, NEIL1, MUTYH, MBD4) genes were analysed at basal level and after a challenge dose of 2.0 Gy at 4 h. Our results did not show any significant effect of chronic LDR on RTL among the individuals from NLNRA and two HLNRA groups (p = 0.195). However, influence of age on RTL was clearly evident among NLNRA and HLNRA individuals. At basal level, TRF1, TRF2, TIN2, MBD4, NEIL1 and RAD17 showed significant up-regulation, whereas XRCC1 was significantly down regulated in HLNRA individuals. After a challenge dose of 2.0 Gy, significant transcriptional up-regulation was observed at telomere specific (TRF2, POT1) and BER (MBD4, NEIL1) genes in HLNRA individuals as compared to NLNRA suggesting their role in RAR. In conclusion, elevated level of natural chronic LDR exposure did not have any adverse effect on telomere length in Kerala coast. Significant transcriptional response at TRF2, MBD4 and NEIL1 at basal level and with a challenge dose of 2.0 Gy suggested their active involvement in efficient repair and telomere maintenance in individuals from HLNRA of Kerala coast.

Low Dose Radiation Therapy Induces Long-Lasting Reduction of Pain and Immune Modulations in the Peripheral Blood - Interim Analysis of the IMMO-LDRT01 Trial.

The treatment of chronic inflammatory and degenerative diseases by low dose radiation therapy (LDRT) is promising especially for patients who were refractory for classical therapies. LDRT aims to reduce pain of patients and to increase their mobility. Although LDRT has been applied since the late 19th century, the immunological mechanisms remain elusive. Within the prospective IMMO-LDRT01 trial (NCT02653079) the effects of LDRT on the peripheral blood immune status, as well as on pain and life quality of patients have been analyzed. Blood is taken before and after every serial irradiation with a single dose per fraction of 0.5Gy, as well as during follow-up appointments in order to determine a detailed longitudinal immune status by multicolor flow cytometry. Here, we report the results of an interim analysis of 125 patients, representing half the number of patients to be recruited. LDRT significantly improved patients' pain levels and induced distinct systemic immune modulations. While the total number of leukocytes remained unchanged in the peripheral blood, LDRT induced a slight reduction of eosinophils, basophils and plasmacytoid dendritic cells and an increase of B cells. Furthermore, activated immune cells were decreased following LDRT. Especially cells of the monocytic lineage correlated to LDRT-induced improvements of clinical symptoms, qualifying these immune cells as predictive biomarkers for the therapeutic success. We conclude that LDRT improves pain of the patients by inducing systemic immune modulations and that immune biomarkers could be defined for prediction by improved patient stratification in the future.

Effects of captopril against radiation injuries in the Göttingen minipig model of hematopoietic-acute radiation syndrome.

Our laboratory has demonstrated that captopril, an angiotensin converting enzyme inhibitor, mitigates hematopoietic injury following total body irradiation in mice. Improved survival in mice is correlated with improved recovery of mature blood cells and bone marrow, reduction of radiation-induced inflammation, and suppression of radiation coagulopathy. Here we investigated the effects of captopril treatment against radiation injuries in the Göttingen mini pig model of Hematopoietic-Acute Radiation Syndrome (H-ARS). Minipigs were given captopril orally (0.96 mg/kg) twice daily for 12 days following total body irradiation (60Co 1.79 Gy, 0.42-0.48 Gy/min). Blood was drawn over a time course following irradiation, and tissue samples were collected at euthanasia (32-35 days post-irradiation). We observed improved survival with captopril treatment, with survival rates of 62.5% in vehicle treated and 87.5% in captopril treated group. Additionally, captopril significantly improved recovery of peripheral blood mononuclear cells, and a trend toward improvement in recovery of red blood cells and platelets. Captopril significantly reduced radiation-induced expression of cytokines erythropoietin and granulocyte-macrophage colony-stimulating factor and suppressed radiation-induced acute-phase inflammatory response cytokine serum amyloid protein A. Using quantitative-RT-PCR to monitor bone marrow recovery, we observed significant suppression of radiation-induced expression of redox stress genes and improved hematopoietic cytokine expression. Our findings suggest that captopril activities in the Göttingen minipig model of hematopoietic-acute radiation syndrome reflect findings in the murine model.

