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High-dose radiation exposure results in hematopoietic (H) and gastrointestinal (GI) acute radiation syndromes (ARS) followed by delayed effects of acute radiation exposure (DEARE), which include damage to lung, heart, and GI. Whereas DEARE includes inflammation and fibrosis in multiple tissues, the molecular mechanisms contributing to inflammation and to the development of fibrosis remain incompletely understood. Reports that radiation dysregulates retinoids and proteins within the retinoid pathway indicate that radiation disrupts essential nutrient homeostasis. An active metabolite of vitamin A, retinoic acid (RA), is a master regulator of cell proliferation, differentiation, and apoptosis roles in inflammatory signaling and the development of fibrosis. As facets of inflammation and fibrosis are regulated by RA, we surveyed radiation-induced changes in retinoids as well as proteins related to and targets of the retinoid pathway in the non-human primate after high dose radiation with minimal bone marrow sparing (12 Gy PBI/BM2.5). Retinoic acid was decreased in plasma as well as in lung, heart, and jejunum over time, indicating a global disruption of RA homeostasis after IR. A number of proteins associated with fibrosis and with RA were significantly altered after radiation. Together these data indicate that a local deficiency of endogenous RA presents a permissive environment for fibrotic transformation.

OBJECTIVE: The goal of this study was to develop rat models of partial body irradiation with bone-marrow sparing (leg-out PBI), to test medical countermeasures (MCM) of both acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE) under the FDA animal rule. METHODS: The leg-out PBI models were developed in female and male WAG/RijCmcr rats at doses of 12.5–14.5 Gy. Rats received supportive care consisting of fluids and antibiotics. Gastrointestinal ARS (GI-ARS) was assessed by lethality to day 7 and diarrhea scoring to day 10. Differential blood counts were analyzed between days 1–42 for the natural history of hematopoietic ARS (H-ARS). Lethality and breathing intervals (BI) were measured between days 28–110 to assess delayed injury to the lung (L-DEARE). Kidney injury (K-DEARE) was evaluated by measuring elevation of blood urea nitrogen (BUN) between days 90–180. RESULTS: The LD(50/30), including both lethality from GI-ARS and H-ARS, for female and male rats is 14.0 Gy and 13.5 Gy respectively, while the LD(50/7) for only GI-ARS is 14.3 Gy and 13.6 Gy respectively. The all-cause mortality, including ARS and L-DEARE, through 120 days (LD(50/120)) is 13.5 Gy and 12.9 Gy respectively. Secondary end points confirmed occurrence of 4 distinct sequelae representing GI, hematopoietic, lung and kidney toxicities after leg-out PBI. CONCLUSION: Adult rat models of leg-out PBI developed showed the acute and long-term sequelae of radiation damage that has been reported in human radiation exposure case studies. Sex-specific differences were observed in the DRR between females and males. These rat models are among the most useful for the development and approval of countermeasures for mitigation of radiation injuries under the FDA animal rule.

Radiation-induced lung injury is a delayed effect of acute radiation exposure resulting in pulmonary pneumonitis and fibrosis. Molecular mechanisms that lead to radiation-induced lung injury remain incompletely understood. Using a non-human primate model of partial body irradiation with minimal bone marrow sparing, lung was analyzed from animals irradiated with 12 Gy at timepoints every 4 d up to 21 d after irradiation and compared to non-irradiated (sham) controls. Tryptic digests of lung tissues were analyzed by liquid chromatography-tandem mass spectrometry followed by pathway analysis. Out of the 3,101 unique proteins that were identified, we found that 252 proteins showed significant and consistent responses across at least three time points post-irradiation, of which 215 proteins showed strong up-regulation while 37 proteins showed down-regulation. Canonical pathways affected by irradiation, changes in proteins that serve as upstream regulators, and proteins involved in key processes including inflammation, fibrosis, and retinoic acid signaling were identified. The proteomic profiling of lung conducted here represents an untargeted systems biology approach to identify acute molecular events in the non-human primate lung that could potentially be initiating events for radiation-induced lung injury.

High-dose radiation exposure results in hematopoietic and gastrointestinal acute radiation syndromes followed by delayed effects of acute radiation exposure, which encompasses multiple organs, including heart, kidney, and lung. Here we sought to further characterize the natural history of radiation-induced heart injury via determination of differential protein and metabolite expression in the heart. We quantitatively profiled the proteome and metabolome of left and right ventricle from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Global proteome profiling identified more than 2,200 unique proteins, with 220 and 286 in the left and right ventricles, respectively, showing significant responses across at least three time points compared to baseline levels. High-throughput targeted metabolomics analyzed a total of 229 metabolites and metabolite combinations, with 18 and 22 in the left and right ventricles, respectively, showing significant responses compared to baseline levels. Bioinformatic analysis performed on metabolomic and proteomic data revealed pathways related to inflammation, energy metabolism, and myocardial remodeling were dysregulated. Additionally, we observed dysregulation of the retinoid homeostasis pathway, including significant post-radiation decreases in retinoic acid, an active metabolite of vitamin A. Significant differences between left and right ventricles in the pathology of radiation-induced injury were identified. This multi-omic study characterizes the natural history and molecular mechanisms of radiation-induced heart injury in NHP exposed to PBI with minimal bone marrow sparing.

