Natural-history Characterization of a Murine Partial-body Irradiation Model System: Establishment of a Multiple-Parameter Based GI-ARS Severity-Scoring System.

The purpose of this investigation was to characterize the natural history of a murine total-abdominal-irradiation exposure model to measure gastrointestinal acute radiation injury. Male CD2F1 mice at 12 to 15 weeks old received total-abdominal irradiation using 4-MV linear accelerator X-rays doses of 0, 11, 13.5, 15, 15.75 and 16.5 Gy (2.75 Gy/min). Daily cage-side (i.e., in the animal housing room) observations of clinical signs and symptoms including body weights on all animals were measured up to 10 days after exposure. Jejunum tissues from cohorts of mice were collected at 1, 3, 7 and 10 days after exposure and radiation injury was assessed by histopathological analyses. Results showed time- and dose-dependent loss of body weight [for example at 7 days: 0.66 (±0.80) % loss for 0 Gy, 6.40 (±0.76) % loss at 11 Gy, 9.43 (±2.06) % loss at 13.5 Gy, 23.53 (± 1.91) % loss at 15 Gy, 29.97 (±1.16) % loss at 15.75 Gy, and 31.79 (±0.76) % loss at 16.5 Gy]. Negligible clinical signs and symptoms, except body weight changes, of radiation injury were observed up to 10 days after irradiation with doses of 11 to 15 Gy. Progressive increases in the severity of clinical signs and symptoms were found after irradiation with doses >15 Gy. Jejunum histology showed a progressive dose-dependent increase in injury. For example, at 7 days postirradiation, the percent of crypts, compared to controls, decreased to 82.3 (±9.5), 69.2 (±12.3), 45.4 (±11.9), 18.0 (±3.4), and 11.5 (± 1.8) with increases in doses from 11 to 16.5 Gy. A mucosal injury scoring system was used that mainly focused on changes in villus morphology damage (i.e., subepithelial spaces near the tips of the villi with capillary congestion, significant epithelial lifting along the length of the villi with a few denuded villus tips). Peak levels of total-abdominal irradiation induced effects on the mucosal injury score were seen 7 days after irradiation for doses ≥15 Gy, with a trend to show a decline after 7 days. A murine multiple-parameter gastrointestinal acute-radiation syndrome severity-scoring system was established based on clinical signs and symptoms that included measures of appearance (i.e., hunched and/or fluffed fur), respiratory rate, general (i.e., decreased mobility) and provoked behavior (i.e., subdued response to stimulation), weight loss, and feces/diarrhea score combined with jejunum mucosal-injury grade score. In summary, the natural-history radio-response for murine partial-body irradiation exposures is important for establishing a well-characterized radiation model system; here we established a multiple-parameter gastrointestinal acute-radiation syndrome severity-scoring system that provides a radiation injury gastrointestinal tissue-based assessment utility.

Feasibility of Reducing Animal Numbers in Radiation Countermeasure Experiments from Historic Levels when using Sample Size Calculations.

Historically, animal numbers have most often been in the hundreds for experiments designed to estimate the dose reduction factor (DRF) of a radiation countermeasure treatment compared to a control treatment. Before 2010, researchers had to rely on previous experience, both from others and their own, to determine the number of animals needed for a DRF experiment. In 2010, a formal sample size formula was developed by Kodell et al. This theoretical work showed that sample sizes for realistic, yet hypothetical, DRF experiments could be less than a hundred animals and still have sufficient power to detect clinically meaningful DRF values. However, researchers have been slow to use the formula for their DRF experiments, whether from ignorance to its existence or hesitancy to depart from "tried and true" sample sizes. Here, we adapt the sample size formula to better fit usual DRF experiments, and, importantly, we provide real experimental evidence from two independent DRF experiments that sample sizes smaller than what have typically been used can still statistically detect clinically meaningful DRF values. In addition, we update a literature review of DRF experiments which can be used to inform future DRF experiments, provide answers to questions that researchers have asked when considering sample size calculations rather than solely relying on previous experience, whether their own or others', and, in the supplementary material, provide R code implementing the formula, along with several exercises to familiarize the user with the adapted formula.

Transcriptomic Profiling and Pathway Analysis of Mesenchymal Stem Cells Following Low Dose-Rate Radiation Exposure.

