Gy Whole Body Irradiation Research Articles

Ferulic acid reduces inflammatory response induced by radiation through Sirt1-NLRP3 pathway

Objective: A model of inflammatory damage was induced by radiation to investigate whether ferulic acid (FA) can reduce the inflammatory response through the Sirt1-NLRP3 inflammatory pathway. This will help discover radiation-protective drugs and elucidate the molecular mechanisms related to radiation-induced inflammatory damage. Methods: A mouse model of radiation-induced immunoinflammatory injury was established to verify the anti-inflammatory effects of FA in vivo. C57BL/6J mice were randomly divided into six groups, and 5 Gy whole-body irradiation was used for modeling. Mice were administered a gastric solvent, amifostine, or 25, 50, or 100 mg/kg FA daily for 12 days, consecutively, before irradiation. The serum of mice was collected 24 hour after irradiation to observe the content of inflammatory factors interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α. The spleen and thymus tissues of mice were weighed and the organ index was calculated for pathological testing and immunofluorescence detection. Results: FA reduced the radiation-induced decrease in the spleen and thymus indices. FA significantly reduced the secretion of inflammatory factors in the serum and reversed the radiation-induced reduction in lymphocytes in the spleen and thymus of mice. FA activated Sirt1 and inhibited the expression of the NLRP3 inflammasome to alleviate the inflammatory response. Conclusions: FA reduced radiation-induced inflammation in animals, possibly by activating Sirt1 and reducing nucleotide oligomerization domain (NOD)-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome expression, thereby reducing the secretion of inflammatory factors.

Microbial-Dependent Recruitment of Immature Myeloid Cells Promotes Intestinal Regeneration

The intestinal epithelium functions both in nutrient absorption and as a barrier, separating the luminal contents from a network of vascular, fibroblastic, and immune cells underneath. After injury to the intestine, multiple cell populations cooperate to drive regeneration of the mucosal barrier, including lymphatic endothelial cells (LECs). A population of granulocytic immature myeloid cells (IMCs), marked by Hdc, participate in regeneration of multiple organs such as the colon and central nervous system, and their contribution to intestinal regeneration was investigated. By using male and female histidine decarboxylase (Hdc) green fluorescent reporter (GFP) mice, we investigated the role of Hdc+ IMCs in intestinal regeneration after exposure to 12 Gy whole-body irradiation. The movement of IMCs was analyzed using flow cytometry and immunostaining. Ablation of Hdc+ cells using the HdcCreERT2 tamoxifen-inducible recombinase Cre system, conditional knockout of Prostaglandin-endoperoxidase synthase 2 (Ptgs2) in Hdc+ cells using HdcCre; Ptgs2 floxed mice, and visualization of LECs using Prox1tdTomato mice also was performed. The role of microbial signals was investigated by knocking down mice gut microbiomes using antibiotic cocktail gavages. We found that Hdc+ IMCs infiltrate the injured intestine after irradiation injury and promote epithelial regeneration in part by modulating LEC activity. Hdc+ IMCs express Ptgs2 (encoding cyclooxygenase-2/COX-2), and enables them to produce prostaglandin E2. Prostaglandin E2 acts on the prostaglandin E2 receptor 4 receptor (EP4) on LECs to promote lymphangiogenesis and induce the expression of proregenerative factors including R-spondin 3. Depletion of gut microbes leads to reduced intestinal regeneration by impaired recruitment of IMCs. Altogether, our results unveil a critical role for IMCs in intestinal repair by modulating LEC activity and implicate gut microbes as mediators of intestinal regeneration.

Honokiol alleviates radiation-induced premature ovarian failure via enhancing Nrf2.