Bile acids (BAs) have been established as ubiquitous regulatory molecules implicated in a large variety of healthy and pathological processes. However, the scope of BA heterogeneity is often underrepresented in current literature. This is due in part to inadequate detection methods, which fail to distinguish the individual constituents of the BA pool. Thus, the primary aim of this study was to develop a method that would allow the simultaneous analysis of specific C24 BA species, and to apply that method to biological systems of interest. Herein, we describe the generation and validation of an LC-MS/MS assay for quantification of numerous BAs in a variety of cell systems and relevant biofluids and tissue. These studies included the first baseline level assessment for planar BAs, including allocholic acid, in cell lines, biofluids, and tissue in a nonhuman primate (NHP) laboratory animal, Macaca mulatta, in healthy conditions. These results indicate that immortalized cell lines make poor models for the study of BA synthesis and metabolism, whereas human primary hepatocytes represent a promising alternative model system. We also characterized the BA pool of M. mulatta in detail. Our results support the use of NHP models for the study of BA metabolism and pathology in lieu of murine models. Moreover, the method developed here can be applied to the study of common and planar C24 BA species in other systems.

Exposure to total- and partial-body irradiation following a nuclear or radiological incident result in the potentially lethal acute radiation syndromes of the gastrointestinal and hematopoietic systems in a dose-and time-dependent manner. Radiation-induced damage to the gastrointestinal tract is observed within days to weeks post-irradiation. Our objective in this study was to evaluate plasma biomarker utility for the gastrointestinal acute radiation syndrome in non-human primate after partial body irradiation with minimal bone marrow sparing through correlation with tissue and histological analyses. Plasma and jejunum samples from non-human primates exposed to partial body irradiation of 12 Gy with bone marrow sparing of 2.5% were evaluated at various time points from day 0 to day 21 as part of a natural history study. Additionally, longitudinal plasma samples from non-human primates exposed to 10 Gy partial body irradiation with 2.5% bone marrow sparing were evaluated at timepoints out to 180 days post-irradiation. Plasma and jejunum metabolites were quantified via liquid chromatography - tandem mass spectrometry and histological analysis consisted of corrected crypt number, an established metric to assess radiation-induced gastrointestinal damage. A positive correlation of metabolite levels in jejunum and plasma was observed for citrulline, serotonin, acylcarnitine and multiple species of phosphatidylcholines. Citrulline levels also correlated with injury and regeneration of crypts in the small intestine. These results expand the characterization of the natural history of gastrointestinal acute radiation syndrome in non-human primates exposed to partial body irradiation with minimal bone marrow sparing and also provide additional data toward the correlation of citrulline with histological endpoints.

Exposure to ionizing radiation results in injuries of the hematopoietic, gastrointestinal, and respiratory systems, which are the leading causes responsible for morbidity and mortality. Gastrointestinal injury occurs as an acute radiation syndrome. To help inform on the natural history of the radiation-induced injury of the partial body irradiation model, we quantitatively profiled the proteome of jejunum from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Jejunum was analyzed by liquid chromatography-tandem mass spectrometry, and pathway and gene ontology analysis were performed. A total of 3,245 unique proteins were quantified out of more than 3,700 proteins identified in this study. Also a total of 289 proteins of the quantified proteins showed significant and consistent responses across at least three time points post-irradiation, of which 263 proteins showed strong upregulations while 26 proteins showed downregulations. Bioinformatic analysis suggests significant pathway and upstream regulator perturbations post-high dose irradiation and shed light on underlying mechanisms of radiation damage. Canonical pathways altered by radiation included GP6 signaling pathway, acute phase response signaling, LXR/RXR activation, and intrinsic prothrombin activation pathway. Additionally, we observed dysregulation of proteins of the retinoid pathway and retinoic acid, an active metabolite of vitamin A, as quantified by liquid chromatography-tandem mass spectrometry. Correlation of changes in protein abundance with a well-characterized histological endpoint, corrected crypt number, was used to evaluate biomarker potential. These data further define the natural history of the gastrointestinal acute radiation syndrome in a non-human primate model of partial body irradiation with minimal bone marrow sparing.

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-day 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.

