Turning anecdotal irradiation-induced anticancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells.

Irradiation (IR) induces immunogenic cell death (ICD) in tumors, but it rarely leads to the abscopal effect (AE); even combining IR with immune checkpoint inhibitors has shown only anecdotal success in inducing AEs. In this study, we aimed to enhance the IR-induced immune response and generate reproducible AEs using the anti-alcoholism drug, disulfiram (DSF), complexed with copper (DSF/Cu) to induce tumor ICD. We measured ICD in vitro and in vivo. In mouse tumor models, DSF/Cu was injected intratumorally followed by localized tumor IR, creating an in situ cancer vaccine. We determined the anticancer response by primary tumor rejection and assessed systemic immune responses by tumor rechallenge and the occurrence of AEs relative to spontaneous lung metastasis. In addition, we analyzed immune cell subsets and quantified proinflammatory and immunosuppressive chemokines/cytokines in the tumor microenvironment (TME) and blood of the vaccinated mice. Immune cell depletion was investigated for its effects on the vaccine-induced anticancer response. The results showed that DSF/Cu and IR induced more potent ICD under hypoxia than normoxia in vitro. Low-dose intratumoral (i.t.) injection of DSF/Cu and IR(12Gy) demonstrated strong anti-primary and -rechallenged tumor effects and robust AEs in mouse models. These vaccinations also increased CD8+ and CD4+ cell numbers while decreasing Tregs and myeloid-derived suppressor cells in the 4T1 model, and increased CD8+, dendritic cells (DC), and decreased Treg cell numbers in the MCa-M3C model. Depleting both CD8+ and CD4+ cells abolished the vaccine's anticancer response. Moreover, vaccinated tumor-bearing mice exhibited increased TNFα levels and reduced levels of immunosuppressive chemokines/cytokines. In conclusion, our novel approach generated an anticancer immune response that results in a lack of or low tumor incidence post-rechallenge and robust AEs, i.e., absence of or decreased spontaneous lung metastasis in tumor-bearing mice. This approach is readily translatable to clinical settings and may increase IR-induced AEs in cancer patients.

Effects of Hemorrhage on Hematopoietic Cell Depletion after a Combined Injury with Radiation: Role of White Blood Cells and Red Blood Cells as Biomarkers.

Combined radiation with hemorrhage (combined injury, CI) exacerbates hematopoietic acute radiation syndrome and mortality compared to radiation alone (RI). We evaluated the effects of RI or CI on blood cell depletion as a biomarker to differentiate the two. Male CD2F1 mice were exposed to 8.75 Gy γ-radiation (60Co). Within 2 h of RI, animals were bled under anesthesia 0% (RI) or 20% (CI) of total blood volume. Blood samples were collected at 4-5 h and days 1, 2, 3, 7, and 15 after RI. CI decreased WBC at 4-5 h and continued to decrease it until day 3; counts then stayed at the nadir up to day 15. CI decreased neutrophils, lymphocytes, monocytes, eosinophils, and basophils more than RI on day 1 or day 2. CI decreased RBCs, hemoglobin, and hematocrit on days 7 and 15 more than RI, whereas hemorrhage alone returned to the baseline on days 7 and 15. RBCs depleted after CI faster than post-RI. Hemorrhage alone increased platelet counts on days 2, 3, and 7, which returned to the baseline on day 15. Our data suggest that WBC depletion may be a potential biomarker within 2 days post-RI and post-CI and RBC depletion after 3 days post-RI and post-CI. For hemorrhage alone, neutrophil counts at 4-5 h and platelets for day 2 through day 7 can be used as a tool for confirmation.

Skin Wound following Irradiation Aggravates Radiation-Induced Brain Injury in a Mouse Model.

Radiation injury- and radiation combined with skin injury-induced inflammatory responses in the mouse brain were evaluated in this study. Female B6D2F1/J mice were subjected to a sham, a skin wound (SW), 9.5 Gy 60Co total-body gamma irradiation (RI), or 9.5 Gy RI combined with a skin puncture wound (RCI). Survival, body weight, and wound healing were tracked for 30 days, and mouse brain samples were collected on day 30 after SW, RI, RCI, and the sham control. Our results showed that RCI caused more severe animal death and body weight loss compared with RI, and skin wound healing was significantly delayed by RCI compared to SW. RCI and RI increased the chemokines Eotaxin, IP-10, MIG, 6Ckine/Exodus2, MCP-5, and TIMP-1 in the brain compared to SW and the sham control mice, and the Western blot results showed that IP-10 and p21 were significantly upregulated in brain cells post-RI or -RCI. RI and RCI activated both astrocytes and endothelial cells in the mouse brain, subsequently inducing blood-brain barrier (BBB) leakage, as shown by the increased ICAM1 and GFAP proteins in the brain and GFAP in the serum. The Doublecortin (DCX) protein, the "gold standard" for measuring neurogenesis, was significantly downregulated by RI and RCI compared with the sham group. Furthermore, RI and RCI decreased the expression of the neural stem cell marker E-cadherin, the intermediate progenitor marker MASH1, the immature neuron cell marker NeuroD1, and the mature neuron cell marker NeuN, indicating neural cell damage in all development stages after RI and RCI. Immunohistochemistry (IHC) staining further confirmed the significant loss of neural cells in RCI. Our data demonstrated that RI and RCI induced brain injury through inflammatory pathways, and RCI exacerbated neural cell damage more than RI.