Low dose-rate radiation exposure can occur in medical imaging, as background from environmental or industrial radiation, and is a hazard of space travel. In contrast with high dose-rate radiation exposure that can induce acute life-threatening syndromes, chronic low-dose radiation is associated with Chronic Radiation Syndrome (CRS), which can alter environmental sensitivity. Secondary effects of chronic low dose-rate radiation exposure include circulatory, digestive, cardiovascular, and neurological diseases, as well as cancer. Here, we investigated 1-2 Gy, 0.66 cGy/h, 60Co radiation effects on primary human mesenchymal stem cells (hMSC). There was no significant induction of apoptosis or DNA damage, and cells continued to proliferate. Gene ontology (GO) analysis of transcriptome changes revealed alterations in pathways related to cellular metabolism (cholesterol, fatty acid, and glucose metabolism), extracellular matrix modification and cell adhesion/migration, and regulation of vasoconstriction and inflammation. Interestingly, there was increased hypoxia signaling and increased activation of pathways regulated by iron deficiency, but Nrf2 and related genes were reduced. The data were validated in hMSC and human lung microvascular endothelial cells using targeted qPCR and Western blotting. Notably absent in the GO analysis were alteration pathways for DNA damage response, cell cycle inhibition, senescence, and pro-inflammatory response that we previously observed for high dose-rate radiation exposure. Our findings suggest that cellular gene transcription response to low dose-rate ionizing radiation is fundamentally different compared to high-dose-rate exposure. We hypothesize that cellular response to hypoxia and iron deficiency are driving processes, upstream of the other pathway regulation.

A Review of Radiation-Induced Alterations of Multi-Omic Profiles, Radiation Injury Biomarkers, and Countermeasures.

Increasing utilization of nuclear power enhances the risks associated with industrial accidents, occupational hazards, and the threat of nuclear terrorism. Exposure to ionizing radiation interferes with genomic stability and gene expression resulting in the disruption of normal metabolic processes in cells and organs by inducing complex biological responses. Exposure to high-dose radiation causes acute radiation syndrome, which leads to hematopoietic, gastrointestinal, cerebrovascular, and many other organ-specific injuries. Altered genomic variations, gene expression, metabolite concentrations, and microbiota profiles in blood plasma or tissue samples reflect the whole-body radiation injuries. Hence, multi-omic profiles obtained from high-resolution omics platforms offer a holistic approach for identifying reliable biomarkers to predict the radiation injury of organs and tissues resulting from radiation exposures. In this review, we performed a literature search to systematically catalog the radiation-induced alterations from multi-omic studies and radiation countermeasures. We covered radiation-induced changes in the genomic, transcriptomic, proteomic, metabolomic, lipidomic, and microbiome profiles. Furthermore, we have covered promising multi-omic biomarkers, FDA-approved countermeasure drugs, and other radiation countermeasures that include radioprotectors and radiomitigators. This review presents an overview of radiation-induced alterations of multi-omics profiles and biomarkers, and associated radiation countermeasures.

RRx-001 Radioprotection: Enhancement of Survival and Hematopoietic Recovery in Gamma-Irradiated Mice.

The present studies evaluate the in vivo prophylactic radioprotective effects of 1-bromoacetyl-3, 3-dinitroazetidine (RRx-001), a phase III anticancer agent that inhibits c-myc and downregulates CD-47, after total body irradiation (TBI), in lethally and sublethally irradiated CD2F1 male mice. A single dose of RRx-001 was administered by intraperitoneal (IP) injection 24 h prior to a lethal or sublethal radiation dose. When irradiated with 9.35 Gy, the dose lethal to 70% of untreated mice at 30 days (LD70/30), only 33% of mice receiving RRx-001 (10 mg/kg) 24 h prior to total body irradiation (TBI) died by day 30, compared to 67% in vehicle-treated mice. The same pretreatment dose of RRx-001 resulted in a significant dose reduction factor of 1.07. In sublethally TBI mice, bone marrow cellularity was increased at day 14 in the RRx-001-treated mice compared to irradiated vehicle-treated animals. In addition, significantly higher numbers of lymphocytes, platelets, percent hematocrit and percent reticulocytes were observed on days 7 and/or 14 in RRx-001-treated mice. These experiments provide proof of principle that systemic administration of RRx-001 prior to TBI significantly improves overall survival and bone marrow regeneration.