The dose response relationship and corresponding values for mid-lethal dose and slope are used to define the dose- and time-dependent parameters of the hematopoietic acute radiation syndrome. The characteristic time course of mortality, morbidity, and secondary endpoints are well defined. The concomitant comorbidities, potential mortality, and other multi-organ injuries that are similarly dose- and time-dependent are less defined. Determination of the natural history or pathophysiology associated with the lethal hematopoietic acute radiation syndrome is a significant gap in knowledge, especially when considered in the context of a nuclear weapon scenario. In this regard, the exposure is likely ill-defined, heterogenous, and nonuniform. These conditions forecast sparing of bone marrow and increased survival from the acute radiation syndrome consequent to threshold doses for the delayed effects of acute radiation exposure due to marrow sparing, medical management, and use of approved medical countermeasures. The intent herein is to provide a composite natural history of the pathophysiology concomitant with the evolution of the potentially lethal hematopoietic acute radiation syndrome derived from studies that focused on total body irradiation and partial body irradiation with bone marrow sparing. The marked differential in estimated LD50/60 from 7.5 Gy to 10.88 Gy for the total body irradiation and partial body irradiation with 5% bone marrow sparing models, respectively, provided a clear distinction between the attendant multiple organ injury and natural history of the two models that included medical management. Total body irradiation was focused on equivalent LD50/60 exposures. The 10 Gy and 11 Gy partial body with 5% bone marrow sparing exposures bracketed the LD50/60 (10.88 Gy). The incidence, progression, and duration of multiple organ injury was described for each exposure protocol within the hematopoietic acute radiation syndrome. The higher threshold doses for the partial body irradiation with bone marrow sparing protocol induced a marked degree of multiple organ injury to include lethal gastrointestinal acute radiation syndrome, prolonged crypt loss and mucosal damage, immune suppression, acute kidney injury, body weight loss, and added clinical comorbidities that defined a complex timeline of organ injury through the acute hematopoietic acute radiation syndrome. The natural history of the acute radiation syndrome presents a 60-d time segment of multi-organ sequelae that is concomitant with the latent period or time to onset of the evolving multi-organ injury of the delayed effects of acute radiation exposure.

Exposure to ionizing radiation following a nuclear or radiological incident results in potential acute radiation syndromes causing sequelae of multi-organ injury in a dose- and time-dependent manner. Currently, medical countermeasures against radiation injury are limited, and no biomarkers have been approved by regulatory authorities. Identification of circulating plasma biomarkers indicative of radiation injury can be useful for early triage and injury assessment and in the development of novel therapies (medical countermeasures). Aims of this study are to (1) identify metabolites and lipids with consensus signatures that can inform on mechanism of injury in radiation-induced multi-organ injury and (2) identify plasma biomarkers in non-human primate (NHP) that correlate with tissues (kidney, liver, lung, left and right heart, jejunum) indicative of radiation injury, assessing samples collected over 3 wk post-exposure to 12 Gy partial body irradiation with 2.5% bone marrow sparing. About 180 plasma and tissue metabolites and lipids were quantified through Biocrates AbsoluteIDQ p180 kit using liquid chromatography and mass spectrometry. System-wide perturbations of specific metabolites and lipid levels and pathway alterations were identified. Citrulline, Serotonin, PC ae 38:2, PC ae 36:2, and sum of branched chain amino acids were identified as potential biomarkers of radiation injury. Pathway analysis revealed consistent changes in fatty acid oxidation and metabolism and perturbations in multiple other pathways.

Medical countermeasure development under the US Food and Drug Administration animal rule requires validated animal models of acute radiation effects. The key large animal model is the non-human primate, rhesus macaque. To date, only the rhesus macaque has been used for both critical supportive data and pivotal efficacy trials seeking US Food and Drug Administration approval. The potential for use of the rhesus for other high priority studies such as vaccine development underscores the need to identify another non-human primate model to account for the current lack of rhesus for medical countermeasure development. The cynomolgus macaque, Macaca fascicularis, has an existing database of medical countermeasure development against the hematopoietic acute radiation syndrome, as well as the use of radiation exposure protocols that mimic the likely nonuniform and heterogenous exposure consequent to a nuclear terrorist event. The review herein describes published studies of adult male cynomolgus macaques that used two exposure protocols-unilateral, nonuniform total-body irradiation and partial-body irradiation with bone marrow sparing-with the administration of subject-based medical management to assess mitigation against the hematopoietic acute radiation syndrome. These studies assessed the efficacy of cytokine combinations and cell-based therapy to mitigate acute radiation-induced myelosuppression. Both therapeutics were shown to mitigate the myelosuppression of the hematopoietic acute radiation syndrome. Additional studies being presented herein further defined the dose-dependent hematopoietic acute radiation syndrome of cynomolgus and rhesus macaques and a differential dose-dependent effect with young male and female cynomolgus macaques. The database supports the investigation of the cynomolgus macaque as a comparable non-human primate for efficacy testing under the US Food and Drug Administration animal rule. Critical gaps in knowledge required to validate the models and exposure protocols are also identified.

