Flow cytometry detection and quantification of circulating leukocyte subpopulations in mice after brain irradiation.

In the context of high-grade gliomas such as glioblastoma (GBM), the immune part of the tumor microenvironment (TME) is involved in tumor growth and tumor recurrence. It is mostly represented by high amount of macrophages and low amount of lymphocytes. GBM in itself as well as x-ray-based radiotherapy, a standard treatment for brain tumors, are also associated with systemic effects like lymphopenia that correlates with a poor prognosis. This contributes to the immune-suppressive nature of the TME and may explain the lack of the anti-tumor immune response. Radiation-induced lymphopenia (RIL) is generally evaluated on CD4+ and CD8+ count or on a CBC (complete blood count), but the heterogeneity of the subtypes prompts us to explore them in detail to better understand the cellular response to brain irradiation. To facilitate and develop the evaluation of x-ray brain exposure on circulating immune cells, we developed a reproducible and reliable method to quantify the variation of lymphoid and myeloid subtypes using flow cytometry after brain irradiation in the rodent.

CYRAD: a translational study assessing immune response to radiotherapy by photons or protons in postoperative head and neck cancer patients through circulating leukocyte subpopulations and cytokine levels.

BACKGROUND: Radiotherapy has both immunostimulant and immunosuppressive effects, particularly in radiation-induced lymphopenia. Proton therapy has demonstrated potential in mitigating this lymphopenia, yet the mechanisms by which different types of radiation affect the immune system function are not fully characterized. The Circulating Immunes Cells, Cytokines and Brain Radiotherapy (CYRAD) trial aims to compare the effects of postoperative X-ray and proton radiotherapy on circulating leukocyte subpopulations and cytokine levels in patients with head and neck (CNS and ear nose throat) cancer. METHODS: CYRAD is a prospective, non-randomized, single-center non interventional study assessing changes in the circulating leukocyte subpopulations and cytokine levels in head and neck cancer patients receiving X-ray or proton radiotherapy following tumor resection. Dosimetry parameters, including dose deposited to organs-at-risk such as the blood and cervical lymph nodes, are computed. Participants undergo 29 to 35 radiotherapy sessions over 40 to 50 days, followed by a 3-month follow-up. Blood samples are collected before starting radiotherapy (baseline), before the 11th (D15) and 30th sessions (D40), and three months after completing radiotherapy. The study will be conducted with 40 patients, in 2 groups of 20 patients per modality of radiotherapy (proton therapy and photon therapy). Statistical analyses will assess the absolute and relative relationship between variations (depletion, recovery) in immune cells, biomarkers, dosimetry parameters and early outcomes. DISCUSSION: Previous research has primarily focused on radiation-induced lymphopenia, paying less attention to the specific impacts of radiation on different lymphoid and myeloid cell types. Early studies indicate that X-ray and proton irradiation may lead to divergent outcomes in leukocyte subpopulations within the bloodstream. Based on these preliminary findings, this study aims to refine our understanding of how proton therapy can better preserve immune function in postoperative (macroscopic tumor-free) head and neck cancer patients, potentially improving treatment outcomes. PROTOCOL VERSION: Version 2.1 dated from January 18, 2023. TRIAL REGISTRATION: The CYRAD trial is registered from October 19, 2021, at the US National Library of Medicine, ClinicalTrials.gov ID NCT05082961.

Changes in Telomere Length in Leukocytes and Leukemic Cells after Ultrashort Electron Beam Radiation.

