Survival efficacy of the PEGylated G-CSFs Maxy-G34 and neulasta in a mouse model of lethal H-ARS, and residual bone marrow damage in treated survivors

In an effort to expand the worldwide pool of available medical countermeasures (MCM) against radiation, the PEGylated G-CSF (PEG-G-CSF) molecules Neulasta and Maxy-G34, a novel PEG-G-CSF designed for increased half-life and enhanced activity compared to Neulasta, were examined in a murine model of the Hematopoietic Syndrome of the Acute Radiation Syndrome (H-ARS), along with the lead MCM for licensure and stockpiling, G-CSF. Both PEG-G-CSFs were shown to retain significant survival efficacy when administered as a single dose 24 h post-exposure, compared to the 16 daily doses of G-CSF required for survival efficacy. Furthermore, 0.1 mg kg of either PEG-G-CSF affected survival of lethally-irradiated mice that was similar to a 10-fold higher dose. The one dose/low dose administration schedules are attractive attributes of radiation MCM given the logistical challenges of medical care in a mass casualty event. Maxy-G34-treated mice that survived H-ARS were examined for residual bone marrow damage (RBMD) up to 9 mo post-exposure. Despite differences in Sca-1 expression and cell cycle position in some hematopoietic progenitor phenotypes, Maxy-G34-treated mice exhibited the same degree of hematopoietic stem cell (HSC) insufficiency as vehicle-treated H-ARS survivors in competitive transplantation assays of 150 purified Sca-1+cKit+lin-CD150+cells. These data suggest that Maxy-G34, at the dose, schedule, and time frame examined, did not mitigate RBMD but significantly increased survival from H-ARS at one-tenth the dose previously tested, providing strong support for advanced development of Maxy-G34, as well as Neulasta, as MCM against radiation

PEGylated G-CSF (BBT-015), GM-CSF (BBT-007), and IL-11 (BBT-059) analogs enhance survival and hematopoietic cell recovery in a mouse model of the hematopoietic syndrome of the acute radiation syndrome

Hematopoietic growth factors (HGF) are recommended therapy for high dose radiation exposure, but unfavorable administration schedules requiring early and repeat dosing limit the logistical ease with which they can be used. In this report, using a previously described murine model of H-ARS, survival efficacy and effect on hematopoietic recovery of unique PEGylated HGF were investigated. The PEGylated-HGFs possess longer half-lives and more potent hematopoietic properties than corresponding non-PEGylated-HGFs. C57BL/6 mice underwent single dose lethal irradiation (7.76-8.72 Gy, Cs, 0.62-1.02 Gy min) and were treated with various dosing regimens of 0.1, 0.3, and 1.0 mg kg of analogs of human PEG-G-CSF, murine PEG-GM-CSF, or human PEG-IL-11. Mice were administered one of the HGF analogs at 24-28 h post irradiation, and in some studies, additional doses given every other day (beginning with the 24-28 h dose) for a total of three or nine doses. Thirty-day (30 d) survival was significantly increased with only one dose of 0.3 mg kg of PEG-G-CSF and PEG-IL-11 or three doses of 0.3 mg kg of PEG-GM-CSF (p ≤ 0.006). Enhanced survival correlated with consistently and significantly enhanced WBC, NE, RBC, and PLT recovery for PEG-G- and PEG-GM-CSF, and enhanced RBC and PLT recovery for PEG-IL-11 (p ≤ 0.05). Longer administration schedules or higher doses did not provide a significant additional survival benefit over the shorter, lower dose, schedules. These data demonstrate the efficacy of BBT's PEG-HGF to provide significantly increased survival with fewer injections and lower drug doses, which may have significant economic and logistical value in the aftermath of a radiation event

Lifelong residual bone marrow damage in murine survivors of the hematopoietic acute radiation syndrome (H-ARS): a compilation of studies comprising the Indiana University experience

Accurate analyses of the delayed effects of acute radiation exposure (DEARE) in survivors of the hematopoietic acute radiation syndrome (H-ARS) are hampered by low numbers of mice for examination due to high lethality from the acute syndrome, increased morbidity and mortality in survivors, high cost of husbandry for long-term studies, biological variability, and inconsistencies of models from different laboratories complicating meta-analyses. To address this, a compilation of 38 similar H-ARS studies conducted over a seven-year period in the authors’ laboratory, comprising more than 1,500 irradiated young adult C57BL/6 mice and almost 600 day-30 survivors, was assessed for hematopoietic DEARE at various times up to 30 months of age. Significant loss of long-term repopulating potential of phenotypically-defined primitive hematopoietic stem cells (HSC) was documented in H-ARS survivors, as well as significant decreases in all hematopoietic lineages in peripheral blood, prominent myeloid skew, significantly decreased bone marrow cellularity and numbers of lineage-negative Sca-1+ cKit+ CD150+ cells (KSLCD150+; the phenotype known to be enriched for HSC), and increased cycling of KSLCD150+ cells. Studies interrogating the phenotype of bone marrow cells capable of initiation of suspension cultures and engraftment in competitive transplantation assays documented the phenotype of HSC in H-ARS survivors to be the same as that in non-irradiated age-matched controls. This compilation study adds rigor and validity to our initial findings of persistent hematopoietic dysfunction in H-ARS survivors that arises at the level of the HSC and which affects all classes of hematopoietic cells for the life of the survivor