PURPOSE: To study the molecular and cellular mechanisms of radiation-induced lung injury (RILI) in a non-human primate model. METHODS AND MATERIALS: Rhesus macaques were irradiated with lethal doses of radiation to the whole thorax. A subset of the irradiated animals was treated with AEOL 10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. Lung tissues were collected at necropsy for molecular and immunohistochemical (IHC) studies. RESULTS: Microarray expression profiling in the irradiated lung tissues identified differentially expressed genes (DEGs) and pathways important in innate immunity. The elevated expression of cytokines (CCL2, CCL11, IL-8), complement factors (CFB, C3), apoptosis-related molecules (p53, PTEN, Bax, p21, MDM2, c-Caspase 3) and adhesion molecules (fibronectin, integrin β6, ICAM-1) were further studied using realtime PCR, Western blot, or IHC. Oxidative stress and pulmonary inflammatory cell infiltration were increased in the irradiated lungs. Treatment with AEOL 10150 significantly decreased oxidative stress and monocyte/macrophage infiltration. CONCLUSIONS: Cytokine/chemokine induced excessive innate immune response after thoracic irradiation plays important roles in RILI. To our knowledge, this is the first study to highlight the role of cytokine/chemokine induced innate immune responses in radiation-induced pulmonary toxicity in a NHP model.

Radiation sequelae is complex and characterized by multiple pathologies, which occur over time and nonuniformly throughout different organs. The study of the mesenteric lymph node (MLN) due to its importance in the gastrointestinal system is of particular interest. Other studies have shown an immediate post-irradiation reduction in cellularity due to the known effects of irradiation on lymphoid cell populations, but the molecular and functional mechanisms that lead to these cellular alterations remain limited. In this work, we show the use of lipidomic, proteomic, and mass spectrometry imaging in the characterization of the effects of acute radiation exposure on the MLN at different time points after ionizing radiation (IR) from 4 d to 21 d after 12 Gy partial body irradiation with 2.5% bone marrow sparing. The combined analyses showed a dysregulation of the lipid and protein composition in the MLN after IR. Protein expression was affected in numerous pathways, including pathways regulating lipids such as LXR/RXR activation and acute phase response. Lipid distribution and abundance was also affected by IR in the MLN, including an accumulation of triacylglycerides, a decrease in polyunsaturated glycerophospholipids, and changes in polyunsaturated fatty acids. Those changes were observed as early as 4 d after IR and were more pronounced for lipids with a higher concentration in the nodules and the medulla of the MLN. These results provide molecular insight into the MLN that can inform on injury mechanism in a non-human primate model of the acute radiation syndrome of the gastrointestinal tract. Those findings may contribute to the identification of therapeutic targets and the development of new medical countermeasures.

High-throughput, targeted metabolomics was used to identify early time point small intestine and plasma metabolite markers of gastrointestinal acute radiation syndrome. The small intestine metabolite markers were cross-correlated to plasma metabolites in order to identify minimally invasive circulating markers. The radiation exposure covered lethal and sub-lethal gastrointestinal acute radiation syndrome. The small intestine and plasma metabolite profiles were generated at 1 and 3 days post-exposure following total-body irradiation. The small intestine and plasma metabolite profiles for mice receiving radiation at day 1 and 3 post-exposure were significantly different from sham-irradiated mice. There were 14 metabolite markers identified at day 1 and 18 metabolite markers at day 3 that were small intestine-specific plasma markers of gastrointestinal acute radiation syndrome. A number of the identified metabolites at day 1 were amino acids. Dysregulation of amino acid metabolism at 24-hours post total-body irradiation provides potential insight into the initial inflammatory response during gastrointestinal acute radiation syndrome.

The acute radiation syndrome of the gastrointestinal tract has been histologically characterized, but the molecular and functional mechanisms that lead to these cellular alterations remain enigmatic. Mass spectrometry imaging is the only technique that enables the simultaneous detection and cellular or regional localization of hundreds of biomolecules in a single experiment. This current study utilized matrix-assisted laser desorption/ionization mass spectrometry imaging for the molecular characterization of the first natural history study of gastrointestinal acute radiation syndrome in the nonhuman primate. Jejunum samples were collected at days 4, 8, 11, 15, and 21 following 12-Gy partial-body irradiation with 2.5% bone marrow sparing. Mass spectrometry imaging investigations identified alterations in lipid species that further understanding of the functional alterations that occur over time in the different cellular regions of the jejunum following exposure to high doses of irradiation. Alterations in phosphatidylinositol species informed on dysfunctional epithelial cell differentiation and maturation. Differences in glycosphingolipids of the villi epithelium that would influence the absorptive capacity and functional structure of the brush border membrane were detected. Dichotomous alterations in cardiolipins indicated altered structural and functional integrity of mitochondria. Phosphatidylglycerol species, known regulators of toll-like receptors, were detected and localized to regions in the lamina propria that contained distinct immune cell populations. These results provide molecular insight that can inform on injury mechanism in a nonhuman primate model of the acute radiation syndrome of the gastrointestinal tract. Findings may contribute to the identification of therapeutic targets and the development of new medical countermeasures.