Celebrating 60 Years of Accomplishments of the Armed Forces Radiobiology Research Institute1.

Chartered by the U.S. Congress in 1961, the Armed Forces Radiobiology Research Institute (AFRRI) is a Joint Department of Defense (DoD) entity with the mission of carrying out the Medical Radiological Defense Research Program in support of our military forces around the globe. In the last 60 years, the investigators at AFRRI have conducted exploratory and developmental research with broad application to the field of radiation sciences. As the only DoD facility dedicated to radiation research, AFRRI's Medical Radiobiology Advisory Team provides deployable medical and radiobiological subject matter expertise, advising commanders in the response to a U.S. nuclear weapon incident and other nuclear or radiological material incidents. AFRRI received the DoD Joint Meritorious Unit Award on February 17, 2004, for its exceptionally meritorious achievements from September 11, 2001 to June 20, 2003, in response to acts of terrorism and nuclear/radiological threats at home and abroad. In August 2009, the American Nuclear Society designated the institute a nuclear historic landmark as the U.S.'s primary source of medical nuclear and radiological research, preparedness and training. Since then, research has continued, and core areas of study include prevention, assessment and treatment of radiological injuries that may occur from exposure to a wide range of doses (low to high). AFRRI collaborates with other government entities, academic institutions, civilian laboratories and other countries to research the biological effects of ionizing radiation. Notable early research contributions were the establishment of dose limits for major acute radiation syndromes in primates, applicable to human exposures, followed by the subsequent evolution of radiobiology concepts, particularly the importance of immune collapse and combined injury. In this century, the program has been essential in the development and validation of prophylactic and therapeutic drugs, such as Amifostine, Neupogen®, Neulasta®, Nplate® and Leukine®, all of which are used to prevent and treat radiation injuries. Moreover, AFRRI has helped develop rapid, high-precision, biodosimetry tools ranging from novel assays to software decision support. New drug candidates and biological dose assessment technologies are currently being developed. Such efforts are supported by unique and unmatched radiation sources and generators that allow for comprehensive analyses across the various types and qualities of radiation. These include but are not limited to both 60Co facilities, a TRIGA® reactor providing variable mixed neutron and γ-ray fields, a clinical linear accelerator, and a small animal radiation research platform with low-energy photons. There are five major research areas at AFRRI that encompass the prevention, assessment and treatment of injuries resulting from the effects of ionizing radiation: 1. biodosimetry; 2. low-level and low-dose-rate radiation; 3. internal contamination and metal toxicity; 4. radiation combined injury; and 5. radiation medical countermeasures. These research areas are bolstered by an educational component to broadcast and increase awareness of the medical effects of ionizing radiation, in the mass-casualty scenario after a nuclear detonation or radiological accidents. This work provides a description of the military medical operations as well as the radiation facilities and capabilities present at AFRRI, followed by a review and discussion of each of the research areas.

CDX-301: a novel medical countermeasure for hematopoietic acute radiation syndrome in mice.

Bone marrow failure and hematopoietic damage is one of the major consequences of irradiation-induced lethality. There is an immediate need to develop medical countermeasures (MCMs) to combat irradiation-induced lethality. We tested the efficacy of CDX-301, developed by Celldex Therapeutics Inc., in mice exposed to Co-60 gamma total body irradiation (TBI). The drug demonstrated its efficacy both as a prophylactic countermeasure and a mitigator in CD2F1 mice exposed to TBI. A single dose of CDX-301 administered 24 h prior to 24 h post-exposure conferred significant survival. Accelerated recovery from irradiation-induced peripheral blood cytopenia, bone marrow damage as well as apoptosis in sternum was observed in mice pre-treated with CDX-301. Analysis of splenocytes revealed alterations in T cell profiles that were dependent on the time of drug administration. Prophylactic treatment of CDX-301 resulted in increased splenic CD3+ T cells, specifically CD4+T helper cells, compared to splenocytes from non-irradiated mice. These results indicate that CDX-301 is a promising radiation countermeasure and demonstrate its capability to protect cells within hematopoietic organs. These data support potential use of CDX-301, both pre- and post-radiation, against hematopoietic acute radiation syndrome with a broad window for medical management in a radiological or nuclear event.

Bone Marrow Endothelial Cells Influence Function and Phenotype of Hematopoietic Stem and Progenitor Cells after Mixed Neutron/Gamma Radiation.