SPM Receptor Expression and Localization in Irradiated Salivary Glands.

Radiation therapy-mediated salivary gland destruction is characterized by increased inflammatory cell infiltration and fibrosis, both of which ultimately lead to salivary gland hypofunction. However, current treatments (e.g., artificial saliva and sialagogues) only promote temporary relief of symptoms. As such, developing alternative measures against radiation damage is critical for restoring salivary gland structure and function. One promising option for managing radiation therapy-mediated damage in salivary glands is by activation of specialized proresolving lipid mediator receptors due to their demonstrated role in resolution of inflammation and fibrosis in many tissues. Nonetheless, little is known about the presence and function of these receptors in healthy and/or irradiated salivary glands. Therefore, the goal of this study was to detect whether these specialized proresolving lipid mediator receptors are expressed in healthy salivary glands and, if so, if they are maintained after radiation therapy-mediated damage. Our results indicate that specialized proresolving lipid mediator receptors are heterogeneously expressed in inflammatory as well as in acinar and ductal cells within human submandibular glands and that their expression persists after radiation therapy. These findings suggest that epithelial cells as well as resident immune cells represent potential targets for modulation of resolution of inflammation and fibrosis in irradiated salivary glands.

Low-Dose Radiation Therapy for COVID-19: Promises and Pitfalls.

The coronavirus disease-2019 (COVID-19) pandemic caused by SARS-CoV-2 has exacted an enormous toll on healthcare systems worldwide. The cytokine storm that follows pulmonary infection is causally linked to respiratory compromise and mortality in the majority of patients. The sparsity of viable treatment options for this viral infection and the sequelae of pulmonary complications have fueled the quest for new therapeutic considerations. One such option, the long-forgotten idea of using low-dose radiation therapy, has recently found renewed interest in many academic centers. We outline the scientific and logistical rationale for consideration of this option and the mechanistic underpinnings of any potential therapeutic value, particularly as viewed from an immunological perspective. We also discuss the preliminary and/or published results of prospective trials examining low-dose radiation therapy for COVID-19.

A simplified extracorporeal photopheresis procedure based on single high-dose ultraviolet A light irradiation shows similar in vitro efficacy.

BACKGROUND: Extracorporeal photopheresis (ECP) is one of the most widely used and effective cell-based therapies for the treatment of T-cell-mediated diseases. The patients' white blood cells (WBCs) are collected by apheresis and exposed to the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet A (UVA) light before retransfusion. The UVA/8-MOP combination has been in use in ECP for more than 4 decades; however, whether ECP can be simplified by UVA light irradiation only has never been analyzed. STUDY DESIGN AND METHODS: Peripheral blood mononuclear cells were treated with classical ECP or different UVA light doses only (UVAonly ). Treatment efficacy was investigated by apoptosis induction in WBC subsets, inhibition of T-cell proliferation, and the ability of monocytes to induce allogeneic T-cell expansion and to respond to lipopolysaccharide and interferon-γ stimulation in vitro. RESULTS: High-dose UVAonly treatment (5 J/cm2 ) was as efficient as ECP to induce apoptosis within 48 hours. UVAonly treatment modulated the composition of the surviving cells by improving monocyte survival and promoting CD8+ T-cell apoptosis. Both ECP and UVAonly treatment inhibited anti-CD3/anti-CD28 triggered T-cell proliferation. Interestingly, whereas ECP-treated monocytes exhibited a markedly reduced capacity to respond to stimulation and to induce allogeneic T-cell proliferation, UVAonly treatment preserved monocyte functionality to some degree. CONCLUSIONS: High-dose UVAonly and standard ECP showed comparable efficacy in inducing apoptosis and inhibiting direct T-cell proliferation. Hence, UVAonly treatment can be a simplified alternative to ECP therapy. Furthermore, increased monocyte survival with partially preserved functionality after UVAonly treatment may provide a novel method for immunoregulation.