Application of laser-generated electron beams in radiotherapy is a recent development. Accordingly, mechanisms of biological response to radiation damage need to be investigated. In this study, telomere length (TL) as endpoint of genetic damage was analyzed in human blood cells (leukocytes) and K562 leukemic cells irradiated with laser-generated ultrashort electron beam. Metaphases and interphases were analyzed in quantitative fluorescence in situ hybridization (Q-FISH) to assess TL. TLs were shortened compared to non-irradiated controls in both settings (metaphase and interphase) after irradiation with 0.5, 1.5, and 3.0 Gy in blood leukocytes. Radiation also caused a significant TL shortening detectable in the interphase of K562 cells. Overall, a negative correlation between TL and radiation doses was observed in normal and leukemic cells in a dose-dependent manner. K562 cells were more sensitive than normal blood cells to increasing doses of ultrashort electron beam radiation. As telomere shortening leads to genome instability and cell death, the results obtained confirm the suitability of this biomarker for assessing genotoxic effects of accelerated electrons for their further use in radiation therapy. Observed differences in TL shortening between normal and K562 cells provide an opportunity for further development of optimal radiation parameters to reduce side effects in normal cells during radiotherapy.

In Vivo Cytotoxic, Genotoxic and Radiosensitizing Effects of Clofarabine.

BACKGROUND/AIM: ClFdA is a second-generation antineoplastic agent that has demonstrated significant anticancer activity, particularly against acute lymphoblastic leukemia and has been shown to have radiosensitizing activity. The aim of the study was to explore the genotoxic, cytotoxic and radiosensitizing effects of clofarabine (ClFdA) on bone marrow cells (BMCs), normoblasts and leukocytes of mice in vivo. MATERIALS AND METHODS: Cytotoxicity was determined by the reduction in reticulocytes (RET), and genotoxicity was determined by the induction of micronucleated reticulocytes (MN-RET) in the peripheral blood and by DNA break induction in leukocytes determined by single-cell gel electrophoresis (SCGE). The radiosensitizing capacity of ClFdA was determined in leukocytes and BMCs by SCGE. RESULTS: Two mechanisms of MN-RET induction were identified according to the antecedents, that could be due to inhibition of DNA synthesis and demethylation of G-C regions, and subsequent chromosome fragility. ClFdA cytotoxicity causes two contiguous peaks, an early peak that seems to inhibit MN-RET induction and a second peak that seems to be caused by ribonucleotide reductase (RR) and/or DNA synthesis inhibitions. ClFdA induced early DNA damage in noncycling leukocytes, and also radiosensitizes leukocytes immediately after treatment. ClFdA-ionizing radiation (IR) causes two time-dependent episodes of DNA damage, the latest after 80 min triggers a major breakage of DNA. In terms of the number of damaged cells, leukocytes and BMCs are similarly sensitive to ionizing radiation; BMCs are slightly more sensitive than leukocytes to ClFdA, but BMCs are doubly sensitive to combined treatment. CONCLUSION: ClFdA causes early DNA damage and radiosensitivity in non-proliferating leukocytes, which rules out the most favored hypotheses of the participation of RR and DNA polymerase inhibition.

Early effects of different brain radiotherapy modalities on circulating leucocyte subpopulations in rodents.