The bone marrow (BM) microenvironment plays a crucial role in the maintenance and regeneration of hematopoietic stem (HSC) and progenitor cells (HSPC). In particular, the vascular niche is responsible for regulating HSC maintenance, differentiation, and migration of cells in and out of the BM. Damage to this niche upon exposure to ionizing radiation, whether accidental or as a result of therapy, can contribute to delays in HSC recovery and/or function. The ability of BM derived-endothelial cells (BMEC) to alter and/or protect HSPC after exposure to ionizing radiation was investigated. Our data show that exposure of BMEC to ionizing radiation resulted in alterations in Akt signaling, increased expression of PARP-1, IL6, and MCP-1, and decreased expression of MMP1 and MMP9. In addition, global analysis of gene expression of HSC and BMEC in response to mixed neutron/gamma field (MF) radiation identified 60 genes whose expression was altered after radiation in both cell types, suggesting that a subset of genes is commonly affected by this type of radiation. Focused gene analysis by RT-PCR revealed two categories of BMEC alterations: (a) a subset of genes whose expression was altered in response to radiation, with no additional effect observed during coculture with HSPC, and (b) a subset of genes upregulated in response to radiation, and altered when cocultured with HSPC. Coculture of BMEC with CD34+ HSPC induced HSPC proliferation, and improved BM function after MF radiation. Nonirradiated HSPC exhibited reduced CD34 expression over time, but when irradiated, they maintained higher CD34 expression. Nonirradiated HSPC cocultured with nonirradiated BMEC expressed lower levels of CD34 expression compared to nonirradiated alone. These data characterize the role of each cell type in response to MF radiation and demonstrate the interdependence of each cell's response to ionizing radiation. The identified genes modulated by radiation and coculture provide guidance for future experiments to test hypotheses concerning specific factors mediating the beneficial effects of BMEC on HSPC. This information will prove useful in the search for medical countermeasures to radiation-induced hematopoietic injury.

Impact of Abbreviated Filgrastim Schedule on Survival and Hematopoietic Recovery after Irradiation in Four Mouse Strains with Different Radiosensitivity.

Filgrastim (Neupogen®, granulocyte-colony stimulating factor) is among the few countermeasures recommended for management of patients in the event of lethal total-body irradiation. Despite the plethora of studies using filgrastim as a radiation countermeasure, relatively little is known about the optimal dose schedule of filgrastim to mitigate radiation lethality. We evaluated the efficacy of filgrastim in improving 30-day survival of CD2F1 mice irradiated with a lethal dose (LD70/30) in the AFRRI cobalt-60 facility. We tested different schedules of 1, 3, 5, 10 or 16 once-daily injections of filgrastim initiated one day after irradiation. Time optimization studies with filgrastim treatment were also performed, beginning 6-48 h postirradiation. Maximum survival was observed with 3 daily doses of 0.17 mg/kg filgrastim. Survival efficacy of the 3-day treatment was compared against the conventional 16-day filgrastim treatment after irradiation in four mouse strains with varying radiation sensitivities: C3H/HeN, C57BL/6, B6C3F1 and CD2F1. Blood indices, bone marrow histopathology and colony forming unit assays were also evaluated. Filgrastim significantly increased 30-day survival (P < 0.001) with a 3-day treatment compared to 16-day treatment. Filgrastim did not prevent cytopenia nadirs, but facilitated faster recovery of white blood cells, neutrophils, red blood cells, platelets, lymphocytes and hematocrits in all four strains. Accelerated hematopoietic recovery was also reflected in faster bone marrow reconstitution and significant increase in hematopoietic progenitors (P < 0.001) in all four mouse strains. These data indicate that prompt and abbreviated filgrastim treatment has potential benefit for triage in the event of a radiological incident for treating acute hematopoietic syndrome.

Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation.