Combined radiation injury and its impacts on radiation countermeasures and biodosimetry.

PURPOSE: Preparedness for medical responses to major radiation accidents and the increasing threat of nuclear warfare worldwide necessitates an understanding of the complexity of combined radiation injury (CI) and identifying drugs to treat CI is inevitably critical. The vital sign and survival after CI were presented. The molecular mechanisms, such as microRNA pathways, NF-κB-iNOS-IL-18 pathway, C3 production, the AKT-MAPK cross-talk, and TLR/MMP increases, underlying CI in relation to organ injury and mortality were analyzed. At present, no FDA-approved drug to protect, mitigate, or treat CI is available. The development of CI-specific medical countermeasures was reviewed. Because of the worsened acute radiation syndrome resulting from CI, diagnostic triage can be problematic. Therefore, biodosimetry and CI are bundled together with the need to establish effective triage methods with CI. CONCLUSIONS: CI mouse model studies at AFRRI are reviewed addressing molecular responses, findings from medical countermeasures, and a proposed plasma proteomic biodosimetry approach based on a panel of radiation-responsive biomarkers (i.e., CD27, Flt-3L, GM-CSF, CD45, IL-12, TPO) negligibly influenced by wounding in an algorithm used for dose predictions is described.

Ciprofloxacin and pegylated G-CSF combined therapy mitigates brain hemorrhage and mortality induced by ionizing irradiation.

Introduction: Brain hemorrhage was found between 13 and 16 days after acute whole-body 9.5 Gy 60Co-γ irradiation (IR). This study tested countermeasures mitigating brain hemorrhage and increasing survival from IR. Previously, we found that pegylated G-CSF therapy (PEG) (i.e., Neulasta®, an FDA-approved drug) improved survival post-IR by 20-40%. This study investigated whether Ciprofloxacin (CIP) could enhance PEG-induced survival and whether IR-induced brain hemorrhage could be mitigated by PEG alone or combined with CIP. Methods: B6D2F1 female mice were exposed to 60Co-γ-radiation. CIP was fed to mice for 21 days. PEG was injected on days 1, 8, and 15. 30-day survival and weight loss were studied in mice treated with vehicles, CIP, PEG, or PEG + CIP. For the early time point study, blood and sternums on days 2, 4, 9, and 15 and brains on day 15 post-IR were collected. Platelet numbers, brain hemorrhage, and histopathology were analyzed. The cerebellum/pons/medulla oblongata were detected with glial fibrillary acidic protein (GFAP), p53, p16, interleukin-18 (IL-18), ICAM1, Claudin 2, ZO-1, and complement protein 3 (C3). Results: CIP + PEG enhanced survival after IR by 85% vs. the 30% improvement by PEG alone. IR depleted platelets, which was mitigated by PEG or CIP + PEG. Brain hemorrhage, both surface and intracranial, was observed, whereas the sham mice displayed no hemorrhage. CIP or CIP + PEG significantly mitigated brain hemorrhage. IR reduced GFAP levels that were recovered by CIP or CIP + PEG, but not by PEG alone. IR increased IL-18 levels on day 4 only, which was inhibited by CIP alone, PEG alone, or PEG + CIP. IR increased C3 on day 4 and day 15 and that coincided with the occurrence of brain hemorrhage on day 15. IR increased phosphorylated p53 and p53 levels, which was mitigated by CIP, PEG or PEG + CIP. P16, Claudin 2, and ZO-1 were not altered; ICAM1 was increased. Discussion: CIP + PEG enhanced survival post-IR more than PEG alone. The Concurrence of brain hemorrhage, C3 increases and p53 activation post-IR suggests their involvement in the IR-induced brain impairment. CIP + PEG effectively mitigated the brain lesions, suggesting effectiveness of CIP + PEG therapy for treating the IR-induced brain hemorrhage by recovering GFAP and platelets and reducing C3 and p53.

Deteriorative Effects of Radiation Injury Combined with Skin Wounding in a Mouse Model.