Radiation-induced lung injury is a characteristic, dose- and time-dependent sequela of potentially lethal, delayed effects of acute radiation exposure. Understanding of these delayed effects to include development of medical countermeasures requires well-characterized and validated animal models that mimic the human response to acute radiation and adhere to the criteria of the US Food and Drug Administration Animal Rule. The objective herein was to establish a nonhuman primate model of whole-thorax lung irradiation in female rhesus macaques. Definition of the dose-response relationship to include key signs of morbidity and mortality in the female macaque served to independently validate the recent model performed with male macaques and importantly, to establish the lack of sex and institutional bias across the dose-response relationship for radiation-induced lung injury. The study design was similar to that described previously, with the exception that female rhesus macaques were utilized. In brief, a computed tomography scan was conducted prior to irradiation and used for treatment planning. Animals in 5 cohorts (n = 8 per cohort) were exposed to a single 6-MV photon exposure focused on the lung as determined by the computed tomography scan and treatment planning at a dose of 9.5, 10, 10.5, 11, or 11.5 Gy. Subject-based supportive care, including administration of dexamethasone, was based on trigger-to-treat criteria. Clearly defined euthanasia criteria were used to determine a moribund condition over the 180-day study duration post-whole-thorax lung irradiation. Percent mortality per radiation dose was 12.5% at 9.5 Gy, 25% at 10 Gy, 62.5% at 10.5 Gy, 87.5% at 11 Gy, and 100% at 11.5 Gy. The resulting probit plot for the whole-thorax lung irradiation model estimated an LD50/180 of 10.28 Gy, which was not significantly different from the published estimate of 10.27 Gy for the male rhesus. The key parameters of morbidity and mortality support the conclusion that there is an absence of a sex influence on the radiation dose-response relationship for whole-thorax lung irradiation in the rhesus macaque. This work also provides a significant interlaboratory validation of the previously published model.

Male rhesus macaques were subjected to partial-body irradiation at 10, 11, or 12 Gy with 5% bone marrow protection. Animals were euthanized when dictated by prospectively determined clinical parameters, or at approximately 180 days following irradiation. Histological sections of jejunum, colon and mesenteric lymph node were stained with hematoxylin and eosin as well as a battery of histochemical and immunohistochemical stains. The immediate post-irradiation histopathological alterations in the jejunum and colon were based primarily on injury to rapidly proliferating crypt epithelial cells, though there was evidence of additional radiation-induced fibrogenic responses. There was substantial resolution of the radiation-related mucosal injury through the observation period, but microscopically visible defects in mucosal structure persisted to the end of the observation period. In the later stages of the observation period the jejunum and colon had overt fibrosis that was most commonly located in the submucosa and serosa, with less microscopically discernible involvement of the mucosa. Mesenteric lymph nodes had an immediate post-irradiation reduction in cellularity due to the known effects of irradiation on lymphoid cell populations. In later stages of the observation period the lymph nodes also developed fibrotic changes, possibly related to transmigration of immunomodulatory cells and/or signaling molecules from the radiation-damaged intestine.

The primary objectives of two companion manuscripts were to assess the natural history of delayed radiation-induced lung injury in a nonhuman primate model of acute high-dose, partial-body irradiation with 5% bone marrow sparing, to include the clinical, radiographic, and histopathological evidence and the effect of Neupogen administration on the morbidity and mortality. Nonhuman primates were exposed to 10.0 or 11.0 Gy with 6 MV linac-derived photons at approximately 0.80 Gy min. All nonhuman primates received subject-based, medical management. Subsets of nonhuman primates were administered Neupogen (10 μg kg) starting on day 1, day 3, or day 5 until recovery (absolute neutrophil count ≥ 1,000 cells μL for three consecutive days). Mortality due to multiple organ injury at 180 d study duration: Mortality at 180 d post either 10.0 Gy or 11.0 Gy was the consequence of concurrent injury due to the acute radiation syndrome (gastrointestinal and hematological) and delayed radiation-induced lung injury. The 180-d all-cause mortality observed in the control cohorts at 10.0 Gy (53%) or 11.0 Gy (86%) did not vary from cohorts that received Neupogen at any administration schedule. Mortality ranged from 43-50% (10 Gy) to 75-100% (11.0 Gy) in the Neupogen-treated cohorts. The study, however, was not powered to detect statistical significant differences between mortality in the control and Neupogen-treated cohorts. Clinical and radiographic evidence of radiation-induced lung injury: The mean nonsedated respiratory rate in the control cohorts exposed to 10 or 11 Gy increased from a baseline value of 37 breaths min to >60 breaths min within 103 d and 94 d postexposure, and the incidence of nonsedated respiratory rate > 80 breaths min was 50% and 70%, respectively. The mean duration of latency to development of clinical pneumonitis and/or fibrosis (nonsedated respiratory rate > 80 breaths min) was not significantly different between the 10.0-Gy or 11.0 Gy-cohorts (range 100-107 d). Neupogen (granulocyte colony-stimulating factor) administration had no apparent effect of the latency, incidence, or severity of nonsedated respiratory rate within either radiation dose or administration schedule. Computed tomography scans were obtained and images were analyzed for evidence of lung injury, e.g., pneumonitis and/or fibrosis, pleural and pericardial effusion. A quantitative, semiautomated method was developed based on differences in radiodensity (Hounsfield units) and lung morphology to extract the volume of pneumonitis/fibrosis and pleural effusion as indexed against total lung at each time point obtained. At both irradiation doses, 100% of the nonhuman primates surviving acute radiation syndrome manifested radiographic evidence of radiation-induced lung injury as pneumonitis and/or fibrosis. There was no apparent effect of Neupogen administration on the latency, incidence, severity, or progression of pneumonitis/fibrosis:total lung volume or pleural effusion:total lung volume at either exposure. A comparative review of the data illustrated the concomitant time course of increased mortality, nonsedated respiratory rate, and pneumonitis/fibrosis:total lung volume and pleural effusion:total lung volume consequent to 10.0-Gy or 11.0-Gy partial-body irradiation with 5% bone marrow sparing. All key parameters proceeded from a latent period of approximately 60 d followed by an increase in all three indices of clinical and radiographic evidence of radiation-induced lung injury within the next 60 d to 120 d postexposure. The subsequent time course and longitudinal analysis was influenced by the persistent progression of radiation-induced lung injury, administration of dexamethasone, and loss of nonhuman primates due to lethality. Companion paper: Lung and Heart Injury in a Nonhuman Primate Model of Partial-body Irradiation With Minimal Bone Marrow Sparing: Histopathological Evidence of Lung and Heart Injury (Parker et al. 2019): Note that the computed tomography-based radiodensity data do not permit differentiation of pneumonitis and fibrosis. The companion paper employed Masson's trichrome, collagen 1, and selected staining to identify the key time and incidence parameters relative to excessive collagen deposition indicative of fibrosis and associated histopathology in the lung. This histological database provided valuable longitudinal analysis in support of the clinical and radiographic evidence associated with the time course of radiation-induced lung injury.