PURPOSES: Lymphopenia is extensively studied, but not circulating leucocyte subpopulations, which however have distinct roles in tumor tolerance. Proton therapy has been shown to have a lesser impact on the immune system than conventional X-ray radiotherapy through lower dose exposure to healthy tissues. We explored the differential effects of brain X-ray and proton irradiation on circulating leucocyte subpopulations. MATERIALS AND METHODS: Leucocyte subpopulation counts from tumor-free mice were obtained 12 hours after 4 fractions of 2.5 Gy. The relationships between irradiation type (X-rays or protons), irradiated volume (whole-brain/hemi-brain) and dose rate (1 or 2 Gy/min) with circulating leucocyte subpopulations (T-CD4+, T-CD8+, B, and NK-cells, neutrophils, and monocytes) were investigated using linear regression and tree-based modeling approaches. Relationships between dose maps (brain, vessels, lymph nodes (LNs)) and leucocyte subpopulations were analyzed and applied to construct the blood dose model, assessing the hypothesis of a direct lymphocyte-killing effect in radiation-induced lymphopenia. RESULTS: Radiation-induced lymphopenia occurred after X-ray but not proton brain irradiation in lymphoid subpopulations (T-CD4+, T-CD8+, B, and NK-cells). There was an increase in neutrophil counts following protons but not X-rays. Monocytes remained unchanged under both X-rays and protons. Besides irradiation type, irradiated volume and dose rate had a significant impact on NK-cell, neutrophil and monocyte levels but not T-CD4+, T-CD8+, and B-cells. The dose to the blood had a heterogeneous impact on leucocyte subpopulations: neutrophil counts remained stable with increasing dose to the blood, while lymphocyte counts decreased with increasing dose (T-CD8+-cells > T-CD4+-cells > B-cells > NK-cells). Direct cell-killing effect of the dose to the blood mildly contributed to radiation-induced lymphopenia. LN exposure significantly contributed to lymphopenia and partially explained the distinct impact of irradiation type on circulating lymphocytes. CONCLUSIONS: Leucocyte subpopulations reacted differently to X-ray or proton brain irradiation. This difference could be partly explained by LN exposure to radiation dose. Further researches and analyses on other biological processes and interactions between leucocyte subpopulations are ongoing. The various mechanisms underlying leucocyte subpopulation changes under different irradiation modalities may have implications for the choice of radiotherapy modalities and their combination with immunotherapy in brain cancer treatment.

Shortened telomere length in peripheral blood leukocytes is associated with cumulative radioactive iodine doses in patients with differentiated thyroid carcinoma.

BACKGROUND: Telomere length is associated with cancer risk and cancer aggressiveness. Radioactive iodine (RAI) therapy for thyroid cancer has raised concerns for second primary malignancy (SPM) in patients with high cumulative doses. The association between RAI dose and peripheral blood leukocyte telomere length was examined. METHODS: A total of 425 patients were included who underwent total thyroidectomy and were followed up for at least 1 year with or without RAI treatment. The relative telomere length (RTL) of the patients was assessed via a quantitative polymerase chain reaction amplification method. RAI doses were divided into five groups on the basis of cumulative dose, and a comparison was made among these groups. RESULTS: The number of patients with RAI treatment was 287 (67.5%), and the cumulative RAI dose was 3.33 GBq (range, 1.11-131.35 GBq). The mean RTL was significantly shorter in the highest RAI group (>22.2 GBq) compared to both the no-RAI and lower dose groups. The association between RAI dose and RTL was positive in the lower RAI group (1.1-3.7 GBq) and negative in the highest RAI group in both univariate and multivariate analyses. We observed 59 (13.9%) SPMs and 20 (4.7%) mortalities, and RTL did not show a significant risk effect for all-cause, thyroid cancer-specific, or SPM-specific mortality. CONCLUSIONS: In patients with thyroid cancer who underwent total thyroidectomy, peripheral blood leukocyte telomere length exhibited a significant association with cumulative RAI dose higher than 22.2 GBq. These results suggest the possibility of telomere length shortening in patients who undergo high-dose RAI treatment.

Low-Energy Laser-Driven Ultrashort Pulsed Electron Beam Irradiation-Induced Immune Response in Rats.

The development of new laser-driven electron linear accelerators, providing unique ultrashort pulsed electron beams (UPEBs) with low repetition rates, opens new opportunities for radiotherapy and new fronts for radiobiological research in general. Considering the growing interest in the application of UPEBs in radiation biology and medicine, the aim of this study was to reveal the changes in immune system in response to low-energy laser-driven UPEB whole-body irradiation in rodents. Forty male albino Wistar rats were exposed to laser-driven UPEB irradiation, after which different immunological parameters were studied on the 1st, 3rd, 7th, 14th, and 28th day after irradiation. According to the results, this type of irradiation induces alterations in the rat immune system, particularly by increasing the production of pro- and anti-inflammatory cytokines and elevating the DNA damage rate. Moreover, such an immune response reaches its maximal levels on the third day after laser-driven UPEB whole-body irradiation, showing partial recovery on subsequent days with a total recovery on the 28th day. The results of this study provide valuable insight into the effect of laser-driven UPEB whole-body irradiation on the immune system of the animals and support further animal experiments on the role of this novel type of irradiation.