Radiation-combined injury (RCI) augments the risk of morbidity and mortality when compared to radiation injury (RI) alone. No FDA-approved medical countermeasures (MCMs) are available for treating RCI. Previous studies implied that RI and RCI elicit differential mechanisms leading to their detrimental effects. We hypothesize that accelerating wound healing improves the survival of RCI mice. In the current study, we examined the effects of RCI at different doses on lethality, weight loss, wound closure delay, and proinflammatory status, and assessed the relative contribution of systemic and local elements to their delayed wound closure. Our data demonstrated that RCI increased the lethality and weight loss, delayed skin wound closure, and induced a systemic proinflammatory status in a radiation dose-dependent manner. We also demonstrated that delayed wound closure did not specifically depend on the extent of hematopoietic suppression, but was significantly influenced by the toxicity of the radiation-induced systemic inflammation and local elements, including the altered levels of proinflammatory chemokines and factors, and the dysregulated collagen homeostasis in the wounded area. In conclusion, the results from our study indicate a close association between delayed wound healing and the significantly altered pathways in RCI mice. This insightful information may contribute to the evaluation of the prognosis of RCI and development of MCMs for RCI.

Female Mice are More Resistant to the Mixed-Field (67% Neutron + 33% Gamma) Radiation-Induced Injury in Bone Marrow and Small Intestine than Male Mice due to Sustained Increases in G-CSF and the Bcl-2/Bax Ratio and Lower miR-34a and MAPK Activation.

In nuclear and radiological incidents, overexposure to ionizing radiation is life-threatening. It is evident that radiation depletes blood cells and increases circulating cytokine/chemokine concentrations as well as mortality. While microglia cells of female mice have been observed to be less damaged by radiation than in male mice, it is unclear whether sex affects physio-pathological responses in the bone marrow (BM) and gastrointestinal system (GI). We exposed B6D2F1 male and female mice to 0, 1.5, 3, or 6 Gy with mixed-field radiation containing 67% neutron and 33% gamma at a dose rate of 0.6 Gy/min. Blood and tissues were collected on days 1, 4, and 7 postirradiation. Radiation increased cytokines/chemokines in the femurs and ilea of female and male mice in a dose-dependent manner. Cytokines and chemokines reached a peak on day 4 and declined on day 7 with the exception of G-CSF which continued to increase on day 7 in female mice but not in male mice. MiR-34a (a Bcl-2 inhibitor), G-CSF (a miR-34a inhibitor), MAPK activation (pro-cell death), and citrulline (a biomarker of entroepithelial proliferation), active caspase-3 (a biomarker of apoptosis) and caspase-1 activated gasdermin D (a pyroptosis biomarker) were measured in the sternum, femur BM and ileum. Sternum histopathology analysis with H&E staining and femur BM cell counts as well as Flt-3L showed that BM cellularity was not as diminished in females, with males showing a 50% greater decline on day 7 postirradiation, mainly mediated by pyroptosis as indicated by increased gasdermin D in femur BM samples. Ileum injury, such as villus height and crypt depth, was also 43% and 30%, respectively, less damaged in females than in males. The severity of injury in both sexes was consistent with the citrulline and active caspase-3 measurements as well as active caspase-1 and gasdermin D measurements, suggesting apoptosis and pyroptosis occurred. On day 7, G-CSF in the ileum of female mice continued to be elevated by sevenfold, whereas G-CSF in the ileum of male mice returned to baseline. Furthermore, G-CSF is known to inhibit miR-34a expression, which in ileum on day 1 displayed a 3- to 4-fold increase in female mice after mixed-field (67% neutron + 33% gamma) irradiation, as compared to a 5- to 9-fold increase in male mice. Moreover, miR-34a blocked Bcl-2 expression. Mixed-field (60% neutron + 33% gamma) radiation induced more Bcl-2 in females than in males. On day 7, AKT activation was found in the ileums of females and males. However, MAPK activation including ERK, JNK, and p38 showed no changes in the ileum of females (by 0-fold; P > 0.05), whereas the MAPK activation was increased in the ileum of males (by 100-fold; P < 0.05). Taken together, the results suggest that organ injury from mixed-field (67% neutron + 33% gamma) radiation is less severe in females than in males, likely due to increased G-CSF, less MAPK activation, low miR-34a and increased Bcl-2/Bax ratio.

From tangled banks to toxic bunnies; a reflection on the issues involved in developing an ecosystem approach for environmental radiation protection.

The objective of this paper is to present the results of discussions at a workshop held as part of the International Congress of Radiation Research (Environmental Health stream) in Manchester UK, 2019. The main objective of the workshop was to provide a platform for radioecologists to engage with radiobiologists to address major questions around developing an Ecosystem approach in radioecology and radiation protection of the environment. The aim was to establish a critical framework to guide research that would permit integration of a pan-ecosystem approach into radiation protection guidelines and regulation for the environment. The conclusions were that the interaction between radioecologists and radiobiologists is useful in particular in addressing field versus laboratory issues where there are issues and challenges in designing good field experiments and a need to cross validate field data against laboratory data and vice versa. Other main conclusions were that there is a need to appreciate wider issues in ecology to design good approaches for an ecosystems approach in radioecology and that with the capture of 'Big Data', novel tools such as machine learning can now be applied to help with the complex issues involved in developing an ecosystem approach.