The ovary is highly sensitive to radiation, and patients receiving radiotherapy are at significant risk of premature ovarian failure (POF). This study aimed to explore the radioprotective effect of honokiol (HKL) on ionizing radiation (IR)-induced POF. Female C57BL/6 mice were administered intraperitoneally with vehicle or HKL once daily for 7 days. On day 7, the mice in the IR and HKL+IR groups were exposed to 3.2Gy whole-body radiation for one hour after the intraperitoneal injection and sacrificed 12 or 72h after radiation exposure. The gonadosomatic index (GSI) was calculated. Blood samples were collected for enzyme-linked immunosorbent assay (ELISA). Ovaries were harvested for histological examination, immunohistochemistry, immunofluorescence, TUNEL, western blot, and qPCR. The fertility assessment was evaluated by calculating live offspring number. The optimum dose of HKL against radiation was 10mg/kg via intraperitoneal injection. POF was induced 72h after irradiation with significantly downregulated proliferating cell nuclear antigen (PCNA). The numbers of primordial and preantral follicles decreased significantly after irradiation (p<.001), whereas the number of atretic follicles increased (p<.001). The serum levels of estradiol (E2 ) and anti-Müllerian hormone (AMH) decreased to 50% of the control group after irradiation (p<.05). Moreover, the GSI after irradiation was 27% lower than in the control group (p<.05). The number of offspring in the IR group dropped by 50% compared with the control group (p<.05). HKL pretreatment protected the animals' fertility, GSI, PCNA, serum levels of E2 and AMH, and the number of primordial and preantral follicles. Significant upregulation of apoptosis-related proteins such as Pho-P53, Bax, Cyto C, C-caspase-3, C-PARP, and pyroptosis-related proteins such as Pho-NF-κB p65, NLRP3, caspase-1, IL-1β, and IL-18 was observed after irradiation, while the expression of Bcl-2 decreased. HKL pretreatment prevented these changes. After irradiation, malondialdehyde (MDA), Nrf2, and HO-1 were upregulated. HKL treatment activated the expression of Nrf2 and HO-1 and promoted the nucleus translocation of Nrf2. Furthermore, HKL did not affect ovarian reserves under physiological conditions. HKL ameliorated IR-induced POF by inhibiting apoptosis and pyroptosis by enhancing Nrf2 expression and translocation.

The effects of human amnion membrane-derived mesenchymal stem cells conditioned medium on ionizing radiation-induced premature ovarian failure and endoplasmic reticulum stress-related apoptosis mechanism

ObjectivesPremature ovarian failure (POF) is defined as the cessation of menstrual periods for at least 4–6 months before the age of 40 years, accompanied by FSH values measuring over 40 IU/L for a month. Radiation therapy, one of the cancer treatment methods, is known to accelerate ovarian aging by reducing and eliminating the number of primordial follicles in the ovarian follicle pool. Ionizing radiation has been reported to cause POF. The objective of this study is to investigate the impact of mesenchymal stem cell conditioned medium (hAMSCs-CM), which is isolated from the amniotic membrane of human placenta, on premature ovarian failure (POF) caused by whole-body irradiation. The study will focus on the ER stress and apoptosis mechanisms in the process. Study DisaynA POF model was created by exposing rats to 7 Gy of whole-body irradiation. Serum-free hAMSCs-CM were then administered via the tail vein. Follicle count was performed on the ovaries, and immunohistochemistry was used to determine the expressions of GRP78, CHOP, IRE-1, caspase-12, caspase-9, caspase-3. TUNEL was also carried out, and levels of serum FSH, LH, E2, AMH, and oxidative stress marker 8-OHdG were measured. Results and ConclusionThe application of hAMSCs-CM has been found to have a positive impact on follicles affected by radiation. After treatment, the number of primordial, primary, secondary, and graafian follicles, which had previously decreased due to radiation, showed an increase. Furthermore, the number of atretic follicles, which had been increasing due to radiation, showed a decrease. ER is one of the targets affected by ionizing radiation. After ionizing radiation, the expressions of ER stress-related markers and apoptosis markers increased in the ovary. After hAMSCs-CM administration, the expressions of these markers and number of TUNEL-positive cells decreased. Following irradiation, anti-mullerian hormone (AMH) and estradiol (E2) levels decreased, while follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels increased. After administration of hAMSCs-CM, AMH and E2 levels increased, while FSH and LH levels decreased.Amnion membrane-derived mesenchymal stem cell conditioned medium can play a therapeutic role in ionizing radiation-induced premature ovarian failure by reducing endoplasmic reticulum stress and apoptosis.

Integrated transcriptomic and metabolomic profiles reveal the protective mechanism of modified Danggui Buxue decoction on radiation-induced leukopenia in mice.

Leukopenia caused by radiation hinders the continuous treatment of cancers. Danggui Buxue Decoction (DBD) has been widely used in clinical owing to low toxicity and definite therapeutic effects to increase leukocytes. Meanwhile, icaritin (ICT) has also been proved to have the effect of boosting peripheral blood cells proliferation. However, there is no study to prove the efficacy of MDBD (Modified Danggui Buxue Decoction), a derivative herbal formula composed of DBD and ICT, in the treatment of radiation-induced leukopenia. In this study, we performed a model of 3.5Gy whole-body radiation to induce leukopenia in mice. The results of pharmacodynamic studies demonstrated that MDBD could significantly increase the white blood cells in peripheral blood by improving the activity of bone marrow nuclear cells, reducing bone marrow damage, modulating spleen index, and regulating hematopoietic factors to alleviate leukopenia. We also analyzed the integrated results of metabolomics and transcriptomics and found that MDBD could relieve leukopenia and alleviate bone marrow damage by targeting steroid biosynthesis and IL-17 signaling pathway, in which the key genes are Jun, Cxcl2 and Egr1. Therefore, our study provides a basis for the effectiveness and compatibility in the combination of traditional Chinese medicine formula and small molecule drugs.