Potential radioprotective properties of arbutin against ionising radiation on human leukocytes in vitro.

Arbutin is a simple phenolic glucoside biosynthesised in many plant families. Some of the everyday foods that contain arbutin are species of the genus Origanum, peaches, cereal products, coffee and tea and Arctostaphyllos uva ursi L. leaves. Arbutin possesses various beneficial effects in the organism, and was confirmed effective in the treatment of urinary tract infections as well as in preventing skin hyperpigmentation. It shows antioxidant and anti-inflammatory properties, and antitumor activity. The aim of this study was to explore potential radioprotective properties of arbutin in concentrations of 11.4 µg/mL, 57 µg/mL, 200 µg/mL and 400 µg/mL administered as a pre-treatment for one hour before exposing human leukocytes to ionising radiation at a therapeutic dose of 2 Gy. The alkaline comet assay was used to establish the levels of primary DNA damage, and cytokinesis-block micronucleus (CBMN) cytome assay to determine the level of cytogenetic damage. None of the tested concentrations of single arbutin showed genotoxic and cytotoxic effects. Even at the lowest tested concentration, 11.4 µg/mL, arbutin demonstrated remarkable potential for radioprotection in vitro, observed both at the level of primary DNA damage, and using CBMN cytome assay. The best dose reduction compared with amifostine was observed after pre-treatment with the highest concentration of arbutin, corresponding to 400 µg/mL. Promising results obtained on the leukocyte model speak in favour of extending similar experiments on other cell and animal models.

More Than Effects in Skin: Ultraviolet Radiation-Induced Changes in Immune Cells in Human Blood.

Cells of the skin and circulation are in constant two-way communication. Following exposure of humans to sunlight or to phototherapy, there are alterations in the number, phenotype and function of circulating blood cells. In this review, only data obtained from human studies are considered, with changes induced by UV radiation (UVR) exposure described for phagocytic leukocytes and peripheral blood mononuclear cells plus their component T and B cells, natural killer cells and dendritic cells. These immune modulations illustrate the potential of UVR to have therapeutic effects beyond the skin, and that sunlight exposure is an important environmental influence on human health.

The 7SK/P-TEFb snRNP controls ultraviolet radiation-induced transcriptional reprogramming.

Conversion of promoter-proximally paused RNA polymerase II (RNAPII) into elongating polymerase by the positive transcription elongation factor b (P-TEFb) is a central regulatory step of mRNA synthesis. The activity of P-TEFb is controlled mainly by the 7SK small nuclear ribonucleoprotein (snRNP), which sequesters active P-TEFb into inactive 7SK/P-TEFb snRNP. Here we demonstrate that under normal culture conditions, the lack of 7SK snRNP has only minor impacts on global RNAPII transcription without detectable consequences on cell proliferation. However, upon ultraviolet (UV)-light-induced DNA damage, cells lacking 7SK have a defective transcriptional response and reduced viability. Both UV-induced release of "lesion-scanning" polymerases and activation of key early-responsive genes are compromised in the absence of 7SK. Proper induction of 7SK-dependent UV-responsive genes requires P-TEFb activity directly mobilized from the nucleoplasmic 7SK/P-TEFb snRNP. Our data demonstrate that the primary function of the 7SK/P-TEFb snRNP is to orchestrate the proper transcriptional response to stress.

Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies.

