Further Characterization of the Mitigation of Radiation Lethality by Protective Wounding

There continues to be a major effort in the United States to develop mitigators for the treatment of mass casualties that received high-intensity acute ionizing radiation exposures from the detonation of an improvised nuclear device during a radiological terrorist attack. The ideal countermeasure should be effective when administered after exposure, and over a wide range of absorbed doses. We have previously shown that the administration of a subcutaneous incision of a defined length, if administered within minutes after irradiation, protected young adult female C57BL/6 mice against radiation-induced lethality, and increased survival after total-body exposure to an LD50/30 X-ray dose from 50% to over 90%. We refer to this approach as "protective wounding". In this article, we report on our efforts to further optimize, characterize and demonstrate the validity of the protective wounding response by comparing the response of female and male mice, varying the radiation dose, the size of the wound, and the timing of wounding with respect to administration of the radiation dose. Both male and female mice that received a subcutaneous incision after irradiation were significantly protected from radiation lethality. We observed that the extent of protection against lethality after an LD50/30 X-ray dose was independent of the size of the subcutaneous cut, and that a 3 mm subcutaneous incision is effective at enhancing the survival of mice exposed to a broad range of radiation doses (LD15-LD100). Over the range of 6.2-6.7 Gy, the increase in survival observed in mice that received an incision was associated with an enhanced recovery of hematopoiesis. The enhanced rate of recovery of hematopoiesis was preceded by an increase in the production of a select group of cytokines. Thus, a thorough knowledge of the timing of the cytokine cascade after wounding could aid in the development of novel pharmacological radiation countermeasures that can be administered several days after the actual radiation exposure

Delayed effects of acute radiation exposure (DEARE) in a murine model of the hematopoietic acute radiation syndrome: Multiple-organ injury consequent to total body irradiation

Introduction. Victims of radiation exposure from terrorist activity, radiation accidents or radiologic warfare will face a variety of acute and chronic organ injuries requiring multi-faceted approaches to treatment. The hematopoietic system is the most sensitive tissue to radiation damage, resulting in the hematopoietic acute radiation syndrome (H-ARS) after exposures of 2-10 Gy in mice. If untreated, H-ARS results in death within weeks from opportunistic infection and/or hemorrhage due to loss of neutrophils and platelets, respectively. However, survivors of ARS are plagued months to years later in life by delayed effects of acute radiation exposure (DEARE), a myriad of chronic illnesses affecting multiple organ systems believed to be due to persistent systemic oxidative stress, inflammation, fibrosis and loss of stem cell self-renewal. Fibrosis and collagen deposition disrupt both normal tissue structure and function and are common to organs with late radiation injury including the kidney and heart after radiation doses >15Gy, but have not been shown to exist after doses as low as those used in the H-ARS model (8Gy). The goal of this study was to determine the extent, if any, of heart and kidney DEARE in survivors of H-ARS. Methods. Mice (male and female C57BL/6) received total body irradiation (TBI; LD50/30 to LD70/30) and kidney and heart were harvested at 9 and 21 months from the H-ARS survivor mice. Tissues were fixed in neutral buffered formalin, paraffin embedded and sectioned, then stained with hematoxylin/eosin (H&E), trichrome, or picosirius red. Serum was collected at 4.3, 9, and 21 months post-TBI and analyzed for blood urea nitrogen (BUN) as an indicator of kidney function. Total RNA was purified from heart and relative changes in NADPH oxidase 2 (Nox2) mRNA expression were assessed by quantitative real-time PCR. Results/Significance. Compared to age-matched non-irradiated controls (NI), renal pathology at 9 months post-TBI was manifest primarily as enlargement of Bowman’s capsule and glomerosclerosis along with limited interstitial fibrosis. By 21 months there was progression of these pathologies as well as extensive interstitial fibrosis, tubular atrophy, cysts, and atubular glomeruli, all of which were more pronounced in TBI mice compared to NI. Consistent with the renal pathology, BUN in TBI mice was significantly increased at 9 and 21 months post-TBI vs. 4.3 months, but normal in NI mice at all time points. In the heart, pericardial, perivascular and interstitial fibrosis were observed at 9 months with increased severity at 21 months post-TBI compared to NI. The perivascular fibrosis was associated with increased medial layer collagen and apparent loss of vascular smooth muscle cells. Nox2 mRNA in heart was increased at 9 and 21 months post-TBI, indicating an increase in oxidant stress. To our knowledge, such striking heart and kidney damage has not been documented after radiation doses as low as those in our H-ARS model (~8Gy) and indicate that DEARE is a concern for individuals exposed to radiation doses previously thought to not elicit late effects