Male rhesus macaques were subjected to partial-body irradiation at 10, 11, or 12 Gy with 5% bone marrow protection. Animals were euthanized when dictated by prospectively determined clinical parameters, or at approximately 180 days following irradiation. Histological sections of lung and heart were stained with hematoxylin and eosin as well as a battery of histochemical and immunohistochemical stains. Histopathological alterations in the lung were centered on fibrosis, inflammation, and reactive/proliferative changes in pneumocytes. These changes were noted in animals necropsied after approximately 85–100 days post-irradiation and extending through the observation period. Interstitial and pleural fibrosis demonstrated by Masson’s trichrome staining were associated with increased alpha smooth muscle actin and collagen 1 immunohistochemical staining. Areas of interstitial fibrosis had reduced microvascular density with CD31 immunohistochemical staining. Accumulations of CD163- and CD206-positive alveolar macrophages were present in areas of interstitial fibrosis. Unidentified cells termed ‘myxoid cells’ in alveolar walls had histochemical and immunohistochemical staining characteristics of epithelial-, endothelial-, or pericyte-mesenchymal transition states that were developing myofibroblast features. Distinctive focal or multifocal alveolar-bronchiolar hyperplasia had microscopic features of pre-neoplastic proliferation. Delayed radiation-associated changes in the heart consisted primarily of myocardial fibrosis, with rare histological evidence of myofiber degeneration.

Male rhesus macaques were subjected to partial-body irradiation at 10, 11, or 12 Gy with 5% bone marrow protection. Animals were euthanized when dictated by prospectively determined clinical parameters, or approximately 180 days following irradiation. Histological sections of kidney were stained with hematoxylin and eosin as well as a battery of histochemical and immunohistochemical stains. Histopathological alterations were centered on glomerular changes and fibrosis of glomeruli and the interstitial compartment. These changes were first noted in animals necropsied approximately 100 days post-irradiation and continued in animals necropsied through the observation period. Glomerular changes included congestion, thrombosis, erythrocyte degeneration, capillary tuft dilation, fibrin deposition, altered quantity and dispersion pattern of von Willebrand factor, increased mesangial matrix, and mesangial deposits of material that stained positively with periodic acid-Schiff staining. Areas of interstitial and glomerular fibrosis, as demonstrated by Masson’s trichrome staining, were topographically associated with increased immunohistochemical staining for connective tissue growth factor, alpha smooth muscle actin and collagen 1, but there was little staining for transforming growth factor beta. Fibrotic glomeruli had reduced microvascularity as demonstrated by reduced CD31 immunohistochemical staining. Vascular congestion was commonly noted in the region of the corticomedullary junction, and proteinaceous casts were commonly noted in cortical and medullary tubules. Longitudinal analysis of histopathological alterations provided evidence defining the latency, severity and progression of delayed radiation-induced kidney injury.