EXPRESSION OF LIPOPROTEIN LIPASE AND c-MYC ONCOGENE IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA AFFECTED BY THE CHORNOBYL ACCIDENT. / EKSPRESIIa GENA LIPOPROTEÏNLIPAZY I ONKOGENA c-MYC U KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEIKEMIIu, IaKI POSTRAZhDALY VNASLIDOK AVARIÏ NA ChORNOBYL'S'KII AES.

OBJECTIVE: to determine the association between the expression of lipoprotein lipase (LPL) and c-MYC genes inperipheral blood cells of chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophedepending on the mutational status of IGHV genes. METHODS: Analysis was performed in the group of 69 CLL patients irradiated due to the Chornobyl NPP accident (58clean-up workers of 1986 year, 6 inhabitants of radionuclide contaminated areas, and 5 evacuees). The IGHV genemutational status was studied by polymerase chain reaction (PCR) followed by direct sequencing. LPL and c-MYCexpression was evaluated by Quantitative Real-time PCR. Data were analyzed with the SPSS software package, version 20.0. RESULTS: Relative LPL expression levels in CLL samples ranged from 0 to 1663.5 (mean 138.47 ± 30.69, median 26.1).A strong correlation between individual LPL expression levels and IGHV mutational status was found (r = 0.684;p < 0.0001). The average relative c-MYC expression level was 5.7 ± 0.87 (median 2.86; range 0-48.5). No association between c-MYC expression and IGHV mutational status was found. Among unmutated IGHV cases, a correlationbetween LPL and c-MYC gene expression levels was identified: r = 0.351; p = 0.013. CONCLUSIONS: Our data confirm the dominant concept that unmutated IGHV CLL cases are more sensitive to the actionof proliferative stimuli compared to mutated IGHV CLL cases. This is manifested by an increase in the expression ofa functionally significant LPL gene, is one for the strongest negative prognostic markers in CLL.

CHANGES OF CYCLIN D1-DEPENDENT REGULATION OF CELL CYCLE IN PERIPHERAL BLOOD LYMPHOCYTES OF CHORNOBYL CLEAN-UP WORKERS AT A REMOTE PERIOD AFTER RADIATION EXPOSURE. / PORUShENNIa TsYKLIN D1-ZALEZhNOÏ REGULIaTsIÏ KLITYNNOGO TsYKLU LIMFOTsYTIV PERYFERYChNOÏ KROVI UChASNYKIV LIKVIDATsIÏ NASLIDKIV AVARIÏ NA ChAES U VIDDALENOMU PERIODI PISLIa OPROMINENNIa.

OBJECTIVE: To study proliferative potential of peripheral blood lymphocytes of Chornobyl clean-up workers by levelof expression of cyclin D1 and quantitative parameters of cell cycle at a remote period after radiation exposure. MATERIALS AND METHODS: The research subject was the peripheral blood lymphocytes (PB) of Chornobyl clean-upworkers 30-33 years after radiation exposure. A total of 207 men were surveyed, 164 of them were clean-up workers exposed in the dose range 10.43-3623.31 mSv and 43 persons of the control group. Analysis of proliferationpotential (cell cycle initiation) and cyclin D1 expression in PB lymphocytes were performed in vitro by a micro methodof whole blood leukocytes culture with phytohemagglutinine-P (PHA). Sample preparation was performed by a standard immunofluorescent assay for intracellular proteins using the FITC labelled Mouse Anti-Human Cyclin D1Antibody Set. Cell distribution by cell cycle phases studied by propidium iodide DNA staining and analysis onFACSCalibur laser flow cytometer in histogram mode with separation of G0/G1-, S- and G2/M-regions and Sub-G0/G1-region (apoptotic cells). RESULTS AND CONCLUSIONS: An increase in the level of spontaneous сyclin D1 expression and disturbance of сyclinD1-dependent regulation of cell cycle of PB lymphocytes after mitogen activation were determined in a remote period after radiation exposure. An increase in the level of cyclin D1 expression was accompanied by increase in pool ofcells in the S- and G2/M-phases of cell cycle which characterizes the high proliferative potential of PB lymphocytes.Mitogen-induced delay of cell cycle of lymphocytes in G1/S check point and reduction of S-phase was revealed.These changes are a manifestation of genomic instability caused by the effect of radiation and depend on the radiation dose. The results confirm the hypothesis about the significance of levels of cyclin D1 expression, as a criterion for manifestations of genome instability and risks of oncogenesis in a remote period after irradiation.