Peripheral blood leucocytes (PBL) have been traditionally used to investigate DNA damage by the comet assay in population studies, but validating alternative non-invasive samples would expand the application of this assay in human biomonitoring. The objectives of this study were (i) to test the validity of salivary leucocytes as a proper biomatrix for the comet assay, (ii) to evaluate the ability of this approach to detect different types of primary and oxidative DNA damage, and (iii) to determine whether frozen salivary leucocytes are still suitable for displaying those types of DNA damage. Fresh and frozen leucocytes isolated from saliva samples (six healthy non-smoking volunteers), were exposed to four genotoxic agents inducing different types of DNA damage, both primary (methyl methanesulfonate, actinomycin-D, ultraviolet radiation) and oxidative (potassium bromate), and standard or enzyme-modified comet assay was conducted. Results were compared with those obtained from PBL. Cells exposed to the four genotoxic agents showed dose-dependent increases of primary and oxidative DNA damage, demonstrating the suitability of all these samples to detect genetic damage from different origin. When comparing baseline levels of DNA damage, just a slight significant increase in primary DNA damage was observed in frozen salivary leucocytes regarding the other biomatrices, but similar results were obtained regarding sensitivity to DNA damage induction by all agents tested. This study demonstrates that salivary leucocytes can be employed in comet assay as an alternative or complement to blood samples. Frozen salivary leucocytes were proved to be a very convenient sample in large biomonitoring studies.

ROS- and Radiation Source-Dependent Modulation of Leukocyte Adhesion to Primary Microvascular Endothelial Cells.

Anti-inflammatory effects of low-dose irradiation often follow a non-linear dose-effect relationship. These characteristics were also described for the modulation of leukocyte adhesion to endothelial cells. Previous results further revealed a contribution of reactive oxygen species (ROS) and anti-oxidative factors to a reduced leukocyte adhesion. Here, we evaluated the expression of anti-oxidative enzymes and the transcription factor Nrf2 (Nuclear factor-erythroid-2-related factor 2), intracellular ROS content, and leukocyte adhesion in primary human microvascular endothelial cells (HMVEC) upon low-dose irradiation under physiological laminar shear stress or static conditions after irradiation with X-ray or Carbon (C)-ions (0-2 Gy). Laminar conditions contributed to increased mRNA expression of anti-oxidative factors and reduced ROS in HMVEC following a 0.1 Gy X-ray and 0.5 Gy C-ion exposure, corresponding to reduced leukocyte adhesion and expression of adhesion molecules. By contrast, mRNA expression of anti-oxidative markers and adhesion molecules, ROS, and leukocyte adhesion were not altered by irradiation under static conditions. In conclusion, irradiation of endothelial cells with low doses under physiological laminar conditions modulates the mRNA expression of key factors of the anti-oxidative system, the intracellular ROS contents of which contribute at least in part to leucocyte adhesion, dependent on the radiation source.

Inhibition of EphA2 by Dasatinib Suppresses Radiation-Induced Intestinal Injury.

Radiation-induced multiorgan dysfunction is thought to result primarily from damage to the endothelial system, leading to a systemic inflammatory response that is mediated by the recruitment of leukocytes. The Eph-ephrin signaling pathway in the vascular system participates in various disease developmental processes, including cancer and inflammation. In this study, we demonstrate that radiation exposure increased intestinal inflammation via endothelial dysfunction, caused by the radiation-induced activation of EphA2, an Eph receptor tyrosine kinase, and its ligand ephrinA1. Barrier dysfunction in endothelial and epithelial cells was aggravated by vascular endothelial-cadherin disruption and leukocyte adhesion in radiation-induced inflammation both in vitro and in vivo. Among all Eph receptors and their ligands, EphA2 and ephrinA1 were required for barrier destabilization and leukocyte adhesion. Knockdown of EphA2 in endothelial cells reduced radiation-induced endothelial dysfunction. Furthermore, pharmacological inhibition of EphA2-ephrinA1 by the tyrosine kinase inhibitor dasatinib attenuated the loss of vascular integrity and leukocyte adhesion in vitro. Mice administered dasatinib exhibited resistance to radiation injury characterized by reduced barrier leakage and decreased leukocyte infiltration into the intestine. Taken together, these data suggest that dasatinib therapy represents a potential approach for the protection of radiation-mediated intestinal damage by targeting the EphA2-ephrinA1 complex.

