Mark H. Whitnall

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 Administrations 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 drugs 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 CBLB502s 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.

Effects of genistein administration on cytokine induction in whole-body gamma irradiated mice.

The development of an effective pharmacological countermeasure is needed to reduce the morbidity and mortality in military and civilian populations associated with possible exposure to ionizing radiation. We previously demonstrated that a single subcutaneous (sc) administration of genistein at a non-toxic dose provided protection against acute radiation injury and that the radioprotective effects were associated with multilineage, hematopoietic progenitor cell recovery. The purpose of this study was to determine whether hematopoietic recovery was preceded by cytokine induction. In mice treated with sc genistein 24 h before irradiation (7 Gy 60Co), we quantified serum cytokine levels by multiplex Luminex and also investigated a larger number of cytokines using cytokine arrays. Genistein administration stimulated serum granulocyte-colony stimulating factor (G-CSF) 4h and 24h after sham irradiation or gamma-irradiation. Interleukin-6 (IL-6) was significantly increased in genistein-treated animals 4h after irradiation. Because G-CSF and IL-6 are important hematopoietic factors, these results support our hypothesis that the previously observed radioprotective efficacy by genistein may be a result of early recovery of hematopoietic cells due to enhanced production of G-CSF and IL-6.

δ-tocotrienol protects mouse and human hematopoietic progenitors from γ-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.

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.

5-Androstenediol Promotes Survival of γ-Irradiated Human Hematopoietic Progenitors through Induction of Nuclear Factor-κB Activation and Granulocyte Colony-Stimulating Factor Expression

5-Androstenediol (5-AED) stimulates hematopoiesis and enhances survival in animals exposed to ionizing radiation (IR), suggesting that this steroid may act on hematopoietic progenitor cells. We used γ-irradiated primary human CD34+ hematopoietic progenitor cells to show that 5-AED protects hematopoietic cells from IR damage, as shown by enhanced cell survival, clonogenicity, proliferation, and differentiation. Unlike in tumor cells, IR did not induce nuclear factor-κB (NFκB) activation in primary progenitors. However, IR stimulated IκBβ release from NFκB/IκB complexes and caused NFκB1 (p50) degradation. 5-AED stabilized NFκB1 in irradiated cells and induced NFκB gene expression and NFκB activation (DNA binding). 5-AED stimulated interleukin-6 and granulocyte colony-stimulating factor (G-CSF) secretion. The survival-enhancing effects of 5-AED on clonogenic cells were abrogated by small interfering RNA inhibition of NFκB gene expression and by neutralization of G-CSF with antibody. The effects of 5-AED on survival and G-CSF secretion were blocked by the NFκB inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). 5-AED had no effect on accumulation of the proapoptotic factor p53 after IR, as determined by Western blot. The results indicate that NFκB1 degradation after IR may be responsible for the radiation sensitivity of CD34+ cells compared with tumor cells. 5-AED exerts survival-enhancing effects on irradiated human hematopoietic progenitor cells via induction, stabilization, and activation of NFκB, which results in increased secretion of hematopoietic growth factor G-CSF.

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.

Hematopoietic Radiation Syndrome in the Gottingen Minipig

Abstract 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 LD50/30 estimate between 1.7 and 1.9 Gy. The criterion of whether platelet counts were <200 × 103/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.

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.

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

PURPOSEnWe 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.nnnMETHODS AND MATERIALSnMale 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnThese 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 60Co 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-1β 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.