CHANGES IN GENE EXPRESSION ASSOCIATED WITH NON-CANCER EFFECTS OF THE CHORNOBYL CLEAN-UP WORKERS IN THE REMOTE PERIOD AFTER EXPOSURE. / ZMINY GENNOÏ EKSPRESIÏ, ASOTsIIOVANI Z NEPUKhLYNNYMY EFEKTAMY VIDDALENOGO PERIODU PISLIa OPROMINENNIa V UChASNYKIV LIKVIDATsIÏ NASLIDKIV AVARIÏ NA ChAES.

OBJECTIVE: to establish the connection of radiation-induced changes in gene expression with the realized pathology of the broncho-pulmonary and cardiovascular systems in Chornobyl clean-up workers. MATERIALS AND METHODS: We examined 314 male Chornobyl clean-up workers (main group; age (58.94 ± 6.82) years(M ± SD); min 33, max 79 years; radiation dose (411.82 ± 625.41) mSv (M ± SD); min 1.74, max 3600 mSv) with various nosological forms of cardiovascular and broncho-pulmonary pathology (BPP) and 50 subjects of the controlgroup: age (50.50 ± 5.73) years (M ± SD); min 41, max 67 years. The relative level of BCL2, CDKN2A, CLSTN2, GSTM1,IFNG, IL1B, MCF2L, SERPINB9, STAT3, TERF1, TERF2, TERT, TNF, TP53, CCND1, CSF2, VEGFA genes expression was determined inperipheral blood leukocytes by real-time PCR (7900 HT Fast Real-Time PCR System (Applied Biosystems, USA)). The«gene-disease¼ association was determined on statistical models stratified separately for each disease and gene.Logistic regression was used to calculate the odds ratio. RESULTS: Increased GSTM1 gene expression and no changes in angiogenesis-related VEGFA gene expression werefound in the main group of patients with coronary heart disease (CHD). It was established overexpression of TP53,VEGF and IFNG genes in the group of patients with arterial hypertension (AH). At combination of these diseases anincrease of expression of СSF2, TERF1, TERF2 genes was established. The detected changes demonstrate an activationof the antioxidative defense system in patients with CHD, while AH is associated with the expression of genes ofangiogenesis and immune inflammation. It was shown an increase in the expression of genes associated with apoptosis and kinase activity (BCL2, CLSTN2, CDKN2), immune inflammation (CSF2, IL1B, TNF) in Chornobyl clean-upworkers with BPP. Expression of TP53 and GSTM1 (gene, associated with the glutathione system) was significantlyupregulated in the group of individuals with chronic bronchitis, whereas in patients with chronic obstructive pulmonary disease, no increase was detected; the expression of SERPINB9 and MCF2L genes was downregulated. CONCLUSIONS: Changes in the expression of genes, associated with the development of somatic pathology in theremote period after irradiation, in particular the genes of the immune response and inflammatory reactions CSF2,IFNG, IL1B, TNF; expression of genes that regulate cell proliferation, aging and apoptosis TP53, BCL2, MCF2L, CDKN2A,SERPINB9, TERF1, TERF2, TERT; genes that regulate cell adhesion and angiogenesis CLSTN2, VEGF.

IRIDIUM-192 RADIOTHERAPY BENEFITS IN THE MANAGEMENT OF GYNECOLOGICAL TUMORS. / PEREVAGY IONIZUIuChOGO VYPROMINIuVANNIa IRYDIIu-192 U PROMENEVOMU LIKUVANNI ONKOGINEKOLOGIChNYKh ZAKhVORIuVAN'.