Lymphoid lineage recovery and involution after exposure to potentially lethal doses of ionizing radiation have not been well defined, especially the long-term effects in aged survivors and with regard to male / female differences. To examine these questions, male and female C57BL/6 mice were exposed to lethal radiation at 12 weeks of age in a model of the Hematopoietic-Acute Radiation Syndrome, and bone marrow, thymus, spleen and peripheral blood examined up to 24 months of age for the lymphopoietic Delayed Effects of Acute Radiation Exposure. Aged mice showed myeloid skewing and incomplete lymphocyte recovery in all lymphoid tissues. Spleen and peripheral blood both exhibited a mono-phasic recovery pattern while thymus demonstrated a bi-phasic pattern. Naïve T cells in blood and spleen and all subsets of thymocytes were decreased in aged irradiated mice compared to age-matched non-irradiated controls. Of interest, irradiated males experienced significantly improved reconstitution of thymocyte subsets and peripheral blood elements compared to females. Bone marrow from aged irradiated survivors was significantly deficient in the primitive lymphoid-primed multipotent progenitors and common lymphoid progenitors, which were only 8–10% of levels in aged-matched non-irradiated controls. Taken together, these analyses define significant age- and sex-related deficiencies at all levels of lymphopoiesis throughout the lifespan of survivors of the Hematopoietic-Acute Radiation Syndrome, and may provide a murine model suitable for assessing the efficacy of potential medical countermeasures and therapeutic strategies to alleviate the severe immune suppression that occurs after radiation exposure.

High-dose radiation exposure results in organ-specific sequelae that occurs in a time- and dose-dependent manner. The partial body irradiation with minimal bone marrow sparing model was developed to mimic intentional or accidental radiation exposures in humans where bone marrow sparing is likely and permits the concurrent analysis of coincident short- and long-term damage to organ systems. To help inform on the natural history of the radiation-induced injury of the partial body irradiation model, we quantitatively profiled the plasma proteome of non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing with 6 MV LINAC-derived photons at 0.80 Gy min(−1) over a time period of three weeks. The plasma proteome was analyzed by liquid chromatography-tandem mass spectrometry. A number of trends were identified in the proteomic data including pronounced protein changes as well as protein changes that were consistently upregulated or downregulated at all time points and dose levels interrogated. Pathway and gene ontology analysis were performed; bioinformatic analysis revealed significant pathway and biological process perturbations post high dose irradiation and shed light on underlying mechanisms of radiation damage. Additionally, proteins were identified that had the greatest potential to serve as biomarkers for radiation exposure.

The development of medical countermeasures against acute and delayed multi-organ injury requires animal models predictive of the human response to radiation and its treatment. Late chronic injury is a well-known feature of radiation nephropathy, but acute kidney injury has not been reported in an appropriate animal model. We have established a single-fraction partial-body irradiation model with minimal marrow sparing in non-human primates. Subject-based medical management was used including parenteral fluids according to prospective morbidity criteria. We show herein that 10 or 11 Gy exposures caused both acute and chronic kidney injury. Acute and chronic kidney injury appear to be dose-independent between 10 and 11 Gy. Acute kidney injury was identified during the first 50 days postirradiation and appeared to resolve before the occurrence of chronic kidney injury, which was progressively more severe up to 180 days postirradiation, which was the end of the study. These findings show that mitigation of the acute radiation syndrome by medical management will unmask delayed late effects that occur months after partial-body irradiation. They further emphasize that both acute and chronic changes in kidney function must be taken into account in the use and timing of mitigators and medical management for acute radiation syndrome and delayed effects of acute radiation exposure (DEARE).

Accurate analyses of the delayed effects of acute radiation exposure in survivors of the hematopoietic acute radiation syndrome 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 hematopoietic acute radiation syndrome studies conducted over a 7-y 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 delayed effects of acute radiation exposure at various times up to 30 mo of age. Significant loss of long-term repopulating potential of phenotypically defined primitive hematopoietic stem cells was documented in hematopoietic acute radiation syndrome 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 (KSL CD150+; the phenotype known to be enriched for hematopoietic stem cells), and increased cycling of KSL CD150+ 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 hematopoietic stem cells in hematopoietic acute radiation syndrome survivors to be the same as that in nonirradiated age-matched controls. This compilation study adds rigor and validity to our initial findings of persistent hematopoietic dysfunction in hematopoietic acute radiation syndrome survivors that arises at the level of the hematopoietic stem cell and which affects all classes of hematopoietic cells for the life of the survivor.