Detonation of an improvised nuclear device near a population center would cause significant casualties from the acute radiation syndrome (ARS) due to exposure to mixed neutron/gamma fields (MF). The pathophysiology of ARS involves inflammation, microvascular damage and alterations in immune function. Interactions between endothelial cells (EC) and hematopoietic cells are important not only for regulating immune cell traffic and function, but also for providing the microenvironment that controls survival, differentiation and migration of hematopoietic stem and progenitor cells in blood-forming tissues. Endothelial cells/leukocyte interactions also influence tumor progression and the results of anticancer therapies. In this study, we hypothesized that irradiation of endothelial cells would modulate their effects on hematopoietic cells and vice versa. Human umbilical vein endothelial cells (HUVEC) and immortalized T lymphocytes (Jurkat cells) were cultured individually and in co-culture after exposure to mixed fields. Effects of nonirradiated cells were compared to effects of irradiated cells and alterations in signaling pathways were determined. Mitogen-activated protein kinases (MAPKs) p38 and p44/42 (ERK1/2) in HUVEC exhibited higher levels of phosphorylated protein after exposure to mixed field radiation. IL-6, IL-8, G-CSF, platelet derived growth factor (PDGF) and angiopoietin 2 (ANG2) protein expression were upregulated in HUVEC by exposure to mixed field radiation. PCR arrays using HUVEC mRNA revealed alterations in gene expression after exposure to mixed fields and/or co-culture with Jurkat cells. The presence of HUVEC also influenced the function of Jurkat cells. Nonirradiated Jurkat cells showed an increase in proliferation when co-cultured with nonirradiated HUVEC, and a decrease in proliferation when co-cultured with irradiated HUVEC. Additionally, nonirradiated Jurkat cells incubated in media from irradiated HUVEC exhibited upregulation of activated caspase 3. Irradiation of Jurkat cells caused a G2/M arrest and increased adherence to HUVEC. When co-cultured with HUVEC, irradiated Jurkat cells exhibited G0/G1 arrest and increased apoptosis. The data indicate that gene expression and cell function of endothelial cells and hematopoietic cells are influenced by radiation and by interactions between the two cell types. These phenomena may affect the success of therapies for ARS and cancer.

Ciprofloxacin modulates cytokine/chemokine profile in serum, improves bone marrow repopulation, and limits apoptosis and autophagy in ileum after whole body ionizing irradiation combined with skin-wound trauma.

Radiation combined injury (CI) is a radiation injury (RI) combined with other types of injury, which generally leads to greater mortality than RI alone. A spectrum of specific, time-dependent pathophysiological changes is associated with CI. Of these changes, the massive release of pro-inflammatory cytokines, severe hematopoietic and gastrointestinal losses and bacterial sepsis are important treatment targets to improve survival. Ciprofloxacin (CIP) is known to have immunomodulatory effect besides the antimicrobial activity. The present study reports that CIP ameliorated pathophysiological changes unique to CI that later led to major mortality. B6D2F1/J mice received CI on day 0, by RI followed by wound trauma, and were treated with CIP (90 mg/kg p.o., q.d. within 2 h after CI through day 10). At day 10, CIP treatment not only significantly reduced pro-inflammatory cytokine and chemokine concentrations, including interleukin-6 (IL-6) and KC (i.e., IL-8 in human), but it also enhanced IL-3 production compared to vehicle-treated controls. Mice treated with CIP displayed a greater repopulation of bone marrow cells. CIP also limited CI-induced apoptosis and autophagy in ileal villi, systemic bacterial infection, and IgA production. CIP treatment led to LD(0/10) compared to LD(20/10) for vehicle-treated group after CI. Given the multiple beneficial activities of CIP shown in our experiments, CIP may prove to be a useful therapeutic drug for CI.

Gamma-tocotrienol, a radiation prophylaxis agent, induces high levels of granulocyte colony-stimulating factor.

Gamma-tocotrienol (GT3), a promising radioprotectant, is shown to protect CD2F1 mice from radiation-induced neutropenia and thrombocytopenia when given 24h prior to total-body irradiation. GT3 also is shown to increase white blood cells (WBC) and absolute neutrophil counts (ANC) transiently in peripheral blood. We hypothesized that increases in WBC and ANC may involve stimulation of hematopoiesis possibly by cytokines and growth factors. To evaluate the effects of GT3 on hematopoietic system, we measured various cytokines, chemokines and growth factors by cytokine array and Bio-Plex assays. Both showed strong induction of various cytokines and chemokines. GT3 treatment resulted in significant increases in G-CSF, IL-1α, IL-1ß, IL-6, IL-12p70, IL-17, MIP-1α, and KC levels. G-CSF levels increased markedly within 12-24h after administration (5441 pg/ml in GT3-treated groups compared to 17 pg/ml in vehicle control). Most of these cytokine levels were elevated in the presence or absence of radiation. Time-course analysis of G-CSF and IL-6 induction showed that both cytokines were induced transiently after GT3 administration, and returned to normal levels by 48 h post-administration. For G-CSF, the peak was observed between 12 and 24h post-administration of GT3; however, the highest levels of IL-6 were obtained between 6 and 12h. These results demonstrate that GT3 induced high levels of G-CSF and other inflammatory cytokines and chemokines within 24h after administration. Survival studies reported showed that the most efficacious time for administering GT3 was 24h prior to irradiation, possibly because it induced key hematopoietic cytokines in that time window. These results also suggest a possible role of GT3-induced G-CSF stimulation in protecting mice from radiation-induced neutropenia and thrombocytopenia.