Brain Damage and Patterns of Neurovascular Disorder after Ionizing Irradiation. Complications in Radiotherapy and Radiation Combined Injury.

Exposure to ionizing radiation, mechanical trauma, toxic chemicals or infections, or combinations thereof (i.e., combined injury) can induce organic injury to brain tissues, the structural disarrangement of interactive networks of neurovascular and glial cells, as well as on arrays of the paracrine and systemic destruction. This leads to subsequent decline in cognitive capacity and decompensation of mental health. There is an ongoing need for improvement in mitigating and treating radiation- or combined injury-induced brain injury. Cranial irradiation per se can cause a multifactorial encephalopathy that occurs in a radiation dose- and time-dependent manner due to differences in radiosensitivity among the various constituents of brain parenchyma and vasculature. Of particular concern are the radiosensitivity and inflammation susceptibility of: 1. the neurogenic and oligodendrogenic niches in the subependymal and hippocampal domains; and 2. the microvascular endothelium. Thus, cranial or total-body irradiation can cause a plethora of biochemical and cellular disorders in brain tissues, including: 1. decline in neurogenesis and oligodendrogenesis; 2. impairment of the blood-brain barrier; and 3. ablation of vascular capillary. These changes, along with cerebrovascular inflammation, underlie different stages of encephalopathy, from the early protracted stage to the late delayed stage. It is evident that ionizing radiation combined with other traumatic insults such as penetrating wound, burn, blast, systemic infection and chemotherapy, among others, can exacerbate the radiation sequelae (and vice versa) with increasing severity of neurogenic and microvascular patterns of radiation brain damage.

PEG-G-CSF and L-Citrulline Combination Therapy for Mitigating Skin Wound Combined Radiation Injury in a Mouse Model.

Radiation combined injury (RCI, radiation exposure coupled with other forms of injury, such as burn, wound, hemorrhage, blast, trauma and/or sepsis) comprises approximately 65% of injuries from a nuclear explosion, and greatly increases the risk of morbidity and mortality when compared to that of radiation injury alone. To date, no U.S. Food and Drug Administration (FDA)-approved countermeasures are available for RCI. Currently, three leukocyte growth factors (Neupogen®, Neulasta® and Leukine®) have been approved by the FDA for mitigating the hematopoietic acute radiation syndrome. However these granulocyte-colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) products have failed to increase 30-day survival of mice after RCI, suggesting a more complicated biological mechanism is in play for RCI than for radiation injury. In the current study, the mitigative efficacy of combination therapy using pegylated (PEG)-G-CSF (Neulasta) and -citrulline was evaluated in an RCI mouse model. L-citrulline is a neutral alpha-amino acid shown to improve vascular endothelial function in cardiovascular diseases. Three doses of PEG-G-CSF at 1 mg/kg, subcutaneously administered on days 1, 8 and 15 postirradiation, were supplemented with oral -citrulline (1 g/kg), once daily from day 1 to day 21 postirradiation. The combination treatment significantly improved the 30-day survival of mice after RCI from 15% (vehicle-treated) to 42%, and extended the median survival time by 4 days, as compared to vehicle controls. In addition, the combination therapy significantly increased body weight and bone marrow stem and progenitor cell clonogenicity in RCI mice, and accelerated recovery from RCI-induced intestinal injury, compared to animals treated with vehicle. Treatment with -citrulline alone also accelerated skin wound healing after RCI. In conclusion, these data indicate that the PEG-G-CSF and -citrulline combination therapy is a potentially effective countermeasure for mitigating RCI, likely by enhancing survival of the hematopoietic stem/progenitor cells and accelerating recovery from the RCI-induced intestinal injury and skin wounds.

Co-Therapy of Pegylated G-CSF and Ghrelin for Enhancing Survival After Exposure to Lethal Radiation.

