Hui Lin Chua

Establishing a murine model of the hematopoietic syndrome of the acute radiation syndrome.

Abstract The authors have developed a murine model of the Hematopoietic Syndrome of the Acute Radiation Syndrome (H-ARS) for efficacy testing of medical countermeasures (MCM) against radiation according to the FDA Animal Rule. Ten- to 12-wk-old male and female C57BL/6 mice were exposed to the LD50/30–LD70/30 dose of total body irradiation (TBI, 137Cs, 0.62–0.67 Gy min−1) in the morning hours when mice were determined to be most radiosensitive, and they were assessed for 30-d survival and mean survival time (MST). Antibiotics were delivered in drinking water on days 4–30 post-TBI at a concentration based on the amount of water that lethally-irradiated mice were found to consume. The fluoroquinolones, ciprofloxacin and levofloxacin, as well as the tetracycline doxycycline, and aminoglycoside neomycin, all significantly increased MST of decedent mice, while ciprofloxacin (p = 0.061) and doxycycline + neomycin (p = 0.005) showed at least some efficacy to increase 30-d survival. Blood sampling (30 &mgr;L/mouse every fifth day) was found to negatively impact 30-d survival. Histopathology of tissues harvested from nonmoribund mice showed expected effects of lethal irradiation, while moribund mice were largely septicemic with a preponderance of enteric organisms. Kinetics of loss and recovery of peripheral blood cells in untreated mice and those treated with two MCM, granulocyte-colony stimulating factor and Amifostine further characterized and validated this model for use in screening studies and pivotal efficacy studies of candidate MCM for licensure to treat irradiated individuals suffering from H-ARS.

Long-term hematopoietic stem cell damage in a murine model of the hematopoietic syndrome of the acute radiation syndrome.

Abstract Residual bone marrow damage (RBMD) persists for years following exposure to radiation and is believed to be due to decreased self-renewal potential of radiation-damaged hematopoietic stem cells (HSC). Current literature has examined primarily sublethal doses of radiation and time points within a few months of exposure. In this study, the authors examined RBMD in mice surviving lethal doses of total body ionizing irradiation (TBI) in a murine model of the Hematopoietic Syndrome of the Acute Radiation Syndrome (H-ARS). Survivors were analyzed at various time points up to 19 mo post-TBI for hematopoietic function. The competitive bone marrow (BM) repopulating potential of 150 purified c-Kit+ Sca-1+ lineage- CD150+ cells (KSLCD150+) remained severely deficient throughout the study compared to KSLCD150+ cells from non-TBI age-matched controls. The minimal engraftment from these TBI HSCs is predominantly myeloid, with minimal production of lymphocytes both in vitro and in vivo. All classes of blood cells as well as BM cellularity were significantly decreased in TBI mice, especially at later time points as mice aged. Primitive BM hematopoietic cells (KSLCD150+) displayed significantly increased cell cycling in TBI mice at all time points, which may be a physiological attempt to maintain HSC numbers in the post-irradiation state. Taken together, these data suggest that the increased cycling among primitive hematopoietic cells in survivors of lethal radiation may contribute to long-term HSC exhaustion and subsequent RBMD, exacerbated by the added insult of aging at later time points.

Recovery from hematopoietic injury by modulating prostaglandin E2 signaling post-irradiation

While high dose total body irradiation (TBI) is used therapeutically, the proliferation of nuclear weapons, increasing use of nuclear power, and worldwide radical terrorism underscore the need to develop countermeasures to a radiological mass casualty event. The hematopoietic syndrome of the acute radiation syndrome (HS-ARS) results from severe compromise to the hematopoietic system, including lymphocytopenia, neutropenia, thrombocytopenia, and possible death from infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow hematopoietic stem cells (HSC) and progenitor cells (HPC) may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E(2) (PGE(2)) has been shown to have pleiotropic effects on hematopoiesis, acting to inhibit apoptosis and promote self-renewal of HSC, while inhibiting HPC proliferation. We assessed the radio-mitigating potential of modulating PGE(2) signaling in a mouse model of HS-ARS. Treatment with the PGE(2) analog 16,16 dimethyl PGE(2) (dmPGE(2)) 6h post-irradiation or inhibition of PGE(2) synthesis via delayed administration of the non-steroidal anti-inflammatory drug (NSAID) Meloxicam resulted in increased survival of lethally irradiated mice. Both early dmPGE(2) and delayed Meloxicam treatment were associated with increased HPC activity 35days following irradiation, demonstrating enhanced recovery of hematopoiesis. Our results define two different treatment modalities that are highly effective and safe to administer, and can be readily available.

Pyk2 regulates megakaryocyte-induced increases in osteoblast number and bone formation

Preclinical and clinical evidence from megakaryocyte (MK)‐related diseases suggests that MKs play a significant role in maintaining bone homeostasis. Findings from our laboratories reveal that MKs significantly increase osteoblast (OB) number through direct MK‐OB contact and the activation of integrins. We, therefore, examined the role of Pyk2, a tyrosine kinase known to be regulated downstream of integrins, in the MK‐mediated enhancement of OBs. When OBs were co‐cultured with MKs, total Pyk2 levels in OBs were significantly enhanced primarily because of increased Pyk2 gene transcription. Additionally, p53 and Mdm2 were both decreased in OBs upon MK stimulation, which would be permissive of cell cycle entry. We then demonstrated that OB number was markedly reduced when Pyk2−/− OBs, as opposed to wild‐type (WT) OBs, were co‐cultured with MKs. We also determined that MKs inhibit OB differentiation in the presence and absence of Pyk2 expression. Finally, given that MK‐replete spleen cells from GATA‐1–deficient mice can robustly stimulate OB proliferation and bone formation in WT mice, we adoptively transferred spleen cells from these mice into Pyk2−/− recipient mice. Importantly, GATA‐1–deficient spleen cells failed to stimulate an increase in bone formation in Pyk2−/− mice, suggesting in vivo the important role of Pyk2 in the MK‐induced increase in bone volume. Further understanding of the signaling pathways involved in the MK‐mediated enhancement of OB number and bone formation will facilitate the development of novel anabolic therapies to treat bone loss diseases.

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.

AbstractIn 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−1 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

AbstractHematopoietic 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, 137Cs, 0.62–1.02 Gy min−1) and were treated with various dosing regimens of 0.1, 0.3, and 1.0 mg kg−1 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−1 of PEG-G-CSF and PEG-IL-11 or three doses of 0.3 mg kg−1 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.

The H-ARS dose response relationship (DRR): Validation and variables

AbstractManipulations 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.

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

AbstractThe 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 137Cs 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−1, 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.