Evaluation of the gamma-H2AX assay for radiation biodosimetry in a swine model.

There is a paucity of large animal models to study both the extent and the health risk of ionizing radiation exposure in humans. One promising candidate for such a model is the minipig. Here, we evaluate the minipig for its potential in γ-H2AX-based biodosimetry after exposure to ionizing radiation using both Cs137 and Co60 sources. γ-H2AX foci were enumerated in blood lymphocytes and normal fibroblasts of human and porcine origin after ex vivo γ-ray irradiation. DNA double-strand break repair kinetics in minipig blood lymphocytes and fibroblasts, based on the γ-H2AX assay, were similar to those observed in their human counterparts. To substantiate the similarity observed between the human and minipig we show that minipig fibroblast radiosensitivity was similar to that observed with human fibroblasts. Finally, a strong γ-H2AX induction was observed in blood lymphocytes following minipig total body irradiation. Significant responses were detected 3 days after 1.8 Gy and 1 week after 3.8 and 5 Gy with residual γ-H2AX foci proportional to the initial radiation doses. These findings show that the Gottingen minipig provides a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans.

Identification of granulocyte colony-stimulating factor and interleukin-6 as candidate biomarkers of CBLB502 efficacy as a medical radiation countermeasure.

Given an ever-increasing risk of nuclear and radiological emergencies, there is a critical need for development of medical radiation countermeasures (MRCs) that are safe, easily administered, and effective in preventing and/or mitigating the potentially lethal tissue damage caused by acute high-dose radiation exposure. Because the efficacy of MRCs for this indication cannot be ethically tested in humans, development of such drugs is guided by the Food and Drug Administration's Animal Efficacy Rule. According to this rule, human efficacious doses can be projected from experimentally established animal efficacious doses based on the equivalence of the drug's effects on efficacy biomarkers in the respective species. Therefore, identification of efficacy biomarkers is critically important for drug development under the Animal Efficacy Rule. CBLB502 is a truncated derivative of the Salmonella flagellin protein that acts by triggering Toll-like receptor 5 (TLR5) signaling and is currently under development as a MRC. Here, we report identification of two cytokines, granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6), as candidate biomarkers of CBLB502's radioprotective/mitigative efficacy. Induction of both G-CSF and IL-6 by CBLB502 1) is strictly TLR5-dependent, 2) occurs in a CBLB502 dose-dependent manner within its efficacious dose range in both nonirradiated and irradiated mammals, including nonhuman primates, and 3) is critically important for the ability of CBLB502 to rescue irradiated animals from death. After evaluation of CBLB502 effects on G-CSF and IL-6 levels in humans, these biomarkers will be useful for accurate prediction of human efficacious CBLB502 doses, a key step in the development of this prospective radiation countermeasure.

Role of radiation-induced granulocyte colony-stimulating factor in recovery from whole body gamma-irradiation.

The purpose of this study was to further elucidate the radioprotective role of granulocyte colony-stimulating factor (G-CSF) induced in response to irradiation. The induction of G-CSF and interleukin-6 (IL-6) in response to radiation exposure was evaluated in mice. The level of cytokine in serum was determined by multiplex Luminex. The role of G-CSF on survival and tissue injury after total body gamma-irradiation was evaluated by administration of neutralizing antibody to G-CSF before radiation exposure. An isotype control was used for comparison and survival was monitored for 30 d after irradiation. Jejunum samples were used for immunohistochemistry. Ionizing radiation exposure induced significant levels of the hematopoietic cytokines G-CSF and IL-6, in mice receiving 9.2 Gy radiation. Maximal levels of G-CSF were observed in peripheral blood of mice 8h after irradiation. IL-6 levels were maximum at 12h after irradiation. Administration of G-CSF antibody significantly enhanced mortality in irradiated mice. G-CSF antibody-treated mice had higher numbers of CD68(+) cells and apoptotic cells in intestinal villi. Our results confirm that radiation exposure induces elevations of circulating G-CSF and IL-6. Neutralizing antibody to G-CSF exacerbates the deleterious effects of radiation, indicating that G-CSF induced in response to irradiation plays an important role in recovery.

Delta-tocotrienol protects mouse and human hematopoietic progenitors from gamma-irradiation through extracellular signal-regulated kinase/mammalian target of rapamycin signaling.