Exposure to ionizing radiation (radiation injury, RI) in nuclear-related episode is evident to be life-threatening. RI occurs at levels of organs, tissues, cytosols, or nucleus. Their mechanisms are still not fully understood. FDA approves pegylated granulocyte colony-stimulating factor (Neulasta™, Peg-G-CSF) for acute hematopoietic syndrome and has been shown to save lives after lethal RI. We aimed to test whether Ghrelin enhanced Peg-G-CSF's efficacy to save more lives after lethal RI. B6D2F1/J female mice were used for the study. They received 9.5 Gy (LD50/30 at 0.4 Gy/min) emitted from the 60Co-γ-photon radiation facility. Peg-G-CSF was injected subcutaneously at 1 mg/kg once on days 1, 8, and 15 after irradiation. Ghrelin contains 28 amino acid and is a hunger peptide that has been shown to stimulate food intake, promote intestinal epithelial cell proliferation, elevates immunity, inhibits brain hemorrhage, and increases stress-coping. Ghrelin was injected subcutaneously at 113 µg/kg once on days 1, 2, and 3 after irradiation. Survival, body weight, water consumption, hematology, spleen weight, splenocytes, bone marrow cells, and histology of bone marrow and ileum were performed. We observed that radiation resulted in 30-days survival by 30%. RI decreased their body weights and water consumption volumes. On the 30th day post-RI, platelets and WBCs such as basophils, eosinophils, monocytes, lymphocytes, neutrophils and leukocytes were still significantly decreased in surviving mice. Likewise, their RBC, hemoglobin, hematocrit, and splenocytes remained low; splenomegaly was found in these mice. Bone marrow in surviving RI animals maintained low cellularity with high counts of fat cells and low counts of megakaryocytes. Meanwhile, ileum histology displayed injury. However, mice co-treated with both drugs 24 h after RI resulted in 30-days survival by 45% above the vehicle group. Additionally, the body-weight loss was mitigated, the acute radiation syndrome was reduced. This co-therapy significantly increased neutrophils, eosinophils, leukocytes, and platelets in circulation, inhibited splenomegaly, and increased bone marrow cells. Histopathological analysis showed significant improvement on bone marrow cellularity and ileum morphology. In conclusion, the results provide a proof of concept and suggest that the co-therapy of Peg-G-CSF and Ghrelin is efficacious to ameliorate RI.

Ghrelin, a novel therapy, corrects cytokine and NF-κB-AKT-MAPK network and mitigates intestinal injury induced by combined radiation and skin-wound trauma.

BACKGROUND: Compared to radiation injury alone (RI), radiation injury combined wound (CI) further enhances acute radiation syndrome and subsequently mortality. We previously reported that therapy with Ghrelin, the 28-amino-acid-peptide secreted from the stomach, significantly increased 30-day survival and mitigated hematopoietic death by enhancing and sustaining granulocyte-colony stimulating factor (G-CSF) and keratinocyte chemoattractant (KC) in the blood and bone marrow; increasing circulating white blood cell depletion; inhibiting splenocytopenia; and accelerating skin-wound healing on day 30 after CI. Herein, we aimed to study the efficacy of Ghrelin on intestinal injury at early time points after CI. METHODS: B6D2F1/J female mice were exposed to 60Co-γ-photon radiation (9.5 Gy, 0.4 Gy/min, bilateral), followed by 15% total-body-surface-area skin wounds. Several endpoints were measured: at 4-5 h and on days 1, 3, 7, and 15. RESULTS: Ghrelin therapy mitigated CI-induced increases in IL-1ß, IL-6, IL-17A, IL-18, KC, and TNF-α in serum but sustained G-CSF, KC and MIP-1α increases in ileum. Histological analysis of ileum on day 15 showed that Ghrelin treatment mitigated ileum injury by increasing villus height, crypt depth and counts, as well as decreasing villus width and mucosal injury score. Ghrelin therapy increased AKT activation and ERK activation; suppressed JNK activation and caspase-3 activation in ileum; and reduced NF-κB, iNOS, BAX and Bcl-2 in ileum. This therapy recovered the tight junction protein and mitigated bacterial translocation and lipopolysaccharides levels. The results suggest that the capacity of Ghrelin therapy to reduce CI-induced ileum injury is mediated by a balanced NF-κB-AKT-MAPK network that leads to homeostasis of pro-inflammatory and anti-inflammatory cytokines. CONCLUSIONS: Our novel results are the first to suggest that Ghrelin therapy effectively decreases intestinal injury after CI.

Radiation: a poly-traumatic hit leading to multi-organ injury.

The range of radiation threats we face today includes everything from individual radiation exposures to mass casualties resulting from a terrorist incident, and many of these exposure scenarios include the likelihood of additional traumatic injury as well. Radiation injury is defined as an ionizing radiation exposure inducing a series of organ injury within a specified time. Severity of organ injury depends on the radiation dose and the duration of radiation exposure. Organs and cells with high sensitivity to radiation injury are the skin, the hematopoietic system, the gastrointestinal (GI) tract, spermatogenic cells, and the vascular system. In general, acute radiation syndrome (ARS) includes DNA double strand breaks (DSB), hematopoietic syndrome (bone marrow cells and circulatory cells depletion), cutaneous injury, GI death, brain hemorrhage, and splenomegaly within 30 days after radiation exposure. Radiation injury sensitizes target organs and cells resulting in ARS. Among its many effects on tissue integrity at various levels, radiation exposure results in activation of the iNOS/NF-kB/NF-IL6 and p53/Bax pathways; and increases DNA single and double strand breaks, TLR signaling, cytokine concentrations, bacterial infection, cytochrome c release from mitochondria to cytoplasm, and possible PARP-dependent NAD and ATP-pool depletion. These alterations lead to apoptosis and autophagy and, as a result, increased mortality. In this review, we summarize what is known about how radiation exposure leads to the radiation response with time. We also describe current and prospective countermeasures relevant to the treatment and prevention of radiation injury.