Radiation-induced DNA double-strand breaks in peripheral leukocytes and therapeutic response of heel spur patients treated by orthovoltage X-rays or a linear accelerator.

PURPOSE: Biodosimetric assessment and comparison of radiation-induced deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by γH2AX immunostaining in peripheral leukocytes of patients with painful heel spur after radiation therapy (RT) with orthovoltage X­rays or a 6-MV linear accelerator (linac). The treatment response for each RT technique was monitored as a secondary endpoint. PATIENTS AND METHODS: 22 patients were treated either with 140-kV orthovoltage X­rays (n = 11) or a 6-MV linac (n = 11) with two weekly fractions of 0.5 Gy for 3 weeks. In both scenarios, the dose was prescribed to the International Commission on Radiation Units and Measurements (ICRU) dose reference point. Blood samples were obtained before and 30 min after the first RT session. γH2AX foci were quantified by immunofluorescence microscopy to assess the yield of DSBs at the basal level and after radiation exposure ex vivo or in vivo. The treatment response was assessed before and 3 months after RT using a five-level functional calcaneodynia score. RESULTS: RT for painful heel spurs induced a very mild but significant increase of γH2AX foci in patients' leukocytes. No difference between the RT techniques was observed. High and comparable therapeutic responses were documented for both treatment modalities. This trial was terminated preliminarily after an interim analysis (22 patients randomized). CONCLUSION: Low-dose RT for painful heel spurs with orthovoltage X­rays or a 6-MV linac is an effective treatment option associated with a very low and comparable radiation burden to the patient, as confirmed by biodosimetric measurements.

Radiation Potentiates Monocyte Infiltration into Tumors by Ninjurin1 Expression in Endothelial Cells.

Radiation is a widely used treatment for cancer patients, with over half the cancer patients receiving radiation therapy during their course of treatment. Considerable evidence from both preclinical and clinical studies show that tumor recurrence gets restored following radiotherapy, due to the influx of circulating cells consisting primarily of monocytes. The attachment of monocyte to endothelial cell is the first step of the extravasation process. However, the exact molecules that direct the transmigration of monocyte from the blood vessels to the tumors remain largely unknown. The nerve injury-induced protein 1 (Ninjurin1 or Ninj1) gene, which encodes a homophilic adhesion molecule and cell surface protein, was found to be upregulated in inflammatory lesions, particularly in macrophages/monocytes, neutrophils, and endothelial cells. More recently Ninj1 was reported to be regulated following p53 activation. Considering p53 has been known to be activated by radiation, we wondered whether Ninj1 could be increased in the endothelial cells by radiation and it might contribute to the recruiting of monocytes in the tumor. Here we demonstrate that radiation-mediated up-regulation of Ninj1 in endothelial cell lines such as human umbilical vein endothelial cells (HUVECs), EA.hy926, and immortalized HUVECs. Consistent with this, we found over-expressed Ninj1 in irradiated xenograft tumors, and increased monocyte infiltration into tumors. Radiation-induced Ninj1 was transcriptionally regulated by p53, as confirmed by transfection of p53 siRNA. In addition, Ninj1 over-expression in endothelial cells accelerated monocyte adhesion. Irradiation-induced endothelial cells and monocyte interaction was inhibited by knock-down of Ninj1. Furthermore, over-expressed Ninj1 stimulated MMP-2 and MMP-9 expression in monocyte cell lines, whereas the MMP-2 and MMP-9 expression were attenuated by Ninj1 knock-down in monocytes. Taken together, we provide evidence that Ninj1 is a key molecule that generates an interaction between endothelial cells and monocytes. This result suggests that radiation-mediated Ninj1 expression in endothelial cells could be involved in the post-radiotherapy recurrence mechanism.