BACKGROUND: Application of the most advanced radiation technologies of brachytherapy featuring the high dose ratesources i.e. 60Co and 192Ir within contemporary management protocols for gynecological cancer provides maximum dosedistribution in the clinical target along with minimal radiation exposure on surrounding organs and tissues. It involvesirradiation of large spaces with delivery of high therapeutic doses at the tolerance bound of «critical¼ organs (bladder,rectum) and tissues. Thus minimization of the early and late radiation complications, life span extent and quality oflife increase remain just the issues in contemporary radiation oncology requiring therefore the elaboration of radiobiological criteria along with substantiation of physiсо-engineering properties of the radiation sources. Taking intoaccount the basic radiobiological patterns will ensure a definitive further progress in the field of radiation oncology. OBJECTIVE: to study and compare the biological effects of 192Ir with the effects of the reference gamma radiation 60Coand increase the effectiveness of brachytherapy using a 192Ir source. MATERIALS AND METHODS: Radiobiological dosimetry on the basis of a test system of peripheral blood lymphocytesfrom the gynecological cancer patients with subsequent cytogenetic analysis of radiation-induced chromosomeaberrations was performed to study and compare the biological effects of 192Ir and reference 60Со γ-radiation, and toenhance the efficiency of 192Ir brachytherapy. RESULTS: Radiation markers, i.e. dicentric chromosomes with an accompanying paired fragment prevailed in thespectrum of radiation-induced damage. Variability of individual cytogenetic parameters of peripheral lymphocytesupon the first fraction of irradiation at the same dose of 5 Gy indicated an individual sensitivity of patients to the192Ir γ-irradiation. Comprehensive conservative treatment with adjuvant radiotherapy was applied to the patients(n = 98) having got secondary vaginal cancer stage II-III, T2-3N0-1M0. The high dose-rate (HDR) brachytherapy using 192Ir radiation sources was applied in the main study group (n = 37), HDR brachytherapy using 60Co radiation sourceswas applied in the control group (n = 35). CONCLUSION: The HDR brachytherapy with 192Ir and 60Co sources on the up-to-date technology intensive devices provides a high accuracy of dose distributions when irradiating the malignant neoplasms with minimized radiationexposure to the «critical¼ tissues. Treatment results are improved therefore. The use of 192Ir radiation sources compared with 60Co ones resulted in an increased throughput of treatment, enhanced tumor regression, and reduced incidence of radiation effects on the critical organs. Currently we perform the radiobiological studies on somatic cellsfrom cancer patients at the genetic, biochemical, biophysical, and cytological levels in order to receive a biologicalindication of radiation damage under the impact of 192Ir isotope. Continuation of clinical trials with radiobiologicalsupport will provide an opportunity to predict the early and late radiation complications and thus to provide a personalized approach in brachytherapy of cancer patients using the 192Ir sources of γ-rays.

Induction of oxidative stress biomarkers following whole-body irradiation in mice.

Dose assessment is an important issue for radiation emergency medicine to determine appropriate clinical treatment. Hematopoietic tissues are extremely vulnerable to radiation exposure. A decrease in blood cell count following radiation exposure is the first quantitative bio-indicator using hematological techniques. We further examined induction of oxidative stress biomarkers in residual lymphocytes to identify new biomarkers for dosimetry. In vivo whole-body radiation to mice exposed to 5 Gy significantly induces DNA double-strand breaks, which were visualized by γ-H2AX in mouse blood cells. Mouse blood smears and peripheral blood mononuclear cells (PBMC) isolated from irradiated mice were used for immunostaining for oxidative biomarkers, parkin or Nrf2. Parkin is the E3 ubiquitin ligase, which is normally localized in the cytoplasm, is relocated to abnormal mitochondria with low membrane potential (ΔΨm), where it promotes clearance via mitophagy. Nrf2 transcription factor controls the major cellular antioxidant responses. Both markers of oxidative stress were more sensitive and persistent over time than nuclear DNA damage. In conclusion, parkin and Nrf2 are potential biomarkers for use in radiation dosimetry. Identification of several biological markers which show different kinetics for radiation response is essential for radiation dosimetry that allows the assessment of radiation injury and efficacy of clinical treatment in emergency radiation incidents. Radiation-induced oxidative damage is useful not only for radiation dose assessment but also for evaluation of radiation risks on humans.

Association between NF-κB Activation in Peripheral Blood Mononuclear Cells and Late Skin and Subcutaneous Fibrosis following Radiotherapy.