Effects of Low-to-Moderate Doses of Gamma Radiation on Mouse Hematopoietic System.

In this study, we investigated the effects of low-to-moderate doses of radiation in mice, given our limited understanding of the health risks associated with these exposures. Here, we demonstrate the different responses of the CD2F1 mouse hematopoietic system to low-to-moderate (0.5, 1, 3 or 5 Gy) doses of gamma radiation. After 3 and 5 Gy of 60Co total-body irradiation (TBI), mouse blood cell counts were decreased and maintained below baseline up to 28-42 days. In contrast, after 0.5 Gy TBI, lymphocyte and monocyte counts increased, and peaked from day 3 to day 14. Radiation doses at 0.5 and 1 Gy did not cause cell death or T-cell subpopulation changes in spleen and thymus, whereas the clonogenicity of mouse bone marrow (BM) progenitor cells was significantly suppressed on the first day after 0.5-5 Gy TBI, and these low levels were maintained up to 42 days. Although a transient recovery in total colony forming units (CFUs) was shown in mouse BM at days 14 and 21 after 0.5 Gy TBI, the early-stage multipotential progenitor colonies (CFU-GEMM) remained at a significantly low level compared to those of the sham-irradiated (0 Gy) controls. Consistently, the level of stem cell factor (SCF) in BM cells was decreased after low-to-moderate TBI. Serum from individual mice was collected after irradiation and 23 cytokines/chemokines were measured; massive releases of cytokines and chemokines were observed at day 3 postirradiation in a dose-dependent manner. When human hematopoietic CD34+ cells were cultured with the serum collected from mice irradiated at different doses, a significant decrease of CFU-GEMM colonies in the CD34+ cells was observed. Our data suggest that low-to-moderate doses of radiation induced cellular responses that are cell type-dependent. The early stage multipotential progenitor cells in mouse BM were the most sensitive cells even to low-dose irradiation compared to spleen and thymic cells, and 0.5 Gy TBI induced hematopoietic cell injury from day 1 to the end of our experiment, day 42 postirradiation. Radiation-induced decrease of SCF in mouse BM and increase in circulating pro-inflammatory factors may be responsible for the enhanced sensitivity of hematopoietic progenitor cells to radiation.

Ghrelin therapy mitigates bone marrow injury and splenocytopenia by sustaining circulating G-CSF and KC increases after irradiation combined with wound.

BACKGROUND: Radiation injury combined wound (CI) enhances acute radiation syndrome and subsequently mortality as compared to radiation injury alone (RI). We previously reported that ghrelin (a 28-amino-acid-peptide secreted from the stomach) treatment significantly increased a 30-day survival, mitigated hematopoietic death, circulating white blood cell (WBC) depletion and splenocytopenia and accelerated skin-wound healing on day 30 after CI. Herein, we aimed to study the ghrelin efficacy at early time points after CI. METHODS: B6D2F1/J female mice were exposed to 60Co-γ-photon radiation at 9.5 Gy (LD50/30) followed by a 15% total-body-surface-area skin wound. Several endpoints were measured at 4-5 h, days 1, 3, 7 and 15. RESULTS: Histological analysis of sternums on day 15 showed that CI induced more adipocytes and less megakaryocytes than RI. Bone marrow cell counts from femurs also indicated CI resulted in lower bone marrow cell counts on days 1, 7 and 15 than RI. Ghrelin treatment mitigated these CI-induced adverse effects. RI and CI decreased WBCs within 4-5 h and continued to decrease to day 15. Ghrelin treatment mitigated decreases in CI mice, mainly from all types of WBCs, but not RBCs, hemoglobin levels and hematocrit values. Ghrelin mitigated the CI-induced thrombocytopenia and splenocytopenia. CI increased granulocyte-colony stimulating factor (G-CSF) and keratinocyte chemoattractant (KC) in blood and bone marrow. Ghrelin therapy was able to enhance and sustain the increases in serum on day 15, probably contributed by spleen and ileum, suggesting the correlation between G-CSF and KC increases and the neutropenia mitigation. Activated caspase-3 levels in bone marrow cells were significantly mitigated by ghrelin therapy on days 3 and 15. CONCLUSIONS: Our novel results are the first to suggest that ghrelin therapy effectively decreases hematopoietic death and splenocytopenia by sustaining circulating G-CSF and KC increases after CI. These results demonstrate efficacy of ghrelin as a radio-mitigator/therapy agent for CI.

Circulating Cytokine/Chemokine Concentrations Respond to Ionizing Radiation Doses but not Radiation Dose Rates: Granulocyte-Colony Stimulating Factor and Interleukin-18.