DNA damage response in peripheral mouse blood leukocytes in vivo after variable, low-dose rate exposure.

Environmental contamination and ingestion of the radionuclide Cesium-137 (137Cs) is a large concern in fallout from a nuclear reactor accident or improvised nuclear device, and highlights the need to develop biological assays for low-dose rate, internal emitter radiation. To mimic low-dose rates attributable to fallout, we have developed a VAriable Dose-rate External 137Cs irradiatoR (VADER), which can provide arbitrarily varying and progressive low-dose rate irradiations in the range of 0.1-1.2 Gy/day, while circumventing the complexities of dealing with radioactively contaminated biomaterials. We investigated the kinetics of mouse peripheral leukocytes DNA damage response in vivo after variable, low-dose rate 137Cs exposure. C57BL/6 mice were placed in the VADER over 7 days with total accumulated dose up to 2.7 Gy. Peripheral blood response including the leukocyte depletion, apoptosis as well as its signal protein p53 and DNA repair biomarker γ-H2AX was measured. The results illustrated that blood leukocyte numbers had significantly dropped by day 7. P53 levels peaked at day 2 (total dose = 0.91 Gy) and then declined; whereas, γ-H2AX fluorescence intensity (MFI) and foci number generally increased with accumulated dose and peaked at day 5 (total dose = 2.08 Gy). ROC curve analysis for γ-H2AX provided a good discrimination of accumulated dose < 2 Gy and ≥ 2 Gy, highlighting the potential of γ-H2AX MFI as a biomarker for dosimetry in a protracted, environmental exposure scenario.

Effects of silymarin and mesenchymal stem cells on hematological and some biochemical changes induced by gamma radiation in albino rats.

Purpose: The present study was undertaken to evaluate the protective and therapeutic effects of silymarin and mesenchymal stem cells (MSCs) to ameliorate the damage caused by gamma radiation.Materials and methods: MSCs were given by intravenous injection to male rats (1.4 × 107 cells), 1 day next to gamma radiation (4Gy). While, silymarin was administered orally at a dose of 70 mg/kg b. wt., 3 days before irradiation and continued for 21 days post irradiation.Results: After 1 and 3 weeks post-irradiation, the results revealed a significant decline in red blood corpuscles (RBCs), white blood corpuscles (WBCs) and platelets count with rising in serum lipid profile [total lipids (TL), total glycerides (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and very low density lipoprotein-cholesterol (VLDL-C) levels] and total bilirubin; while significant decreases in serum total protein and high density lipoprotein-cholesterol (HDL-C) levels were observed. In irradiated animals receiving double treatment with MSCs and silymarin; amelioration of the changes observed in hematological and biochemical parameters when comparing with the irradiated group.Conclusions: Treatment with a radio-protector (such as silymarin) in addition to MSCs transplantation was recommended to protect against gamma radiation injury.

Analysis of molecular switch between leukocyte and substrate adhesion in bone marrow endothelial cells.

AIM: Endothelial cell damage is critical to understand since its presence in the entire body makes the damage widespread instead of being localized. Being a major component of stem cell niche in bone marrow, deems it essential to gain knowledge of the damage to endothelium associated with bone marrow. Since radiation exposure has become common to numerous therapeutic modalities, its effects on bone marrow and its endothelial cells are crucial to understand. MATERIAL & METHODS: Microarray analysis was performed on irradiated human bone marrow endothelial cells (hBMECs) with and without prior treatment with radioprotectant amifostine to assess the effects of radiation on signalling pathways and the subsequent changes in pathways when treated with radioprotectant prior to radiation exposure. KEY FINDINGS: It was seen that adhesion pathways that were usually inactivated under normal circumstances were stimulated post radiation. However, where in the case of radiation exposure, these adhesion pathways included leukocyte adhesion and migration; in the case of radioprotected conditions the pathways revolve around cell-substrate adhesion and cell spreading. Genes like ROCK1, FLNA, RAC1, PRKCZ and MAP3K8 were seen to regulate the molecular switch between leukocyte-cell adhesion to cell-substrate adhesion. SIGNIFICANCE: Our study demonstrated that irradiated endothelium supports leukocyte adhesion and migration but shifts to substrate adhesion dependent cell spreading under radioprotected conditions in order to repair the monolayer damage from the radiation. The genes responsible for the shift were identified and can be employed to manipulate cell adhesion characteristics for the treatment of diseases caused by radiation or inflammation.