BACKGROUND: Exposure to γ-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this study, we demonstrated that natural δ-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of δ-tocotrienol on hematopoietic progenitor cell survival after γ-irradiation in both in vivo and in vitro experiments. DESIGN AND METHODS: CD2F1 mice and human hematopoietic progenitor CD34(+) cells were treated with δ-tocotrienol or vehicle control 24 h before or 6 h after γ-irradiation. Effects of δ-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity studies, flow cytometry, and bone marrow histochemical staining. δ-tocotrienol and γ-irradiation-induced signal regulatory activities were assessed by immunofluorescence staining, immunoblotting and short-interfering RNA assay. RESULTS: δ-tocotrienol displayed significant radioprotective effects. A single injection of δ-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. δ-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage(-)/Sca-1(+)/ckit(+) stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34(+) cells from radiation-induced damage. δ-tocotrienol activated extracellular signal-related kinase 1/2 phosphorylation and significantly inhibited formation of DNA-damage marker γ-H2AX foci. In addition, δ-tocotrienol up-regulated mammalian target of rapamycin and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6, which is responsible for cell survival and growth. Inhibition of extracellular signal-related kinase 1/2 expression by short interfering RNA abrogated δ-tocotrienol-induced mammalian target of rapamycin phosphorylation and clonogenicity, and increased γ-H2AX foci formation in irradiated CD34(+) cells. CONCLUSIONS: Our data indicate that δ-tocotrienol protects mouse bone marrow and human CD34(+) cells from radiation-induced damage through extracellular signal-related kinase activation-associated mammalian target of rapamycin survival pathways.

Evaluation of EUK-189, a synthetic superoxide dismutase/catalase mimetic as a radiation countermeasure.

EUK-189, a salen-manganese complex and superoxide dismutase/catalase mimetic, was administered subcutaneously (sc; 30 or 70 mg/kg) to mice at - 24, - 1, +1, or +6 h relative to whole-body cobalt-60 gamma irradiation (LD(90/30) dose), and survival was monitored for 30 days. Cell counts and cytokines in circulation were measured in sublethally irradiated mice treated with EUK-189. EUK-189 (70 mg/kg, - 24 h) enhanced 30-day survival with a dose reduction factor (DRF) of 1.15 (p = 0.047, 95% confidence limits: 1.053, 1.244). LD(50/30)s were 7.96 and 9.13 Gy for saline- and EUK-189-treated groups, respectively. Drug treatment was associated with elevations in numbers of total white blood cells, eosinophils, lymphocytes, and platelets in irradiated mice, compared to vehicle-injected, irradiated controls. EUK-189 did not stimulate production of any cytokine or chemokine tested.

The Toll-Like Receptor 5 Agonist Entolimod Mitigates Lethal Acute Radiation Syndrome in Non-Human Primates.

There are currently no approved medical radiation countermeasures (MRC) to reduce the lethality of high-dose total body ionizing irradiation expected in nuclear emergencies. An ideal MRC would be effective even when administered well after radiation exposure and would counteract the effects of irradiation on the hematopoietic system and gastrointestinal tract that contribute to its lethality. Entolimod is a Toll-like receptor 5 agonist with demonstrated radioprotective/mitigative activity in rodents and radioprotective activity in non-human primates. Here, we report data from several exploratory studies conducted in lethally irradiated non-human primates (rhesus macaques) treated with a single intramuscular injection of entolimod (in the absence of intensive individualized supportive care) administered in a mitigative regimen, 1-48 hours after irradiation. Following exposure to LD50-70/40 of radiation, injection of efficacious doses of entolimod administered as late as 25 hours thereafter reduced the risk of mortality 2-3-fold, providing a statistically significant (P<0.01) absolute survival advantage of 40-60% compared to vehicle treatment. Similar magnitude of survival improvement was also achieved with drug delivered 48 hours after irradiation. Improved survival was accompanied by predominantly significant (P<0.05) effects of entolimod administration on accelerated morphological recovery of hematopoietic and immune system organs, decreased severity and duration of thrombocytopenia, anemia and neutropenia, and increased clonogenic potential of the bone marrow compared to control irradiated animals. Entolimod treatment also led to reduced apoptosis and accelerated crypt regeneration in the gastrointestinal tract. Together, these data indicate that entolimod is a highly promising potential life-saving treatment for victims of radiation disasters.

Protection against gamma-irradiation with 5-androstenediol.