Exposure to ionizing radiation is a crucial life-threatening factor in nuclear and radiological incidents. It is known that ionizing radiation affects cytokine/chemokine concentrations in the blood of B6D2F1 mice. It is not clear whether radiation dose rates would vary the physiological response. Therefore, in this study we utilized data from two experiments using B6D2F1 female mice exposed to six different dose rates ranging from low to high rates. In one experiment, mice received a total dose of 8 Gy (LD0/30) of 60Co gamma radiation at four dose rates: 0.04, 0.15, 0.30 and 0.47 Gy/min. Blood samples from mice were collected at 24 and 48 h postirradiation for cytokine/chemokine measurements, including interleukin (IL)-1ß, IL-6, IL-10, keratinocyte cytokine (KC), IL-12p70, IL-15, IL-17A, IL-18, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage (GM)-CSF, macrophage (M)-CSF, monokine induced by gamma interferon (MIG), tumor necrosis factor (TNF)-α, fibroblast growth factor (FGF)-basic, vascular endothelial growth factor (VEGF) and platelet-derived growth factor basic (PDGF-bb). At 24 h after ionizing irradiation at dose rate of 0.04 Gy/min, significant increases were observed only in G-CSF and M-CSF ( P < 0.05). At 0.15 Gy/min, IL-10, IL-17A, G-CSF and GM-CSF concentrations were increased. At 0.3 Gy/min, IL-15, IL-18, G-CSF, GM-CSF, M-CSF, MCP-1, MIP-2, MIG, FGF-basic, VEGF and PDGF-bb were significantly elevated ( P < 0.05). At 0.47 Gy/min, IL-6, KC, IL-10, MCP-1, G-CSF, GM-CSF and M-CSF were significantly increased. At 48 h postirradiation, all cytokines/chemokines except MCP-1 returned to or were below their baselines, suggesting these increases are transient at LD0/30 irradiation. Of note, there is a limitation on day 2 because cytokines/chemokines are either at or below their baselines. Other parameters such as fms-like tyrosine kinase receptor-3 ligand (Flt-3 ligand) concentrations and lymphocyte counts, which have proven to be unaffected by radiation dose rates, can be used instead for assessing the radiation dose. However, in a separate radiation dose and time-course experiment, increases in IL-18 and G-CSF depended on the radiation doses but showed no significant differences between 0.58 and 1.94 Gy/min ( P > 0.05) at 3 and 6 Gy but not 12 Gy. G-CSF continued to increase up to day 7, whereas IL-18 increased on day 4 and remained above baseline level on day 7. Therefore, time after irradiation at different doses should be taken into consideration. To our knowledge, these results are the first to suggest that ionizing radiation, even at a very low-dose-rate (0.04 Gy/min), induces circulating G-CSF increases but not others for selected time points; radiation-induced increases in IL-18 at radiation dose rates between 0.15 and 1.94 Gy/min are also not in a radiation dose-rate-dependent manner. C-CSF, lymphocyte counts and circulating Flt-3 ligand should be explored further as possible biomarkers of radiation exposure at early time points. IL-18 is also worthy of further study as a potential biomarker at later time points.

Combined Therapy of Pegylated G-CSF and Alxn4100TPO Improves Survival and Mitigates Acute Radiation Syndrome after Whole-Body Ionizing Irradiation Alone and Followed by Wound Trauma.

Exposure to ionizing radiation alone or combined with traumatic tissue injury is a crucial life-threatening factor in nuclear and radiological incidents. Radiation injuries occur at the molecular, cellular, tissue and systemic levels; their mechanisms, however, remain largely unclear. Exposure to radiation combined with skin wounding, bacterial infection or burns results in greater mortality than radiation exposure alone in dogs, pigs, rats, guinea pigs and mice. In the current study we observed that B6D2F1/J female mice exposed to 60Co gamma-photon radiation followed by 15% total-body-surface-area skin wounds experienced an increment of 25% higher mortality over a 30-day observation period compared to those subjected to radiation alone. Radiation exposure delayed wound healing by approximately 14 days. On day 30 post-injury, bone marrow and ileum in animals from both groups (radiation alone or combined injury) still displayed low cellularity and structural damage. White blood cell counts, e.g., neutrophils, lymphocytes, monocytes, eosinophils, basophils and platelets, still remained very low in surviving irradiated alone animals, whereas only the lymphocyte count was low in surviving combined injury animals. Likewise, in surviving animals from radiation alone and combined injury groups, the RBCs, hemoglobin, hematocrit and platelets remained low. We observed, that animals treated with both pegylated G-CSF (a cytokine for neutrophil maturation and mobilization) and Alxn4100TPO (a thrombopoietin receptor agonist) at 4 h postirradiation, a 95% survival (vehicle: 60%) over the 30-day period, along with mitigated body-weight loss and significantly reduced acute radiation syndrome. In animals that received combined treatment of radiation and injury that received pegylated G-CSF and Alxn4100TPO, survival was increased from 35% to 55%, but did not accelerate wound healing. Hematopoiesis and ileum showed significant improvement in animals from both groups (irradiation alone and combined injury) when treated with pegylated G-CSF and Alxn4100TPO. Treatment with pegylated G-CSF alone increased survival after irradiation alone and combined injury by 33% and 15%, respectively, and further delayed wound healing, but increased WBC, RBC and platelet counts after irradiation alone, and only RBCs and platelets after combined injury. Treatment with Alxn4100TPO alone increased survival after both irradiation alone and combined injury by 4 and 23%, respectively, and delayed wound healing after combined injury, but increased RBCs, hemoglobin concentrations, hematocrit values and platelets after irradiation alone and only platelets after combined injury. Taken together, the results suggest that combined treatment with pegylated G-CSF and Alxn4100TPO is effective for mitigating effects of both radiation alone and in combination with injury.