Wound trauma alters ionizing radiation dose assessment.

BACKGROUND: Wounding following whole-body γ-irradiation (radiation combined injury, RCI) increases mortality. Wounding-induced increases in radiation mortality are triggered by sustained activation of inducible nitric oxide synthase pathways, persistent alteration of cytokine homeostasis, and increased susceptibility to bacterial infection. Among these factors, cytokines along with other biomarkers have been adopted for biodosimetric evaluation and assessment of radiation dose and injury. Therefore, wounding could complicate biodosimetric assessments. RESULTS: In this report, such confounding effects were addressed. Mice were given 60Co γ-photon radiation followed by skin wounding. Wound trauma exacerbated radiation-induced mortality, body-weight loss, and wound healing. Analyses of DNA damage in bone-marrow cells and peripheral blood mononuclear cells (PBMCs), changes in hematology and cytokine profiles, and fundamental clinical signs were evaluated. Early biomarkers (1 d after RCI) vs. irradiation alone included significant decreases in survivin expression in bone marrow cells, enhanced increases in γ-H2AX formation in Lin+ bone marrow cells, enhanced increases in IL-1ß, IL-6, IL-8, and G-CSF concentrations in blood, and concomitant decreases in γ-H2AX formation in PBMCs and decreases in numbers of splenocytes, lymphocytes, and neutrophils. Intermediate biomarkers (7 - 10 d after RCI) included continuously decreased γ-H2AX formation in PBMC and enhanced increases in IL-1ß, IL-6, IL-8, and G-CSF concentrations in blood. The clinical signs evaluated after RCI were increased water consumption, decreased body weight, and decreased wound healing rate and survival rate. Late clinical signs (30 d after RCI) included poor survival and wound healing. CONCLUSION: Results suggest that confounding factors such as wounding alters ionizing radiation dose assessment and agents inhibiting these responses may prove therapeutic for radiation combined injury and reduce related mortality.

Efficacy of radiation countermeasures depends on radiation quality.

The detonation of a nuclear weapon or a nuclear accident represent possible events with significant exposure to mixed neutron/γ-radiation fields. Although radiation countermeasures generally have been studied in subjects exposed to pure photons (γ or X rays), the mechanisms of injury of these low linear energy transfer (LET) radiations are different from those of high-LET radiation such as neutrons, and these differences may affect countermeasure efficacy. We compared 30-day survival in mice after varying doses of pure γ and mixed neutron/γ (mixed field) radiation (MF, Dn/Dt = 0.65), and also examined peripheral blood cells, bone marrow cell reconstitution, and cytokine expression. Mixed-field-irradiated mice displayed prolonged defects in T-cell populations compared to mice irradiated with pure γ photons. In mouse survival assays, the growth factor granulocyte colony-stimulating factor (G-CSF) was effective as a (post-irradiation) mitigator against both γ-photons and mixed-field radiation, while the thrombopoietin (TPO) mimetic ALXN4100TPO was effective only against γ irradiation. The results indicate that radiation countermeasures should be tested against radiation qualities appropriate for specific scenarios before inclusion in response plans.

Wound trauma increases radiation-induced mortality by activation of iNOS pathway and elevation of cytokine concentrations and bacterial infection.