We showed previously that treatment of gamma-irradiated female B6D2F1 mice with 5-androstenediol (AED) enhanced survival, stimulated myelopoiesis, and ameliorated radiation-induced decreases in circulating neutrophils and platelets. We have now tested survival in male CD2F1 mice, and we have investigated molecular and functional effects on neutrophils and bone marrow stromal cells and screened for toxicity in female B6D2F1 mice. AED (160 mg/kg, subcutaneously, 24 hours before irradiation) enhanced survival in male CD2F1 mice with a dose-reduction factor of 1.23, similar to the dose-reduction factor of 1.26 found previously for female B6D2F1 mice. Expression of CD11b, a developmental marker, was reduced on circulating neutrophils after either in vivo AED administration or whole-body gamma-irradiation (3 Gy), but neutrophil peroxidase activity was unchanged. Stromal cell progenitors (fibroblastoid colony-forming units) were reduced in marrow 5 days after AED injection in nonirradiated mice. Clinical chemistry, histopathology, and behavioral assays showed no evidence of toxicity. We conclude that AED and related steroids are attractive candidates to explore as countermeasures to high- and low-level ionizing radiation.

Effects of anesthesia and vehicle injection on circulating blood elements in C3H/HeN male mice.

The induction and maintenance of general anesthesia can lead to profound alterations of many organ systems, especially the cardiovascular, pulmonary, and nervous systems. Moreover, distinct from their cardiopulmonary effects, certain anesthetics can induce physiologic and behavioral changes, which may persist after recovery from anesthesia. Knowledge of the effects of anesthesia and anesthetic agents on hematologic measurements is important. Although the effects of anesthesia were clinically unapparent, the effect on levels of circulating blood elements was an important determinant for the results of our study. We sought to evaluate the effect of anesthesia and vehicle injection on the levels of circulating blood elements in C3H/HeN male mice. We used an automated hematology system to obtain complete blood counts with differentials in anesthetized and unanesthetized mice receiving subcutaneous injections of polyethylene glycol (PEG-400). Two days after a 30-min exposure to isoflurane anesthesia, mean white blood cell counts had deceased by 15.4%, mean neutrophil counts had decreased by 26.9%, and mean platelet counts by 11.2% compared with levels in unanesthetized mice. Our results indicate that the effect of anesthesia is an important consideration when circulating blood elements in mice must be measured.

Accelerated hematopoietic syndrome after radiation doses bridging hematopoietic (H-ARS) and gastrointestinal (GI-ARS) acute radiation syndrome: early hematological changes and systemic inflammatory response syndrome in minipig.

PURPOSE: To characterize acute radiation syndrome (ARS) sequelae at doses intermediate between the bone marrow (H-ARS) and full gastrointestinal (GI-ARS) syndrome. METHODS: Male minipigs, approximately 5 months old, 9-12 kg in weight, were irradiated with Cobalt-60 (total body, bilateral gamma irradiation, 0.6 Gy/min). Endpoints were 10-day survival, gastrointestinal histology, plasma citrulline, bacterial translocation, vomiting, diarrhea, vital signs, systemic inflammatory response syndrome (SIRS), febrile neutropenia (FN). RESULTS: We exposed animals to doses (2.2-5.0 Gy) above those causing H-ARS (1.6-2.0 Gy), and evaluated development of ARS. Compared to what was observed during H-ARS (historical data: Moroni et al. 2011a , 2011c ), doses above 2 Gy produced signs of increasingly severe pulmonary damage, faster deterioration of clinical conditions, and faster increases in levels of C-reactive protein (CRP). In the range of 4.6-5.0 Gy, animals died by day 9-10; signs of the classic GI syndrome, as measured by diarrhea, vomiting and bacterial translocation, did not occur. At doses above 2 Gy we observed transient reduction in circulating citrulline levels, and animals exhibited earlier depletion of blood elements and faster onset of SIRS and FN. CONCLUSIONS: An accelerated hematopoietic subsyndrome (AH-ARS) is observed at radiation doses between those producing H-ARS and GI-ARS. It is characterized by early onset of SIRS and FN, and greater lung damage, compared to H-ARS.

Gastrointestinal acute radiation syndrome in Göttingen minipigs (Sus scrofa domestica).