Ghrelin Therapy Decreases Incidents of Intracranial Hemorrhage in Mice after Whole-Body Ionizing Irradiation Combined with Burn Trauma.

Nuclear industrial accidents and the detonation of nuclear devices cause a variety of damaging factors which, when their impacts are combined, produce complicated injuries challenging for medical treatment. Thus, trauma following acute ionizing irradiation (IR) can deteriorate the IR-induced secondary reactive metabolic and inflammatory impacts to dose-limiting tissues, such as bone marrow/lymphatic, gastrointestinal tissues, and vascular endothelial tissues, exacerbating the severity of the primary injury and decreasing survival from the exposure. Previously we first reported that ghrelin therapy effectively improved survival by mitigating leukocytopenia, thrombocytopenia, and bone-marrow injury resulting from radiation combined with burn trauma. This study was aimed at investigating whether radiation combined with burn trauma induced the cerebro-vascular impairment and intracranial hemorrhage that could be reversed by ghrelin therapy. When B6D2F1 female mice were exposed to 9.5 Gy Cobalt-60 γ-radiation followed by 15% total skin surface burn, cerebro-vascular impairment and intracranial hemorrhage as well as platelet depletion were observed. Ghrelin treatment after irradiation combined with burn trauma significantly decreased platelet depletion and brain hemorrhage. The results suggest that ghrelin treatment is an effective therapy for ionizing radiation combined with burn trauma.

Thrombopoietin Receptor Agonist Mitigates Hematopoietic Radiation Syndrome and Improves Survival after Whole-Body Ionizing Irradiation Followed by Wound Trauma.

Ionizing radiation combined with trauma tissue injury (combined injury, CI) results in greater mortality and H-ARS than radiation alone (radiation injury, RI), which includes thrombocytopenia. The aim of this study was to determine whether increases in numbers of thrombocytes would improve survival and mitigate H-ARS after CI. We observed in mice that WBC and platelets remained very low in surviving RI animals that were given 9.5 Gy 60Co-γ-photon radiation, whereas only lymphocytes and basophils remained low in surviving CI mice that were irradiated and then given skin wounds. Numbers of RBC and platelets, hemoglobin concentrations, and hematocrit values remained low in surviving RI and CI mice. CI induced 30-day mortality higher than RI. Radiation delayed wound healing by approximately 14 days. Treatment with a thrombopoietin receptor agonist, Alxn4100TPO, after CI improved survival, mitigated body-weight loss, and reduced water consumption. Though this therapy delayed wound-healing rate more than in vehicle groups, it greatly increased numbers of platelets in sham, wounded, RI, and CI mice; it significantly mitigated decreases in WBC, spleen weights, and splenocytes in CI mice and decreases in RBC, hemoglobin, hematocrit values, and splenocytes and splenomegaly in RI mice. The results suggest that Alxn4100TPO is effective in mitigating CI.

Exacerbation of Mild Hypoxia on Acute Radiation Syndrome and Subsequent Mortality.

Mild hypoxia induced by 20% hemorrhage results in increases in few cytokine concentrations and sclerostin levels in blood, but shows no changes in bone formation, bone marrow cellularity, and gastrointestinal (GI) integrity and no systemic bacterial infection as well as no subsequent mortality. On the other hand, severe hypoxia induced by 40% hemorrhage causes significant increases in most cytokine concentrations, GI injury, lung injury, systemic bacterial infection, cellular ATP reduction and subsequent mortality. The severe hypoxia drastically damages GI and lung morphology, elevates cytokine concentrations in blood and increases inducible nitric oxide synthase (iNOS) expression in cells that is mediated by transcription factors NF-κB/NF-IL6, subsequently producing free radicals that disrupt mitochondria. ATP depletion, p53 phosphorylation, and caspase-3 activation are found, suggesting cell apoptosis. As a result, mortality occurs. However, when mild hypoxia follows ionizing radiation, the mild hypoxia significantly enhances radiation-induced mortality and acute radiation syndrome, including injury of bone marrow, GI, kidney, and lung. The synergism also occurs at the molecular level, resulting in alteration of microRNAs, amplification of iNOS expression, cytokine increases, sepsis, and ATP depletion. This is the first demonstration of synergistic effects between mild hypoxia and ionizing radiation.