Extracorporeal photochemotherapy induces bona fide immunogenic cell death.

Extracorporeal photochemotherapy (ECP) is employed for the management of cutaneous T cell lymphoma (CTCL). ECP involves the extracorporeal exposure of white blood cells (WBCs) to a photosensitizer, 8-methoxypsoralen (8-MOP), in the context of ultraviolet A (UVA) radiation, followed by WBC reinfusion. Historically, the therapeutic activity of ECP has been attributed to selective cytotoxicity on circulating CTCL cells. However, only a fraction of WBCs is exposed to ECP, and 8-MOP is inactive in the absence of UVA light, implying that other mechanisms underlie the anticancer effects of ECP. Recently, ECP has been shown to enable the physiological differentiation of monocytes into dendritic cells (DCs) that efficiently cross-present tumor-associated antigens (TAAs) to CD8+ T lymphocytes to initiate cognate immunity. However, the source of TAAs and immunostimulatory signals for such DCs remains to be elucidated. Here, we demonstrate that 8-MOP plus UVA light reduces melanoma cell viability along with the emission of ICD-associated danger signals including calreticulin (CALR) exposure on the cell surface and secretion of ATP, high mobility group box 1 (HMGB1) and type I interferon (IFN). Consistently, melanoma cells succumbing to 8-MOP plus UVA irradiation are efficiently engulfed by monocytes, ultimately leading to cross-priming of CD8+ T cells against cancer. Moreover, malignant cells killed by 8-MOP plus UVA irradiation in vitro vaccinate syngeneic immunocompetent mice against living cancer cells of the same type, and such a protection is lost when cancer cells are depleted of calreticulin or HMGB1, as well as in the presence of an ATP-degrading enzyme or antibodies blocking type I IFN receptors. ECP induces bona fide ICD, hence simultaneously providing monocytes with abundant amounts of TAAs and immunostimulatory signals that are sufficient to initiate cognate anticancer immunity.

Effect of long-term occupational exposure to extremely low-frequency electromagnetic fields on proinflammatory cytokine and hematological parameters.

Purpose: The present study aimed to investigate the effect of extremely low-frequency electromagnetic fields (ELF-EMFs) on proinflammatory cytokines and hematological parameters, among the employees of a power plant, which are one of the most important occupational groups exposed to ELF-EMFs extensively.Materials and methods: The studied population included 112 employees of a power plant as the exposed group and 138 unexposed employees who were enrolled based on inclusion and exclusion criteria. The magnetic flux density and the strength of the electric field were determined by spot measurements and according to the IEEE C95.3.1 standard. Proinflammatory cytokines including serum interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α); and hematologic parameters of all subjects were measured.Results: The mean level of IL-1ß and IL-6, white blood cell count (WBC) and red blood cell count (RBC), lymphocyte percentage (Lym%), mean corpuscular volume (MCV), platelet count (PLT) and procalcitonin (PCT) were significantly more in the exposed group, than the unexposed group. The mean serum levels of IL-6, IL-1ß and some of the hematological parameters including WBC, lymphocyte, RBC and hematocrit were higher in technicians which had the highest level of exposure to magnetic fields compared to other groups and these relations were linear.Conclusions: Long-term exposure to ELF-EMFs probably affects immune responses, by stimulating the production of proinflammatory cytokines, and increasing some hematological parameters.