In the absence of supportive care, exposing Göttingen minipigs to γ-radiation doses of less than 2 Gy achieves lethality due to hematopoietic acute radiation syndrome. Doses of 2 to 5 Gy are associated with an accelerated hematopoietic syndrome, characterized by villus blunting and fusion, the beginning of sepsis, and a mild transient reduction in plasma citrulline concentration. We exposed male Göttingen minipigs (age, 5 mo; weight, 9 to 11 kg) to γ-radiation doses of 5 to 12 Gy (total body; (60)Co, 0.6 Gy/min) to test whether these animals exhibit classic gastrointestinal acute radiation syndrome (GI-ARS). After exposure, the minipigs were monitored for 10 d by using clinical signs, CBC counts, and parameters associated with the development of the gastrointestinal syndrome. Göttingen minipigs exposed to γ radiation of 5 to 12 Gy demonstrate a dose-dependent occurrence of all parameters classically associated with acute GI-ARS. These results suggest that Göttingen minipigs may be a suitable model for studying GI-ARS after total body irradiation, but the use of supportive care to extend survival beyond 10 d is recommended. This study is the first step toward determining the feasibility of using Göttingen minipigs in testing the efficacy of candidate drugs for the treatment of GI-ARS after total body irradiation.

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.

The Gottingen minipig is a model of the hematopoietic acute radiation syndrome: G-colony stimulating factor stimulates hematopoiesis and enhances survival from lethal total-body γ-irradiation.

PURPOSE: We are characterizing the Gottingen minipig as an additional large animal model for advanced drug testing for the acute radiation syndrome (ARS) to enhance the discovery and development of novel radiation countermeasures. Among the advantages provided by this model, the similarities to human hematologic parameters and dynamics of cell loss/recovery after irradiation provide a convenient means to compare the efficacy of drugs known to affect bone marrow cellularity and hematopoiesis. METHODS AND MATERIALS: Male Gottingen minipigs, 4 to 5 months old and weighing 9 to 11 kg, were used for this study. We tested the standard off-label treatment for ARS, rhG-CSF (Neupogen, 10 µg/kg/day for 17 days), at the estimated LD70/30 total-body γ-irradiation (TBI) radiation dose for the hematopoietic syndrome, starting 24 hours after irradiation. RESULTS: The results indicated that granulocyte colony stimulating factor (G-CSF) enhanced survival, stimulated recovery from neutropenia, and induced mobilization of hematopoietic progenitor cells. In addition, the administration of G-CSF resulted in maturation of monocytes/macrophages. CONCLUSIONS: These results support continuing efforts toward validation of the minipig as a large animal model for advanced testing of radiation countermeasures and characterization of the pathophysiology of ARS, and they suggest that the efficacy of G-CSF in improving survival after total body irradiation may involve mechanisms other than increasing the numbers of circulating granulocytes.

Delta-tocotrienol protects mice from radiation-induced gastrointestinal injury.

We recently demonstrated that natural delta-tocotrienol (DT3) significantly enhanced survival in total-body irradiated (TBI) mice, and protected mouse bone marrow cells from radiation-induced damage through Erk activation-associated mTOR survival pathways. Here, we further evaluated the effects and mechanisms of DT3 on survival of radiation-induced mouse acute gastrointestinal syndrome. DT3 (75-100 mg/kg) or vehicle was administered as a single subcutaneous injection to CD2F1 mice 24 h before 10-12 Gy (60)Co total-body irradiation at a dose rate of 0.6 Gy/min and survival was monitored. In a separate group of mice, jejunum sections were stained with hematoxylin and eosin and the surviving crypts in irradiated mice were counted. Apoptosis in intestinal epithelial cells was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and bacterial translocation from gut to heart, spleen and liver in irradiated mice were evaluated. DT3 (75 mg/kg) significantly enhanced survival in mice that received 10, 10.5, 11 or 12 Gy TBI. Administration of DT3 protected intestinal tissue, decreased apoptotic cells in jejunum and inhibited gut bacterial translocation in irradiated mice. Furthermore, DT3 significantly inhibited radiation-induced production of pro-inflammatory factors interleukin-1ß and -6 and suppressed expression of protein tyrosine kinase 6 (PTK6), a stress-induced kinase that promotes apoptosis in mouse intestinal cells. Our data demonstrate that administration of DT3 protected mice from radiation-induced gastrointestinal system damage.

A review of radiation countermeasure work ongoing at the Armed Forces Radiobiology Research Institute.