Novel treatment and insight for irradiation-induced injuries: Dibucaine ameliorates irradiation-induced testicular injury by inhibiting fatty acid oxidation in primary Leydig cells

BackgroundMale infertility is a worldwide problem but few treatments, especially irradiation-induced testicular injury. The aim of this research was to investigate novel drugs for the treatment of irradiation-induced testicular injury. MethodsWe administered dibucaine (0.8 mg/kg) intraperitoneally to male mice (6 mice per group) after five consecutive daily 0.5 Gy whole-body irradiation, and evaluated its ameliorating efficacy by testicular HE staining and morphological measurements. Drug affinity responsive target stability assay (Darts) were used to find target protein and pathway; mouse primary Leydig cells were isolated and to explore the mechanism (Flow cytometry, Western blot, and Seahorse palmitate oxidative stress assays); finally rescue experiments were completed by combining dibucaine with fatty acid oxidative pathway inhibitors and activators. ResultsThe testicular HE staining and morphological measurements in dibucaine treatment group was significantly better than that in irradiation group (P < 0.05); sperm motility and mRNA levels of spermatogenic cell markers were also higher than those in the latter (P < 0.05). Darts and Western blot results showed that dibucaine targets CPT1A and downregulate fatty acid oxidation. Flow cytometry, Western blot, and Palmitate oxidative stress assays of primary Leydig cells demonstrated that dibucaine inhibits fatty acid oxidation in Leydig cells. Dibucaine combined with etomoxir/baicalin confirmed that its inhibition of fatty acid oxidation was beneficial in ameliorating irradiation-induced testicular injury. ConclusionsIn conclusion, our data suggest that dibucaine ameliorates irradiation-induced testicular injury in mice by inhibiting fatty acid oxidation in Leydig cells. This will provide novel ideas for the treatment of irradiation-induced testicular injury.

Monitoring TGFβ signaling in irradiated tumors.

Transforming growth factor β (TGFβ) is exquisitely regulated under physiological conditions but its activity is highly dysregulated in cancer. All cells make TGFβ and have receptors for the ligand, which is sequestered in the extracellular matrix in a latent form. Ionizing radiation elicits rapid release of TGFβ from these stores, so-called activation, over a wide range of doses and exposures, including low dose (<1Gy) whole-body irradiation, creating an extraordinarily potent signal in the irradiated tissue or tumor. Hence, accurate evaluation of TGFβ activity is complicated because of its ubiquitous distribution as a latent complex. Here we describe conditions for assays that reveal TGFβ activity in situ using either tissue preparations or functional imaging.

Radiomitigation by Melatonin in C57BL/6 Mice: Possible Implications as Adjuvant in Radiotherapy and Chemotherapy.

Melatonin (N-acetyl-5-methoxytryptamine), a chief secretory molecule of the pineal gland, has multiple properties, and numerous clinical investigations regarding its actions are in progress. This study investigated the radiomitigative role of melatonin in C57BL/6 mice. Melatonin (100 mg/kg) was orally administered once daily starting at 1 h on day 1 and subsequently every 24 h until day 7 after whole-body irradiation (WBI) and survival was monitored for 30 days. The bone marrow, spleen, and intestine were studied to evaluate the mitigative potential of melatonin after radiation-induced damage. Melatonin significantly improved the survival upto 60% and 90% after 9 Gy (lethal) and 7.5 Gy (sub-lethal) WBI, respectively. Melatonin alleviated WBI-induced myelosuppression and pancytopenia, and increased white blood cell, red blood cell, platelet, and lymphocyte (CD4+ and CD8+) counts in peripheral blood. Bone marrow and spleen cellularity were restored through enhanced haematopoiesis. Melatonin ameliorated the damage in the small intestine, and promoted recovery of villi length, crypts number, and goblet cell count. Melatonin mitigates the radiation-induced injury in the gastrointestinal and haematopoietic systems. The observed radiomitigative properties of melatonin can also be useful in the context of adjuvant therapy for cancer and radiotherapy.