BACKGROUND: This study aimed at evaluating the association between the speed of nuclear factor-kappa B (NF-κB) activation in peripheral blood mononuclear cells (PBMCs) and late skin and subcutaneous fibrosis in patients with head and neck squamous cell carcinoma (HNSCC) after radiotherapy. METHODS: The speed of NF-κB activation was represented by the nuclear p65 expression ratio before and after irradiation. The optimal time point to measure the ratio was determined by Western blot in the PBMCs from healthy outpatients ranging from 0 to 12 hours after ex vivo irradiation. We recruited patients with HNSCC who had received ratiotherapy and who were under regular follow-up care. We assessed the association between the risk of developing ≥grade 2 late fibrosis and the nuclear p65 expression ratio in the PBMCs after ex vivo irradiation in these patients. RESULTS: The maximum nuclear p65 ratio was observed at 1 hour after ex vivo irradiation in the PBMCs from the healthy outpatients. The speed of NF-κB activation was then represented by the nuclear p65 ratio in the PBMCs before and 1 hour after ex vivo irradiation. A total of 200 patients with HNSCC were recruited, 32.50% (n = 65) of which presented with ≥grade 2 late fibrosis. There was a significant association between the speed of NF-κB activation in the PBMCs and an increased risk of developing ≥grade 2 late fibrosis in these patients (P = 0.004). Subgroup analysis suggested that this finding was independent of the known clinical characteristics. CONCLUSIONS: The speed of NF-κB activation might be a potential predictor of late toxicity in cancer patients after radiotherapy. Prospective studies are needed for validation.

Triterpenoid CDDO-Me induces ROS generation and up-regulates cellular levels of antioxidative enzymes without induction of DSBs in human peripheral blood mononuclear cells.

Ionizing radiation produces reactive oxygen species (ROS) leading to cellular DNA damage. Therefore, patients undergoing radiation therapy or first responders in radiological accident scenarios could both benefit from the identification of specifically acting pharmacological radiomitigators. The synthetic triterpenoid bardoxolone-methyl (CDDO-Me) has previously been shown to exert antioxidant, anti-inflammatory and anticancer activities in several cell lines, in part by enhancing the DNA damage response. In our study, we examined the effect of nanomolar concentrations of CDDO-Me in human peripheral blood mononuclear cells (PBMC). We observed increased cellular levels of the antioxidative enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase (quinone1) and mitochondrial superoxide dismutase 2 by immunoblotting. Surprisingly, we found increased intracellular ROS-levels using imaging flow-cytometry. However, the radiation-induced DNA double-strand break (DSB) formation using the γ-H2AX + 53BP1 DSB focus assay and the cytokinesis-block micronucleus assay both revealed, that nanomolar CDDO-Me pre-treatment of PBMC for 2 h or 6 h ahead of X irradiation with 2 Gy did neither significantly affect γ-H2AX + 53BP1 DSB foci formation nor the frequency of micronuclei. CDDO-Me treatment also failed to alter the nuclear division index and the frequency of IR-induced PBMC apoptosis as investigated by Annexin V-labeled live-cell imaging. Our results indicate that pharmacologically increased cellular concentrations of antioxidative enzymes might not necessarily exert radiomitigating short-term effects in IR-exposed PBMC. However, the increase of antioxidative enzymes could also be a result of a defensive cellular mechanism towards elevated ROS levels.

Radio-adaptive response, individual radio-sensitivity and correlation of base excision repair gene polymorphism (hOGG1, APE1, XRCC1, and LIGASE1) in human peripheral blood mononuclear cells exposed to gamma radiation.

Radio-adaptive response (RAR) is a biological mechanism, where cells primed with a low dose exhibit reduced DNA damage with a high challenging dose. Single nucleotide polymorphisms (SNPs) of DNA repair genes including base excision repair (BER) pathway are known to be associated with radio-sensitivity but involvement in RAR is not yet understood. In the present study, attempt was made to correlate genotype frequencies of four BER SNPs [hOGG1(Ser326Cys), XRCC1(Arg399Gln), APE1(Asp148Glu) and LIGASE1(A/C)] with DNA damage, repair and mRNA expression level among 20 healthy donors (12 adaptive and 8 nonadaptive). Our results revealed that LIGASE1 (p = .002) showed significant correlation with DNA damage and mRNA expression level with increasing dose. hOGG1 (Ser326Cys), XRCC1 (Arg399Gln) and LIGASE1(A/C) polymorphisms showed significant difference with DNA damage (%T) and mRNA expression profile in primed cells among adaptive donors. In conclusion, BER gene polymorphisms play important role in identifying donors with radio-sensitivity and RAR in human cells.