An Update on Cancer Cluster Activities at the Centers for Disease Control and Prevention

The Centers for Disease Control and Prevention (CDC) continues to be aware of the need for response to public concern as well as to state and local agency concern about cancer clusters. In 1990 the CDC published the “Guidelines for Investigating Clusters of Health Events,” in which a four-stage process was presented. This document has provided a framework that most state health departments have adopted, with modifications pertaining to their specific situations, available resources, and philosophy concerning disease clusters. The purpose of this present article is not to revise the CDC guidelines; they retain their original usefulness and validity. However, in the past 15 years, multiple cluster studies as well as scientific and technologic developments have affected cluster science and response (improvements in cancer registries, a federal initiative in environmental public health tracking, refinement of biomarker technology, cluster identification using geographic information systems software, and the emergence of the Internet). Thus, we offer an addendum for use with the original document. Currently, to address both the needs of state health departments as well as public concern, the CDC now a) provides a centralized, coordinated response system for cancer cluster inquiries, b) supports an electronic cancer cluster listserver, c) maintains an informative web page, and d) provides support to states, ranging from laboratory analysis to epidemiologic assistance and expertise. Response to cancer clusters is appropriate public health action, and the CDC will continue to provide assistance, facilitate communication among states, and foster the development of new approaches in cluster science

The H-ARS Dose Response Relationship (DRR): Validation and Variables

Manipulations of lethally-irradiated animals, such as for administration of pharmaceuticals, blood sampling, or other laboratory procedures, have the potential to induce stress effects that may negatively affect morbidity and mortality. To investigate this in a murine model of the hematopoietic acute radiation syndrome, 20 individual survival efficacy studies were grouped based on the severity of the administration (Admn) schedules of their medical countermeasure (MCM) into Admn 1 (no injections), Admn 2 (1-3 injections), or Admn 3 (29 injections or 6-9 oral gavages). Radiation doses ranged from LD30/30 to LD95/30. Thirty-day survival of vehicle controls in each group was used to construct radiation dose lethality response relationship (DRR) probit plots, which were compared statistically to the original DRR from which all LDXX/30 for the studies were obtained. The slope of the Admn 3 probit was found to be significantly steeper (5.190) than that of the original DRR (2.842) or Admn 2 (2.009), which were not significantly different. The LD50/30 for Admn 3 (8.43 Gy) was less than that of the original DRR (8.53 Gy, p < 0.050), whereas the LD50/30 of other groups were similar. Kaplan-Meier survival curves showed significantly worse survival of Admn 3 mice compared to the three other groups (p = 0.007). Taken together, these results show that stressful administration schedules of MCM can negatively impact survival and that dosing regimens should be considered when constructing DRR to use in survival studies

Cardiac and Renal Delayed Effects of Acute Radiation Exposure: Organ Differences in Vasculopathy, Inflammation, Senescence and Oxidative Balance

We have previously shown significant pathology in the heart and kidney of murine hematopoietic-acute radiation syndrome (H-ARS) survivors of 8.7-9.0 Gy total-body irradiation (TBI). The goal of this study was to determine temporal relationships in the development of vasculopathy and the progression of renal and cardiovascular delayed effects of acute radiation exposure (DEARE) at TBI doses less than 9 Gy and to elucidate the potential roles of senescence, inflammation and oxidative stress. Our results show significant loss of endothelial cells in coronary arteries by 4 months post-TBI (8.53 or 8.72 Gy of gamma radiation). This loss precedes renal dysfunction and interstitial fibrosis and progresses to abnormalities in the arterial media and adventitia and loss of coronary arterioles. Major differences in radiation-induced pathobiology exist between the heart and kidney in terms of vasculopathy progression and also in indices of inflammation, senescence and oxidative imbalance. The results of this work suggest a need for different medical countermeasures for multiple targets in different organs and at various times after acute radiation injury to prevent the progression of DEARE

Prostaglandin E2 Enhances Aged Hematopoietic Stem Cell Function

Aging of hematopoiesis is associated with increased frequency and clonality of hematopoietic stem cells (HSCs), along with functional compromise and myeloid bias, with donor age being a significant variable in survival after HSC transplantation. No clinical methods currently exist to enhance aged HSC function, and little is known regarding how aging affects molecular responses of HSCs to biological stimuli. Exposure of HSCs from young fish, mice, nonhuman primates, and humans to 16,16-dimethyl prostaglandin E2 (dmPGE2) enhances transplantation, but the effect of dmPGE2 on aged HSCs is unknown. Here we show that ex vivo pulse of bone marrow cells from young adult (3 mo) and aged (25 mo) mice with dmPGE2 prior to serial competitive transplantation significantly enhanced long-term repopulation from aged grafts in primary and secondary transplantation (27 % increase in chimerism) to a similar degree as young grafts (21 % increase in chimerism; both p < 0.05). RNA sequencing of phenotypically-isolated HSCs indicated that the molecular responses to dmPGE2 are similar in young and old, including CREB1 activation and increased cell survival and homeostasis. Common genes within these pathways identified likely key mediators of HSC enhancement by dmPGE2 and age-related signaling differences. HSC expression of the PGE2 receptor EP4, implicated in HSC function, increased with age in both mRNA and surface protein. This work suggests that aging does not alter the major dmPGE2 response pathways in HSCs which mediate enhancement of both young and old HSC function, with significant implications for expanding the therapeutic potential of elderly HSC transplantation