Role of melatonin mediated G-CSF induction in hematopoietic system of gamma-irradiated mice

AimsHematopoietic acute radiation syndrome (H-ARS) can cause lethality, and therefore, the necessity of a safe radioprotector. The present study was focused on investigating the role of melatonin in granulocytes colony-stimulating factor (G-CSF) and related mechanisms underlying the reduction of DNA damage in hematopoietic system of irradiated mice. Main methodsC57BL/6 male mice were exposed to 2, 5, and 7.5Gy of whole-body irradiation (WBI), 30 min after intra-peritoneal administration of melatonin with different doses. Mice were sacrificed at different time intervals after WBI, and bone marrow, splenocytes, and peripheral blood lymphocytes were isolated for studying various parameters including micronuclei (MN), cell cycle, comet, γ-H2AX, gene expression, amino acid profiling, and hematology. Key findingsMelatonin100mg/kg ameliorated radiation (7.5Gy and 5Gy) induced MN frequency and cell death in bone marrow without mortality. At 24 h of post-WBI (2Gy), the frequency of micronucleated polychromatic erythrocytes (mnPCE) with different melatonin doses revealed 20 mg/kg as optimal i.p. dose for protecting the hematopoietic system against radiation injury. In comet assay, a significant reduction in radiation-induced % DNA tail (p ≤ 0.05) was observed at this dose. Melatonin reduced γ-H2AX foci/cell and eventually reached to the control level. Melatonin also decreased blood arginine levels in mice after 24 h of WBI. The gene expression of G-CSF, Bcl-2-associated X protein (BAX), and Bcl2 indicated the role of melatonin in G-CSF regulation and downstream pro-survival pathways along with anti-apoptotic activity. SignificanceThe results revealed that melatonin recovers the hematopoietic system of irradiated mice by inducing G-CSF mediated radioprotection.

Zingerone Alleviate Toxicity Induced by Cisplatin or Gamma Radiation in Rats by Modulating Pro-Inflammatory Mediators

Background: Zingerone is one of the active components of ginger that possesses multiple biological activities and anti-inflammatory properties against either radiation effect or cisplatin toxicity. Purpose: to examine the protective effect of zingerone against gamma radiation (IR) or cisplatin-induced immunotoxicity. Material and Methods: 48 rats were divided into six groups as follows: (group-1); normal control group received distilled water; (group-2); rats received Zingerone orally at a dose of 25 mg/kg b.wt. Once / day for 14 consecutive days (Zing.). (group-3); Rats were given a single injection of Cisplatin at a dose of 7.5 mg/kg b.wt. intraperitoneally (Cispl.). (group-4); Rats exposed to a single dose of 6 Gy whole-body gamma irradiation using 137Cesium source in a Gamma cell 40 (Rad.). (group-5); rats received same dose of Zingerone then they were exposed to gamma radiation as in group 4 (Zing+Rad.). (group-6); rats received Zingerone followed by single injection of Cisplatin at the dose of 7.5 mg/kg b.wt. Intraperitoneally (Zing+Cisp.). Results: Exhibited a significant increase in expression of NF-κB, IL-10, caspase-3, and gene expression of TNF-α as well as oxidative stress biomarkers (MDA and NO) levels accompanied with a reduced level of SOD in either whole body-irradiated or cisplatin-received group. Conversely, pro-inflammatory cytokines levels were significantly decreased with an improvement of oxidative stress in groups that received zingerone. Conclusion: It could be concluded that zingerone exerts its antioxidative activity and immunomodulatory effects through inhibition of pro-inflammatory mediators induced by whole body-gamma irradiation or cisplatin administration at two time interval early and late stage of radiation exposure (after 2 h and one week).Therefore, further studies are required to elucidate the molecular signaling pathway concerning zingerone.

Transcriptomic Response in the Spleen after Whole-Body Low-Dose X-Ray Irradiation

As the use of medical radiation procedures continues to rise, it is imperative to further our understanding of the effects of this exposure. The spleen is not known as a particularly radiosensitive organ, although its tolerance to radiation is not well understood. Low-dose radiation exposure has been implicated in beneficial responses, particularly in cell death and DNA damage repair. In this study, adult male rats received 2, 20, 200 mGy or 4 Gy whole-body X-ray irradiation and the transcriptional response in the spleen was analyzed at 0.5, 4 and 24 h postirradiation. We analyzed expression of genes involved in apoptosis, cell cycle progression and DNA damage repair. As expected, 4 Gy irradiated animals demonstrated elevated expression of genes related to apoptosis at 0.5, 4 and 24 h postirradiation in the spleen. These animals also showed upregulation of DNA damage repair genes at 24 h postirradiation. Interestingly, the spleens of 20 mGy irradiated animals showed reduced apoptosis and cell cycle arrest compared to the spleens of sham-irradiated animals. These results further reveal that the cellular response in the spleen to whole-body irradiation differs between low- and high-dose irradiation.