Abstract Although it is documented that concurrent wounding increases mortality from radiation injury, the molecular mechanism of combined injury is unknown. In this study, mice were exposed to gamma radiation followed by skin wounding. Wound trauma exacerbated radiation-induced mortality, reducing the LD(50/30) from 9.65 Gy to 8.95 Gy. Analyses of histopathology, inducible nitric oxide synthase (iNOS), and serum cytokines were performed on mouse ileum and skin at various times after 9.75 Gy and/or wounding. In the ileum, the villi were significantly shortened 3 days postirradiation but not after wounding; combined injury resulted in decreased villus width and tunica muscularis thickness. The skin of mice subjected to combined injury was less cellular and had a smaller healing bud than the skin of mice subjected to wounding alone. Combined injury significantly delayed wound closure times; it also prolonged the increased levels of iNOS protein in the skin and ileum. iNOS up-regulation was correlated with increases in transcription factors, including NF-kappaB and NF-IL6. The increase in NF-IL6 may be due to increases in cytokines, including IL-1beta, -6, -8, -9, -10 and -13, G-CSF, eotaxin, INF-gamma, MCP-1, MIP-1alpha and MIP-1beta. Combined injury resulted in early detection of bacteria in the blood of the heart and liver, whereas radiation alone resulted in later detection of bacteria; only a transient bacteremia occurred after wounding alone. Results suggest that enhancement of iNOS, cytokines and bacterial infection triggered by combined injury may contribute to mortality. Agents that inhibit these responses may prove to be therapeutic for combined injury and may reduce related mortality.

Epidermal growth factor receptor pathway substrate 8 is overexpressed in human pituitary tumors: role in proliferation and survival.

Based on prior work showing that human pituitary tumors overexpress epidermal and fibroblast growth factor receptors, we hypothesized that downstream components of growth factor signaling pathways may also be dysregulated. Epidermal growth factor pathway substrate number 8 (Eps8) was identified as a transcript overexpressed (5.9-fold) in human pituitary tumors compared with normal pituitary by DNA microarrays. Eps8 mRNA up-regulation was confirmed by semiquantitative RT-PCR. Immunoblot analysis showed that Eps8 protein levels and its downstream target phosphorylated ERK were also up-regulated in human pituitary tumors. Stable overexpression of Eps8 in LbetaT2 gonadotrope pituitary cells augmented colony formation in soft agar at d 21. Eps8 cells proliferated more robustly compared with controls in growth factor replete as well as growth-restricted conditions. In addition, the Eps8 overexpressing cells were protected from serum withdrawal-induced apoptosis compared with controls as assessed by caspase-3 cleavage. Epidermal growth factor activated a robust amplification of ERK and modest up-regulation of Akt in Eps8-overexpressing pituitary cells compared with vector controls. MAPK kinase inhibition or silencing of Eps8 blunted the proliferation of the cells in response to growth factor stimulation. Blockade of the phosphatidylinositol 3-kinase pathway or silencing of Eps8 resulted in a loss of the Eps8 protection from growth factor withdrawal-induced apoptosis. Together these data support a role of Eps8 in amplifying growth factor receptor signaling in human pituitary tumors to promote proliferation and cell survival.

Bone morphogenetic protein and retinoic acid-inducible neural specific protein-3 is expressed in gonadotrope cell pituitary adenomas and induces proliferation, migration, and invasion.

Pituitary tumors are common intracranial neoplasms that often result in endocrine dysfunction due to hormone overproduction or deficiencies from mass effects. Gonadotrope cell or gonadotropinomas are tumors that produce LH and/or FSH and represent 40% of macroadenomas. Little is known about their underlying pathogenic mechanisms. We compared expression profiles of 10 gonadotropinomas with nine normal pituitaries by cDNA array and identified bone morphogenetic protein- and retinoic acid-inducible neural-specific protein-3 (BRINP3) as overexpressed in tumors, compared with normals. BRINP3 is a novel, normally brain restricted protein of unknown function. BRINP3 mRNA was expressed selectively in gonadotropinomas. Subcellular localization studies showed that BRINP3 was targeted to the mitochondria, but BRINP3 overexpression was unable to protect pituitary cells against programmed cell death induced by growth factor withdrawal. However, BRINP3 overexpression in pituitary gonadotrope cells promoted proliferation, migration, and invasion. A BRINP3 antibody was raised that demonstrated clustered expression of BRINP3 protein in gonadotropinomas and not in normal human pituitary samples. Thus, BRINP3 is a mitochondrially localized protein that is selectively up-regulated in human gonadotropinomas. Its actions to increase proliferation, migration, and invasion suggest it may play an important role in pituitary tumorigenesis.