PURPOSE: The hazard of exposure to ionizing radiation is a serious public and military health concern that has justified substantial efforts to develop medically effective radiation countermeasure approaches, including radiation protectors, mitigators, and therapeutics. Although such efforts were initiated more than half a century ago, no safe and effective radiation countermeasure has been approved by the United States Food and Drug Administration (FDA) for the acute radiation syndrome. This situation has prompted intensified research among government laboratories, academic institutions, and pharmaceutical companies to identify a new generation of countermeasures. In this communication we discuss selected promising radiation countermeasures at advanced stages of development. CONCLUSION: Other than granulocyte colony-stimulating factor, which has an Emergency Use Investigational New Drug (IND) status, four countermeasures have FDA IND status and other promising countermeasures are in development. Here we review primarily the in vivo efficacy of selected countermeasures in animal models and clinical studies.

5-AED enhances survival of irradiated mice in a G-CSF-dependent manner, stimulates innate immune cell function, reduces radiation-induced DNA damage and induces genes that modulate cell cycle progression and apoptosis.

The steroid androst-5-ene-3ß,17ß-diol (5-androstenediol, 5-AED) elevates circulating granulocytes and platelets in animals and humans, and enhances survival during the acute radiation syndrome (ARS) in mice and non-human primates. 5-AED promotes survival of irradiated human hematopoietic progenitors in vitro through induction of Nuclear Factor-κB (NFκB)-dependent Granulocyte Colony-Stimulating Factor (G-CSF) expression, and causes elevations of circulating G-CSF and interleukin-6 (IL-6). However, the in vivo cellular and molecular effects of 5-AED are not well understood. The aim of this study was to investigate the mechanisms of action of 5-AED administered subcutaneously (s.c.) to mice 24 h before total body γ- or X-irradiation (TBI). We used neutralizing antibodies, flow cytometric functional assays of circulating innate immune cells, analysis of expression of genes related to cell cycle progression, DNA repair and apoptosis, and assessment of DNA strand breaks with halo-comet assays. Neutralization experiments indicated endogenous G-CSF but not IL-6 was involved in survival enhancement by 5-AED. In keeping with known effects of G-CSF on the innate immune system, s.c. 5-AED stimulated phagocytosis in circulating granulocytes and oxidative burst in monocytes. 5-AED induced expression of both bax and bcl-2 in irradiated animals. Cdkn1a and ddb1, but not gadd45a expression, were upregulated by 5-AED in irradiated mice. S.c. 5-AED administration caused decreased DNA strand breaks in splenocytes from irradiated mice. Our results suggest 5-AED survival enhancement is G-CSF-dependent, and that it stimulates innate immune cell function and reduces radiation-induced DNA damage via induction of genes that modulate cell cycle progression and apoptosis.

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.

Vascular access port implantation and serial blood sampling in a Gottingen minipig (Sus scrofa domestica) model of acute radiation injury.

Threats of nuclear and other radiologic exposures have been increasing, but no countermeasure for acute radiation syndrome has been approved by regulatory authorities. Because of their similarity to humans in regard to physiology and anatomy, we are characterizing Gottingen minipigs as a model to aid the development of radiation countermeasures. Irradiated minipigs exhibit immunosuppression, severe thrombocytopenia, vascular leakage, and acute inflammation. These complications render serial acquisition of blood samples problematic. Vascular access ports (VAP) facilitate serial sampling, but their use often is complicated by infections and fibrin deposition. We demonstrate here the successful use of VAP for multiple blood samplings in irradiated minipigs. Device design and limited postoperative prophylactic antimicrobial therapy before irradiation were key to obtaining serial sampling, reducing swelling, and eliminating infection and skin necrosis at the implantation site. Modifications of previous protocols included the use of polydioxanone sutures instead of silk; eliminating chronic port access; single-use, sterile, antireflux prefilled syringes for flushing; strict aseptic weekly maintenance of the device, and acclimating animals to reduce stress. VAP remained functional in 19 of 20 irradiated animals for as long as 3 mo. The remaining VAP failed due to a small leak in the catheter, leading to clot formation. VAP-related sepsis occurred in 2 minipigs. Blood sampling did not cause detectable stress in nonanesthetized sham-irradiated animals, according to leukograms and clinical signs.

Hematopoietic radiation syndrome in the Gottingen minipig.