Sclerostin antibody increases trabecular bone and bone mechanical properties by increasing osteoblast activity damaged by whole-body irradiation in mice.

Irradiation therapy causes bone deterioration and increased risk for skeletal-related events. Irradiation interferes with trabecular architecture through increased osteoclastic activity, decreased osteoblastic activity, and increased adipocyte expansion in the bone marrow (BM), which further compounds bone-related disease. Neutralizing antibodies to sclerostin (Scl-Ab) increase bone mass and strength by increasing bone formation and reducing bone resorption. We hypothesized that treatment with Scl-Ab would attenuate the adverse effects of irradiation by increasing bone volume and decreasing BM adipose tissue (BMAT), resulting in better quality bone. In this study, 12-week-old female C57BL/6J mice were exposed to 6Gy whole-body irradiation or were non-irradiated, then administered Scl-Ab (25mg/kg) or vehicle weekly for 5weeks. Femoral μCT analysis confirmed that the overall effect of IR significantly decreased trabecular bone volume/total volume (Tb.BV/TV) (2-way ANOVA, p<0.0001) with a -43.8% loss in Tb.BV/TV in the IR control group. Scl-Ab independently increased Tb.BV/TV by 3.07-fold in non-irradiated and 3.6-fold in irradiated mice (2-way ANOVA, p<0.0001). Irradiation did not affect cortical parameters, although Scl-Ab increased cortical thickness and area significantly in both irradiated and non-irradiated mice (2-way ANOVA, p<0.0001). Femoral mechanical testing confirmed Scl-Ab significantly increased bending rigidity and ultimate moment independently of irradiation (2-way ANOVA, p<0.0001). Static and dynamic histomorphometry of the femoral metaphysis revealed osteoblast vigor, not number, was significantly increased in the irradiated mice treated with Scl-Ab. Systemic alterations were assessed through serum lipidomic analysis, which showed that Scl-Ab normalized lipid profiles in the irradiated group. This data supports the theory of sclerostin as a novel contributor to the regulation of osteoblast activity after irradiation. Overall, our data support the hypothesis that Scl-Ab ameliorates the deleterious effects of whole-body irradiation on bone and adipose tissue in a mouse model. Our findings suggest that future research into localized and systemic therapies after irradiation exposure is warranted.

Coenzyme Q10 attenuates inflammation and fibrosis implicated in radiation enteropathy through suppression of NF-kB/TGF-β/MMP-9 pathways

Radiation enteropathy is one the most common clinical issue for patients receiving radiotherapy for abdominal/pelvic tumors which severely affect the quality of life of cancer patients due to dysplastic lesions (ischemia, ulcer, or fibrosis) that aggravate the radiation damage. Herein, this study demonstrated the prophylactic role of coenzyme Q10 (CoQ10), a powerful antioxidant, against radiotherapy-induced gastrointestinal injury. Male Sprague Dawley rats were divided into four groups: group 1 was defined as control, and group 2 was the irradiated group. Group 3 and 4 were CoQ10 control and radiation plus CoQ10 groups, respectively. CoQ10 (10 mg/kg) was orally administered for 10 days before 10 Gy whole-body radiation and was continued for 4 days post-irradiation. CoQ10 administration protected rats delivered a lethal dose of ϒ-radiation from changes in crypt-villus structures and promoted regeneration of the intestinal epithelium. CoQ10 attenuated radiation-induced oxidative stress by decreasing lipid peroxidation and increasing the antioxidant enzyme catalase activity and reduced glutathione level. CoQ10 also counteracts inflammatory response mediated after radiation exposure through downregulating intestinal NF-ĸB expression which subsequently decreased the level of inflammatory cytokine IL-6 and the expression of COX-2. Radiation-induced intestinal fibrosis confirmed via Masson's trichrome staining occurred through upregulating transforming growth factor (TGF)-β1 and matrix metalloproteinase (MMP)-9 expression, while CoQ10 administration significantly diminishes these effects which further confirmed the anti-fibrotic property of CoQ10. Therefore, CoQ10 is a promising radioprotector that could prevent intestinal complications and enhance the therapeutic ratio of radiotherapy in patients with pelvic tumors through suppressing the NF-kB/TGF-β1/MMP-9 signaling pathway.