Delayed effects of acute radiation exposure on the cardiovascular system using a murine model of the hematopoietic acute radiation syndrome

poster abstractIntroduction. Exposure to high level radiation from accidents or belligerent activities results in acute and chronic organ damage. The hematopoietic system is the most sensitive organ to radiation damage (2-10 Gy) and results in the hematopoietic acute radiation syndrome (H-ARS). Survivors of H-ARS are plagued months to years later with delayed effects of acute radiation exposure (DEARE), characterized by chronic illnesses affecting multiple organ systems. Previous results using the murine H-ARS model showed numerous kidney and heart DEARErelated pathologies similar to humans, including tissue fibrosis and elevated blood urea nitrogen. The goal of this study was to utilize the murine H-ARS model to determine possible roles for abnormal iron metabolism, inflammation, oxidant stress, and senescence in the development of cardiac DEARE. Methods. Mice (C57BL/6; 12 week-old) received total body irradiation (TBI: ~8.5-8.7 Gy, 137Cs, LD50to LD70) and hearts were harvested at various times post-TBI from H-ARS survivors. Paraffin tissue sections were stained with hematoxylin/eosin or Perls Prussian Blue, or reacted with a macrophage-specific antibody (F4/80). Total RNA was purified from fresh tissue and changes in mRNA expression were assessed by real-time PCR for the senescence marker p16 and NADPH oxidase subunits Nox2, Nox4, or p47phox. Results/Significance. Compared to age-matched non-irradiated controls (NI), tissue iron deposits were increased in irradiated (IR) hearts at 4 months, and progressively declined with time post-TBI. Numbers of macrophages were greater in IR vs. NI sections at all time points and decreased with time post-TBI. Nox2 and Nox4 mRNA expression was increased at both 9 and 21 months post-TBI, but p47phox increased only at 21 months. Expression of p16 in IR heart was increased at 7, but not at 22 months post-TBI. Taken together, the results indicate abnormal iron metabolism, inflammation, oxidant stress, and early senescence may contribute to development of cardiac DEARE

Aging-Related Reduced Expression of CXCR4 on Bone Marrow Mesenchymal Stromal Cells Contributes to Hematopoietic Stem and Progenitor Cell Defects

Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging, however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS), DNA damage, senescence, and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC, as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically, CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress, as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC), improved their niche supporting activity, and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production, driving an HSC aging phenotype. Thus, modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function

Deficiency of Src family kinases compromises the repopulating ability of hematopoietic stem cells

OBJECTIVE: Src family kinases (SFK) have been implicated in regulating growth factor and integrin-induced proliferation, migration, and gene expression in multiple cell types. However, little is known about the role of these kinases in the growth, homing, and engraftment potential of hematopoietic stem and progenitor cells. RESULTS: Here we show that loss of hematopoietic-specific SFKs Hck, Fgr, and Lyn results in increased number of Sca-1(+)Lin(-) cells in the bone marrow, which respond differentially to cytokine-induced growth in vitro and manifest a significant defect in the long-term repopulating potential in vivo. Interestingly, a significant increase in expression of adhesion molecules, known to coincide with the homing potential of wild-type bone marrow cells is also observed on the surface of SFK(-/-) cells, although, this increase did not affect the homing potential of more primitive Lin(-)Sca-1(+) SFK(-/-) cells. The stem cell-repopulating defect observed in mice transplanted with SFK(-/-) bone marrow cells is due to the loss of Lyn Src kinase, because deficiency of Lyn, but not Hck or Fgr, recapitulated the long-term stem cell defect observed in mice transplanted with SFK(-/-) bone marrow cells. CONCLUSIONS: Taken together, our results demonstrate an essential role for Lyn kinase in positively regulating the long-term and multilineage engraftment of stem cells, which is distinct from its role in mature B cells and myeloid cells

Prognostic Biomarkers for Acute Graft-versus-Host Disease Risk after Cyclophosphamide–Fludarabine Nonmyeloablative Allotransplantation