Radiation Exposure of Peripheral Mononuclear Blood Cells Alters the Composition and Function of Secreted Extracellular Vesicles.

Normal tissue toxicity is a dose-limiting factor in radiation therapy. Therefore, a detailed understanding of the normal tissue response to radiation is necessary to predict the risk of normal tissue toxicity and to development strategies for tissue protection. One component of normal tissue that is continuously exposed during therapeutic irradiation is the circulating population of peripheral blood mononuclear cells (PBMC). PBMCs are highly sensitive to ionizing radiation (IR); however, little is known about how IR affects the PBMC response on a systemic level. It was the aim of this study to investigate whether IR was capable to induce changes in the composition and function of extracellular vesicles (EVs) secreted from PBMCs after radiation exposure to different doses. Therefore, whole blood samples from healthy donors were exposed to X-ray radiation in the clinically relevant doses of 0, 0.1, 2 or 6 Gy and PBMC-secreted EVs were isolated 72 h later. Proteome and miRNome analysis of EVs as well as functional studies were performed. Secreted EVs showed a dose-dependent increase in the number of significantly deregulated proteins and microRNAs. For both, proteome and microRNA data, principal component analysis showed a dose-dependent separation of control and exposed groups. Integrated pathway analysis of the radiation-regulated EV proteins and microRNAs consistently predicted an association of deregulated molecules with apoptosis, cell death and survival. Functional studies identified endothelial cells as an efficient EV recipient system, in which irradiation of recipient cells further increased the uptake. Furthermore an apoptosis suppressive effect of EVs from irradiated PBMCs in endothelial recipient cells was detected. In summary, this study demonstrates that IR modifies the communication between PBMCs and endothelial cells. EVs from irradiated PBMC donors were identified as transmitters of protective signals to irradiated endothelial cells. Thus, these data may lead to the discovery of biomarker candidates for radiation dosimetry and even more importantly, they suggest EVs as a novel systemic communication pathway between irradiated normal, non-cancer tissues.

Protective effects of new aryl sulfone derivatives against radiation-induced hematopoietic injury.

The hematopoietic system is sensitive to radiation. In this research, new aryl sulfone derivatives (XH-201 and XH-202) containing a nitrogen heterocycle were designed and synthesized and their radio-protective efficacies with regard to the hematopoietic system were evaluated. XH-201 administration significantly increased the survival rate of mice after 8.0 Gy total body irradiation (TBI). The results showed that XH-201 treatment not only increased the white blood cells, platelets counts and the percentage of hematopoietic progenitor cells and hematopoietic stem cells in mice exposed to 4.0 Gy TBI but also decreased DNA damage, as determined by flow cytometric analysis of histone H2AX phosphorylation. In addition, our data demonstrated that XH-201 decreased the mitochondrial reactive oxygen species (ROS) levels in hematopoietic cells. Overall, these data suggest that XH-201 is beneficial for the protection of the hemoatopoietic system against radiation-induced injuries.

Irradiation with Polychromatic Incoherent Low Energy Radiation of Human Peripheral Blood Mononuclear Cells In Vitro: Effects on Cytokine Production.

(1) Background: Physical stimuli may activate peripheral blood mononuclear cells (PBMCs) to secrete cytokines, which may favor pro-inflammatory responses or trigger reparative phenomena. The purpose of this study is to evaluate the action of Polarized Polychromatic Incoherent Low Energy Radiation (PILER) on human in vitro PBMCs, by detection of the possible effects on cytokine production; (2) Methods: isolated PBMCs were irradiated with a PILER lamp at different exposure times, at a distance of 10 cm, before incubation. The supernatants were collected after 24 h and 48 h and cytokines evaluated by ELISA; (3) Results: Our results showed a decrease in the levels of pro-inflammatory IL-12p70, IL-17A, IFN-γ, and TNF-α cytokines, whereas IL-10 and TGF-ß1 with regulatory activity increased; (4) Conclusions: PILER irradiation affected the cytokine production by isolated PBMCs driving the immune response toward an anti-inflammatory/reparative profile.