Inhibition of Radioactive Depletion of Hemocytes and Antitumor Effects of Flavonoid

Natural products are able to inhibit radiation effects and exert an antitumor effect with fewer adverse reactions; however, their antitumor effects are less than those of widely-used synthetic drugs. Flavonoid is a natural material that has attracting attention, and we extracted this material with alcohol and investigated the effect of continuous flavonoid administration on radioactivity-induced reduction of hemocytes, in addition to the antioxidant and antitum or effects of flavonoid. Following a 1-week adjustment period, flavonoid was administered intraperitoneally to male ICR mice at a dose of 100 mg/kg every other day for 2 weeks. Following administration, 2 Gy whole-body irradiation was performed and the counts of leukocytes, lymphocytes, and granulocytes and monocytes in the peripheral blood were determined 1, 3, 7, 15 and 30 days after irradiation. These cells were considered since they are closely associated with immunity to radioactivity. In a second experiment, flavonoid was similarly administered to the mice for 2 weeks after a 1-week adjustment period, and 2 Gy whole-body irradiation was performed. The antioxidant effects in hemocytes were then investigated using 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), a radical generator. In a third experiment, 1×106 Sarcoma-180 cells were inoculated into the right thigh of mice, which were divided into four groups: control, flavonoid-treated, 6 Gy irradiated and Flavonoid-treated + 6 Gy irradiated groups, and changes in tumor size were measured for 20 days. Statistical analysis was conducted using ANOVA for multiple groups. In the three experiments, administration of Flavonoid inhibited the reduction of hemocytes caused by whole-body irradiation, showed antioxidant effects against radioactivity, and inhibited tumor growth, respectively. In conclusion, our data suggest that the antioxidant effect of Flavonoid inhibits hemocyte reduction caused by whole-body irradiation and enhances immunological inhibition of tumor growth.

Xenogeneic transplantation of human WJ-MSCs rescues mice from acute radiation syndrome via Nrf-2-dependent regeneration of damaged tissues.

There is an unmet medical need for radiation countermeasures that can be deployed for treatment of exposed individuals during ionizing radiation (IR) accidents or terrorism. Wharton's jelly mesenchymal stem cells (WJ-MSCs) from human umbilical cord have been shown to avoid allorecognition and induce a tissue-regenerating microenvironment, which makes them an attractive candidate for mitigating IR injury. We found that WJ-MSCs protected mice from a lethal dose of IR even when transplanted up to 24hours after irradiation, and a combination of WJ-MSCs and antibiotic (tetracycline) could further expand the window of protection offered by WJ-MSCs. This combinatorial approach mitigated IR-induced damage to the hematopoietic and gastrointestinal system. WJ-MSCs increased the serum concentration of the cytoprotective cytokines granulocyte colony-stimulating factor (G-CSF) and IL-6 in mice. Knockdown of G-CSF and IL-6 in WJ-MSCs before injection to lethally irradiated mice or transplantation of WJ-MSCs to lethally irradiated Nrf-2 knockout mice significantly nullified the therapeutic protective efficacy. Hence, WJ-MSCs could be a potential cell-based therapy for individuals accidentally exposed to radiation.

ATF2 and ATF7 Are Critical Mediators of Intestinal Epithelial Repair.

Background & AimsActivation factor-1 transcription factor family members activating transcription factors 2 and 7 (ATF2 and ATF7) have highly redundant functions owing to highly homologous DNA binding sites. Their role in intestinal epithelial homeostasis and repair is unknown. Here, we assessed the role of these proteins in these conditions in an intestine-specific mouse model.MethodsWe performed in vivo and ex vivo experiments using Villin-CreERT2Atf2fl/flAtf7ko/ko mice. We investigated the effects of intestinal epithelium-specific deletion of the Atf2 DNA binding region in Atf7-/- mice on cellular proliferation, differentiation, apoptosis, and epithelial barrier function under homeostatic conditions. Subsequently, we exposed mice to 2% dextran sulfate sodium (DSS) for 7 days and 12 Gy whole-body irradiation and assessed the response to epithelial damage.ResultsActivating phosphorylation of ATF2 and ATF7 was detected mainly in the crypts of the small intestine and the lower crypt region of the colonic epithelium. Under homeostatic conditions, no major phenotypic changes were detectable in the intestine of ATF mutant mice. However, on DSS exposure or whole-body irradiation, the intestinal epithelium showed a clearly impaired regenerative response. Mutant mice developed severe ulceration and inflammation associated with increased epithelial apoptosis on DSS exposure and were less able to regenerate colonic crypts on irradiation. In vitro, organoids derived from double-mutant epithelium had a growth disadvantage compared with wild-type organoids, impaired wound healing capacity in scratch assay, and increased sensitivity to tumor necrosis factor-α–induced damage.ConclusionsATF2 and ATF7 are dispensable for epithelial homeostasis, but are required to maintain epithelial regenerative capacity and protect against cell death during intestinal epithelial damage and repair.