AbstractFive candidate plasma biomarkers (suppression of tumorogenesis 2 [ST2], regenerating islet-derived-3α [REG3α], elafin, tumor necrosis factor receptor 1 [TNFR1], and soluble IL-2 receptor-alpha [sIL2Rα]) were measured at specific time points after cyclophosphamide/fludarabine-based nonmyeloablative allotransplantation (NMAT) in patients who did or did not develop acute graft-versus-host disease (aGVHD). Plasma samples from 34 patients were analyzed at days +7, +14, +21, and +30. At a median follow-up of 358 days, 17 patients had experienced aGVHD with a median time to onset at day +36. Risk of aGVHD was associated with elevated plasma ST2 concentrations at day +7 (c-statistic = .72, P = .03), day +14 (c-statistic = .74, P = .02), and day +21 (c-statistic = .75, P = .02); elevated plasma REG3α concentrations at day +14 (c-statistic = .73, P = .03), day +21 (c-statistic = .76, P = .01), and day +30 (c-statistic = .73, P = .03); and elevated elafin at day +14 (c-statistic = .71, P = .04). Plasma concentrations of TNFR1 and sIL2Rα were not associated with aGVHD risk at any of the time points studied. This study identified ST2, REG3α, and elafin as prognostic biomarkers to evaluate risk of aGVHD after cyclophosphamide/fludarabine-based NMAT. These results need to be confirmed in an independent validation cohort

Increased Incidence of Lymphosarcoma in Long-Term Murine Survivors of Lethal Radiation: A Classification of Subtypes

poster abstractResidual bone marrow damage (RBMD) persists for years following exposure to radiation and is thought to be due to decreased self-renewal of hematopoietic stem cells (HSC). We previously examined RBMD in murine survivors of lethal radiation modeling a terrorist event [800cGy total-body irradiation (TBI)]. We reported severely deficient HSC potential up to 20mo post-TBI compared to non-TBI age-matched controls, evidenced by minimal engraftment skewed to myeloid cells. CBC and BM cellularity were decreased in TBI mice, most dramatically in old age (>16mo). The percentage of some hematopoietic progenitors was consistently increased in TBI mice (~1.4x higher than non-TBI) possibly due to an increased cell cycling rate compared to non-TBI cells. Of interest, we now report the occurrence of a thymic mass developing in 13-24% of TBI mice 2-19 months post-TBI, compared to <1% of non-TBI. We characterized the Lymphosarcoma into the following groups based on the St. Jude pathology subclassification: Diffuse Lymphosarcoma involving multiple organs, Thymic lymphoma (usually associated with thymic and around the heart), Lymphosarcoma (potentially starting in the spleen and peri-pancreatic lymph nodes (Ab=abdomen)), and follicular lymphoma seen as a diffuse proliferation of lymphocytes in the white pulp area in the spleen. Thymic lymphomas were the most common, followed by Lymphosarcoma (Ab), follicular lymphoma (restricted to white pulp area in the spleen) and diffuse Lymphosarcoma. Immunostain markers revealed the thymic lymphomas were from T-cell lineage and the abdominal Lymphosarcoma were mainly from B-cell lineage. A few mice had disease involving the bone marrow. Taken together, these data suggest that the increased cycling among primitive hematopoietic cells in survivors of lethal radiation may contribute to stem cell exhaustion and subsequent RBMD, as well as predispose survivors to hematopoietic neoplasias

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

A Potential Role for Excess Tissue Iron in Development of Cardiovascular Delayed Effects of Acute Radiation Exposure

Murine hematopoietic-acute radiation syndrome (H-ARS) survivors of total body radiation (TBI) have a significant loss of heart vessel endothelial cells, along with increased tissue iron, as early as 4 months post-TBI. The goal of the current study was to determine the possible role for excess tissue iron in the loss of coronary artery endothelial cells. Experiments utilized the H-ARS mouse model with gamma radiation exposure of 853 cGy (LD50/30) and time points from 1 to 12 weeks post-TBI. Serum iron was elevated at 1 week post-TBI, peaked at 2 weeks, and returned to non-irradiated control values by 4 weeks post-TBI. A similar trend was seen for transferrin saturation, and both results correlated inversely with red blood cell number. Perls’ Prussian Blue staining used to detect iron deposition in heart tissue sections showed myocardial iron was present as early as 2 weeks following irradiation. Pretreatment of mice with the iron chelator deferiprone decreased tissue iron, but not serum iron, at 2 weeks. Coronary artery endothelial cell density was significantly decreased as early as two weeks vs. non-irradiated controls (P<0.05), and the reduced density persisted to 12 weeks after irradiation. Deferiprone treatment of irradiated mice prevented the decrease in endothelial cell density at 2 and 4 weeks post-TBI compared to irradiated, non-treated mice (P<0.03). Taken together, the results suggest excess tissue iron contributes to endothelial cell loss early following TBI and may be a significant event impacting the development of delayed effects of acute radiation exposure

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