Generation of a Transcriptional Radiation Exposure Signature in Human Blood Using Long-Read Nanopore Sequencing.

In the event of a large-scale event leading to acute ionizing radiation exposure, high-throughput methods would be required to assess individual dose estimates for triage purposes. Blood-based gene expression is a broad source of biomarkers of radiation exposure which have great potential for providing rapid dose estimates for a large population. Time is a crucial component in radiological emergencies and the shipment of blood samples to relevant laboratories presents a concern. In this study, we performed nanopore sequencing analysis to determine if the technology can be used to detect radiation-inducible genes in human peripheral blood mononuclear cells (PBMCs). The technology offers not only long-read sequencing but also a portable device which can overcome issues involving sample shipment, and provide faster results. For this goal, blood from nine healthy volunteers was 2 Gy ex vivo X irradiated. After PBMC isolation, irradiated samples were incubated along with the controls for 24 h at 37°C. RNA was extracted, poly(A)+ enriched and reverse-transcribed before sequencing. The data generated was analyzed using a Snakemake pipeline modified to handle paired samples. The sequencing analysis identified a radiation signature consisting of 46 differentially expressed genes (DEGs) which included 41 protein-coding genes, a long non-coding RNA and four pseudogenes, five of which have been identified as radiation-responsive transcripts for the first time. The genes in which transcriptional expression is most significantly modified after radiation exposure were APOBEC3H and FDXR, presenting a 25- and 28-fold change on average, respectively. These levels of transcriptional response were comparable to results we obtained by quantitative polymerase chain reaction (qPCR) analysis. In vivo exposure analyses showed a transcriptional radioresponse at 24 h postirradiation for both genes together with a strong dose-dependent response in blood irradiated ex vivo. Finally, extrapolating from the data we obtained, the minimum sequencing time required to detect an irradiated sample using APOBEC3H transcripts would be less than 3 min for a total of 50,000 reads. Future improvements, in sample processing and bioinformatic pipeline for specific radiation-responsive transcript identification, will allow the provision of a portable, rapid, real-time biodosimetry platform based on this new sequencing technology. In summary, our data show that nanopore sequencing can identify radiation-responsive genes and can also be used for identification of new transcripts.

RNA-Seq analysis of peripheral blood mononuclear cells reveals unique transcriptional signatures associated with radiotherapy response of nasopharyngeal carcinoma and prognosis of head and neck cancer.

Radiotherapy is the main treatment for nasopharyngeal carcinoma (NPC); however, radioresistance limits the therapeutic efficacy and prognosis of patients with NPC. Here, we plan to identify the genes involved in radiotherapy response. Peripheral blood mononuclear cells (PBMC) from three paired NPC patients with pre-radiotherapy and post-radiotherapy were extracted. Next-generation deep sequencing was then performed to identify the PBMCs transcripts profiles in response to radiotherapy. Data of gene chip GSE48501 was obtained from Gene Expression Omnibus (GEO) database. The gene integration of differentially expressed genes identified from RNA-Seq data and gene chip was performed using "RobustRankAggreg" package. RNA-Seq data from 44 normal and 519 Head and neck squamous cell carcinoma (HNSCC) tissues (downloaded from TCGA) was integrated into the analysis to further support our study. Cox regression was used to identify risk factors impacting survival. Total of 45 genes were identified to be associated with radiotherapy response. Significantly enriched Gene Ontology (GO) terms and pathways were enriched. Univariate and multivariate analysis suggested the dysregulated genes, CHAC2, CLEC9A, GNG10, JCHAIN, KLRB1, NOG, OLR1, PRELID2, SYT1, VWCE, ZNF443 were associated with survival in HNSCC patients. Our data provide an overview of the profiles of radiotherapy-associated genes, which will facilitate future investigations into the function of radiotherapy resistance.