Additional large animal models for the acute radiation syndrome (ARS) would facilitate countermeasure development. We demonstrate here that Gottingen minipigs develop hematopoietic ARS symptoms similar to those observed in canines, non-human primates (NHPs) and humans. Dosimetry for whole-body γ irradiation (0.6 Gy/min) was performed using electronic paramagnetic resonance (EPR) with alanine; National Institute of Standards and Technology (NIST)-calibrated alanine pellets and water-filled Plexiglas phantoms were used. After irradiations of 1.6-2.0 Gy, blood pancytopenia was observed for several weeks, accompanied by the characteristic ARS stages: prodromal symptoms, latent period, illness and recovery or morbidity. Morbidity occurred between days 14 and 27, with a preliminary LD(50/30) estimate between 1.7 and 1.9 Gy. The criterion of whether platelet counts were <200 × 10(3)/µl 7 days postirradiation predicted whether animals would survive in 18 out of 20 cases. The degree of granulocytosis 3 h postirradiation was inversely correlated with survival. Animals euthanized based on preset morbidity criteria displayed signs of multi-organ dysfunction, including widespread internal hemorrhage and alterations in organ function reflected in blood chemistry. Circulating C-reactive protein (CRP), a marker for inflammation, became elevated within hours after irradiation, subsided after several days, and increased again after 14 days. The results support further development of the Gottingen minipig as a model for ARS.

Hematological changes as prognostic indicators of survival: similarities between Gottingen minipigs, humans, and other large animal models.

BACKGROUND: The animal efficacy rule addressing development of drugs for selected disease categories has pointed out the need to develop alternative large animal models. Based on this rule, the pathophysiology of the disease in the animal model must be well characterized and must reflect that in humans. So far, manifestations of the acute radiation syndrome (ARS) have been extensively studied only in two large animal models, the non-human primate (NHP) and the canine. We are evaluating the suitability of the minipig as an additional large animal model for development of radiation countermeasures. We have previously shown that the Gottingen minipig manifests hematopoietic ARS phases and symptoms similar to those observed in canines, NHPs, and humans. PRINCIPAL FINDINGS: We establish here the LD50/30 dose (radiation dose at which 50% of the animals succumb within 30 days), and show that at this dose the time of nadir and the duration of cytopenia resemble those observed for NHP and canines, and mimic closely the kinetics of blood cell depletion and recovery in human patients with reversible hematopoietic damage (H3 category, METREPOL approach). No signs of GI damage in terms of diarrhea or shortening of villi were observed at doses up to 1.9 Gy. Platelet counts at days 10 and 14, number of days to reach critical platelet values, duration of thrombocytopenia, neutrophil stress response at 3 hours and count at 14 days, and CRP-to-platelet ratio were correlated with survival. The ratios between neutrophils, lymphocytes and platelets were significantly correlated with exposure to irradiation at different time intervals. SIGNIFICANCE: As a non-rodent animal model, the minipig offers a useful alternative to NHP and canines, with attractive features including ARS resembling human ARS, cost, and regulatory acceptability. Use of the minipig may allow accelerated development of radiation countermeasures.

Multiple parameter radiation injury assessment using a nonhuman primate radiation model-biodosimetry applications.

There are urgent needs to establish capability to rapidly assess radiation injury in mass casualty and population monitoring scenarios. This study's objective was to evaluate several currently available biomarkers that can provide early diagnostic triage information after radiation exposure. Hematology and blood chemistry measurements were performed on samples derived from a nonhuman primate (Macaca mulatta; n = 8) total-body irradiation (TBI) model (6.5-Gy Co gamma rays at 0.6 Gy min). The results from this study demonstrate: a) time course for changes in C-reactive protein (CRP) (-2 d to 15 d after TBI); b) time-dependent (-2 d, 1-4 d after TBI) changes in blood cell counts [i.e., lymphocytes decrease to 5-8% of pre-study levels at 1 to 4 d after TBI; ratio of neutrophil to lymphocytes increases by 44 +/- 18 (p = 0.016), 12 +/- 4 (p = 0.001), 8 +/- 2 (p = 0.0020), and 5.0 +/- 2 (p = 0.002) fold at 1, 2, 3, and 4 days after TBI, respectively]; and c) 4.5 +/- 0.8 (p = 0.002)-fold increases in serum amylase activity 1 d after TBI. Plasma CRP levels at 1 d after exposure were 22 +/- 13 (p = 0.0005) (females) and 44 +/- 11 (p = 0.0004) (males)-fold elevated above baseline levels. One hundred percent successful separation of samples from exposed macaques (24 h after TBI) vs. samples from the same macaque taken before irradiation using a discriminant analysis based on four biomarkers (i.e., lymphocytes, neutrophils, ratio of neutrophils to lymphocytes, and serum amylase activity) was demonstrated. These results demonstrate the practical use of multiple parameter biomarkers to enhance the discrimination of exposed vs. non-exposed individuals and justify a follow-on rhesus macaque dose-response study.