Differentiation of Heterogeneous Radiation Exposure Using Hematology and Blood Chemistry.

In the aftermath of a nuclear incident, survivors will suffer the deleterious effects from acute radiation exposure. The majority of those affected would have received heterogeneous radiation exposure, reflected in hematological metrics and blood chemistry. Here, we investigated the acute and long-term changes in kinetics and magnitude of pancytopenia and blood chemistry in rats irradiated using varying degrees of body shielding. We hypothesized that, although a single blood count may not be able to differentiate the degree of radiation exposure, a combination of measurements from complete blood cell counts (CBCs) and blood chemistry tests is able to do so. Male Sprague Dawley rats, 8-10 weeks of age, received single-dose 7.5 Gy (160 kVp, 25 mA, 1.16 Gy/min) whole-body irradiation (WBI, LD100/30) or partial-body irradiation (PBI), as follows: one leg shielded (1LS, LD0/30), two legs shielded (2LS, LD0/30) or the upper half of the body shielded (UHS, LD0/30). Animal morbidity and weights were measured. Blood was drawn at 1, 5, 10, 20 and 30 days postirradiation (n = 4-11). For kidney and liver function measurements, CBC and blood chemistry analyses were performed. WBI animals on average survived 9 ± 0.4 days postirradiation. In contrast, all PBI animals survived the 30-day study period. CBC analysis revealed that both white blood cell (WBC) and platelet counts were most affected after irradiation. While WBC counts were significantly lower in all irradiated groups on days 1, 5 and 10, platelets were only significantly lower on days 5 and 10 postirradiation. In addition, on day 5 postirradiation both WBC and platelet counts were able to differentiate WBI (non-survivors) from PBI 2LS and UHS animals (survivors). Using four blood parameters (platelets, percentage lymphocytes, percentage neutrophils and percentage monocytes) on day 5 after 7.5 Gy irradiation and a linear discrimination analysis (LDA), we were able to predict the degree of body exposure in animals with a 95.8% accuracy. Alkaline phosphatase (ALP) was significantly lower in all groups on days 5 and 10 postirradiation compared to baseline. Furthermore, ALP was significantly higher in the UHS than WBI animals. The AST:ALT ratio was significantly higher than baseline in all irradiated groups on day 1 postirradiation. In conclusion, four CBC parameters, on day 5 after receiving a 7.5 Gy dose of radiation, can be employed in a LDA to differentiate various degrees of exposure (shielding). The characterization presented in this work paves the way for further studies in differences caused by heterogeneous body exposure to radiation and a new metric for biodosimetry.

Protective effect of the antioxidative peptide SS31 on ionizing radiation-induced hematopoietic system damage in mice

Ionizing radiation (IR) causes severe damage to the hematopoietic system; thus, it is necessary to explore agents or compounds that can reduce this damage. SS31 is a mitochondria-targeted peptide that can scavenge cellular reactive oxygen species (ROS) and inhibit the production of mitochondrial ROS. Therefore, in this study, we discuss the protective effect of SS31 on IR-induced hematopoietic system damage. Our results showed that treatment with 6 mg/kg SS31 elevated the survival rate of lethally irradiated mice and increased the numbers of white blood cells, red blood cells, hemoglobin and platelets in mice exposed to 4 Gy whole-body irradiation. In addition, SS31 administration improved the number of hematopoietic stem/progenitor cells (HSPCs) and the self-renewal and reconstitution abilities of these cells in irradiated mice. The elevation of ROS levels is the main cause of IR-induced hematopoietic system damage, and SS31 can effectively reduce the ROS level in HSPCs. The above results suggest that SS31 can protect the hematopoietic system from radiation-induced damage by reducing cellular ROS levels.

Age Dependence of Radiation-Induced Genomic Instability in Mouse Hematopoietic Stem Cells.