Delayed Effects of Acute Radiation Exposure in a Murine Model of the H-ARS: Multiple-Organ Injury Consequent to <10 Gy Total Body Irradiation

The threat of radiation exposure from warfare or radiation accidents raises the need for appropriate animal models to study the acute and chronic effects of high dose rate radiation exposure. The goal of this study was to assess the late development of fibrosis in multiple organs (kidney, heart, and lung) in survivors of the C57BL/6 mouse model of the hematopoietic-acute radiation syndrome (H-ARS). Separate groups of mice for histological and functional studies were exposed to a single uniform total body dose between 8.53 and 8.72 Gy of gamma radiation from a Cs radiation source and studied 1-21 mo later. Blood urea nitrogen levels were elevated significantly in the irradiated mice at 9 and 21 mo (from ∼22 to 34 ± 3.8 and 69 ± 6.0 mg dL, p < 0.01 vs. non-irradiated controls) and correlated with glomerosclerosis (29 ± 1.8% vs. 64 ± 9.7% of total glomeruli, p < 0.01 vs. non-irradiated controls). Glomerular tubularization and hypertrophy and tubular atrophy were also observed at 21 mo post-total body irradiation (TBI). An increase in interstitial, perivascular, pericardial and peribronchial fibrosis/collagen deposition was observed from ∼9-21 mo post-TBI in kidney, heart, and lung of irradiated mice relative to age-matched controls. Echocardiography suggested decreased ventricular volumes with a compensatory increase in the left ventricular ejection fraction. The results indicate that significant delayed effects of acute radiation exposure occur in kidney, heart, and lung in survivors of the murine H-ARS TBI model, which mirrors pathology detected in larger species and humans at higher radiation doses focused on specific organs

A Single Radioprotective Dose of Prostaglandin E2 Blocks Irradiation-Induced Apoptotic Signaling and Early Cycling of Hematopoietic Stem Cells

Ionizing radiation exposure results in acute and delayed bone marrow suppression. Treatment of mice with 16,16-dimethyl prostaglandin E2 (dmPGE2) prior to lethal ionizing radiation (IR) facilitates survival, but the cellular and molecular mechanisms are unclear. In this study we show that dmPGE2 attenuates loss and enhances recovery of bone marrow cellularity, corresponding to a less severe hematopoietic stem cell nadir, and significantly preserves long-term repopulation capacity and progenitor cell function. Mechanistically, dmPGE2 suppressed hematopoietic stem cell (HSC) proliferation through 24 h post IR, which correlated with fewer DNA double-strand breaks and attenuation of apoptosis, mitochondrial compromise, oxidative stress, and senescence. RNA sequencing of HSCs at 1 h and 24 h post IR identified a predominant interference with IR-induced p53-downstream gene expression at 1 h, and confirmed the suppression of IR-induced cell-cycle genes at 24 h. These data identify mechanisms of dmPGE2 radioprotection and its potential role as a medical countermeasure against radiation exposure

Development of a Model of the Acute and Delayed Effects of High Dose Radiation Exposure in Jackson Diversity Outbred Mice; Comparison to Inbred C57BL/6 Mice

Development of medical countermeasures against radiation relies on robust animal models for efficacy testing. Mouse models have advantages over larger species due to economics, ease of conducting aging studies, existence of historical databases, and research tools allowing for sophisticated mechanistic studies. However, the radiation dose-response relationship of inbred strains is inherently steep and sensitive to experimental variables, and inbred models have been criticized for lacking genetic diversity. Jackson Diversity Outbred (JDO) mice are the most genetically diverse strain available, developed by the Collaborative Cross Consortium using eight founder strains, and may represent a more accurate model of humans than inbred strains. Herein, models of the Hematopoietic-Acute Radiation Syndrome and the Delayed Effects of Acute Radiation Exposure were developed in JDO mice and compared to inbred C57BL/6. The dose response relationship curve in JDO mice mirrored the more shallow curves of primates and humans, characteristic of genetic diversity. JDO mice were more radioresistant than C57BL/6 and differed in sensitivity to antibiotic countermeasures. The model was validated with pegylated-G-CSF, which provided significantly enhanced 30-d survival and accelerated blood recovery. Long-term JDO survivors exhibited increased recovery of blood cells and functional bone marrow hematopoietic progenitors compared to C57BL/6. While JDO hematopoietic stem cells declined more in number, they maintained a greater degree of quiescence compared to C57BL/6, which is essential for maintaining function. These JDO radiation models offer many of the advantages of small animals with the genetic diversity of large animals, providing an attractive alternative to currently available radiation animal models