Age at exposure is a critical factor that influences the risk of radiation-induced leukemia. Accumulating evidence suggests that ionizing radiation can induce genomic instability and promote leukemogenesis in hematopoietic stem cells (HSCs); however, the influence of age on this phenomenon has not been elucidated. In this study, infant (1-week-old) or adult (14-week-old) C3H/He mice received sham or 4 Gy whole-body irradiation, and bone marrow cells were transplanted to recipients at day 1 or 60 postirradiation. Twelve days after bone marrow transplant, we analyzed the radiation-induced genomic instability by scoring the frequency of DNA damage and micronucleus formation in colony-forming units-spleen (CFU-Ss). We observed significant increases in DNA damage and micronucleus formation in CFU-Ss of the 4 Gy irradiated adult cells transplanted at day 1 or 60 postirradiation. However, the frequency of DNA damage focus and micronucleus formation in CFU-Ss of 4 Gy irradiated infant cells transplanted at day 1 or 60 postirradiation was relatively decreased. Quantitative differences in the reactive oxygen species and cells expressing inducible nitric oxide synthase in CFU-Ss suggested that age-dependent radiation-induced genomic instability may result from chronic oxidative stress by pro-inflammatory states in HSC descendants after radiation exposure.

Abstract LB-105: Combining CD4+ T cell transfer and immune checkpoint blockade demonstrates the power of combined immunotherapies for treatment of progressed solid carcinomas in mice

Abstract Tumor and immune cells express inhibitory immune checkpoints (ICP) that paralyze tumor infiltrating T cells. ICP-specific antibodies (mAbs) can block and restore T cell functions and promote anti-tumoral effects. Also, tumor-antigen specific IFN-y secreting CD4+ T cells (TA-Th1) mediate strong anti-tumoral effects and can induce senescence in cancer cells. Here, we report on the development and immunological characterization of a highly efficient TA-Th1 and checkpoint-inhibitor based combined immunotherapy (CIT) in a progressed endogenous cancer model. Mice bearing progressed pancreatic islet carcinomas (RIP1-Tag2) and symptomatic low blood glucose levels (BGL; ~80 mg/dl) were treated weekly with TA-Th1 and αPD-L1+αLAG-3 mAbs (PDL1/LAG3) after an initial preparative 2 Gy-whole body radiation. Therapy was monitored by BGL measurements. Additionally, we conducted baseline and follow-up 18F-FDG PET/MRI scans to uncover the splenic glucose metabolism as a consequence of immune cell activation. We performed ex vivo flow cytometry (FC), immunohistochemistry (IHC), and fluorescent microscopy (FM) of tumor tissue and lymphatic organs at early (1 wk) and late (3-4 wks; endpoint of SHAM treated mice) time point of treatment focusing on the immune cell composition, activation patterns and senescence induction. Treatment exclusively with TA-Th1 prolonged the median survival of the mice from 14 to 18 wks (n=14) while ICP blockade without TA-Th1 cells was not efficient at all (PDL1/LAG3; median survival=14wks, n=11). The combination of TA-Th1 and PDL1/LAG3 (CIT) was highly efficient and significantly extended the median survival to 20 wks (n=15, p=0.001). Thus, exclusively CIT-treated mice revealed very small tumors and a strong lymphocytic infiltrate. FC analysis demonstrated early and specific homing of highly activated (CD69) TA-Th1 into the draining LN without impairment of the endogenous CD8+ T cell population. After 4 wks of treatment, we determined an increase in endogenous effector CD4+ and CD8+ T cells exclusively in mice receiving TA-Th1, indicating cross-priming as a possible mechanism of T cell activation. Analysis of p16/Ki67 expression in tumors revealed a strongly enhanced p16 and reduced Ki67 expression exclusively in CIT-treated mice indicating tumor senescence which was not observed in control groups. 18F-FDG-PET/MRI of the spleen showed a significantly increased glucose metabolism in CIT treated mice when compared to SHAM-treatment. In conclusion, our newly developed CIT (2 Gy+TA-Th1+PDL1/LAG3) strongly promoted an anti-tumor immune response in mice with progressed solid cancer. Most importantly, only CIT was able to induce TA-Th1 mediated tumor senescence whereas TA-Th1 or PDL1/LAG3 mAbs alone were inefficient. Moreover, 18F-FDG-PET/MRI of the spleen might represent a novel powerful tool to stratify ICP-blocked responders. Thus, ICP-blockade is applicable to reinforce Th1-cell based immunotherapies and to prolong the lifespan of mice with progressed solid carcinomas. Citation Format: Barbara F. Schörg, Dominik Sonanini, Johannes Schwenck, Christoph Griessinger, Birgit Fehrenbacher, Martin Schaller, Ursula Kohlhofer, Leticia Quintanilla-Martinez, Christian La Fourgère, Martin Röcken, Bernd J. Pichler, Manfred Kneilling. Combining CD4+ T cell transfer and immune checkpoint blockade demonstrates the power of combined immunotherapies for treatment of progressed solid carcinomas in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-105.