A nonhuman primate model of acute, partial-body, high-dose irradiation with minimal (2.5%) bone marrow sparing was used to assess endogenous gastrointestinal and hematopoietic recovery and the ability of Neulasta (pegylated granulocyte colony-stimulating factor) or Neupogen (granulocyte colony-stimulating factor) to enhance recovery from myelosuppression when administered at an increased interval between exposure and initiation of treatment. A secondary objective was to assess the effect of Neulasta or Neupogen on mortality and morbidity due to the hematopoietic acute radiation syndrome and concomitant gastrointestinal acute radiation syndrome. Nonhuman primates were exposed to 10.0 Gy, 6 MV, linear accelerator-derived photons delivered at 0.80 Gy min. All nonhuman primates received subject-based medical management. Nonhuman primates were dosed daily with control article (5% dextrose in water), initiated on day 1 postexposure; Neulasta (300 μg kg), administered on days 1, 8, and 15 or days 3, 10, and 17 postexposure; or Neupogen (10 μg kg), administered daily postexposure following its initiation on day 1 or day 3 until neutrophil recovery (absolute neutrophil count ≥1,000 cells μL for 3 consecutive days). Mortality in the irradiated cohorts suggested that administration of Neulasta or Neupogen on either schedule did not affect mortality due to gastrointestinal acute radiation syndrome or mitigate mortality due to hematopoietic acute radiation syndrome (plus gastrointestinal damage). Following 10.0 Gy partial-body irradiation with 2.5% bone marrow sparing, the mean duration of neutropenia (absolute neutrophil count

Acute and chronic kidney injury may occur after accidental prompt radiation exposures. We have modeled their occurrence in a nonhuman primate model. Subjects who are exposed to more than 5-Gy prompt irradiation are apt to show blood cell cytopenias and be treated with granulocyte colony-stimulating factors such as Neupogen® or Neulasta® to mitigate the hematologic injury of the acute radiation syndrome. Neupogen or Neulasta are now approved by the US Food and Drug Administration for this indication. This will significantly increase the number of survivors of acute radiation exposures who will be at risk for delayed effects of radiation exposure, which includes acute and chronic kidney injury. The primary objectives of the present two companion manuscripts were to assess natural history of delayed radiation-induced renal injury in a nonhuman primate model of acute, high-dose, partial-body irradiation with 5% bone marrow sparing to include the clinical and histopathological evidence and the effect of Neupogen administration on morbidity and mortality. In this study, 88 nonhuman primates underwent 10- or 11-Gy partial-body irradiation with 5% bone marrow sparing, of which 36 were treated with Neupogen within 1, 3, or 5 d postirradiation. All animals were followed up to 180 d after irradiation. Renal function and histology end points showed early acute and later chronic kidney injury. These end points were not affected by use of Neupogen. We conclude that use of Neupogen to mitigate against the hematopoietic acute radiation syndrome has no impact on acute or chronic kidney injury.

Chronic respiratory diseases are among the leading causes of deaths worldwide and major contributors of morbidity and global disease burden. To appropriately investigate lung disease, the respiratory airways must be fixed in their physiological orientation and should be inflated prior to investigations. We present an inflation-fixation method that enables lipidomic investigations of whole lung samples and resected biopsy specimens by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Formalin-inflation enables sample preparation to parallel standard clinical and surgical procedures, in addition to greatly reducing the complexity of analysis, by decreasing the number of analytes in the MALDI plume and reducing adduct formation in the resulting mass spectra. The reduced complexity increased sensitivity and enabled high-resolution imaging acquisitions without any loss in analyte detection at 10 and 20 μm scans. We present a detailed study of over 100 lipid ions detected in positive and negative ion modes covering the conducting and respiratory airways and parts of the peripheral nervous tissue running through the lungs. By defining the resolution required for clear definition of the alveolar space and thus the respiratory airways we have provided a guideline for MSI investigations of respiratory diseases involving the airways, including the interstitium. This study has provided a detailed map of lipid species and their localization within larger mammalian lung samples, for the first time, thus categorizing the lipidome for future MALDI-MSI studies of pulmonary diseases.

A nonhuman primate (NHP) model of acute high-dose, partial-body irradiation with 5% bone marrow (PBI/BM5) sparing was used to assess the effect of Neupogen(®) (granulocyte colony stimulating factor [G-CSF]) to mitigate the associated myelosuppression when administered at an increasing interval between exposure and initiation of treatment. A secondary objective was to assess the effect of Neupogen(®) on the mortality or morbidity of the hematopoietic (H)- acute radiation syndrome (ARS) and concurrent acute gastrointestinal radiation syndrome (GI-ARS). NHP were exposed to 10.0 or 11.0 Gy with 6 MV LINAC-derived photons at approximately 0.80 Gy min(−1). All NHP received medical management. NHP were dosed daily with control article (5% dextrose in water) initiated on day 1 post-exposure or Neupogen(®) (10 μg kg(−1)) initiated on day 1, day 3, or day 5 until recovery (absolute neutrophil count [ANC] ≥ 1,000 cells μL(−1) for 3 consecutive days). MORTALITY DUE TO GI- AND H-ARS: Mortality in both the 10.0 Gy and 11.0 Gy cohorts suggested that early administration of Neupogen(®) at day 1 post exposure may affect acute GI-ARS mortality, while Neupogen(®) appeared to mitigate mortality due to the H-ARS. However, the study was not powered to detect statistically significant differences in survival. NEUTROPHIL RECOVERY: The ability of Neupogen(®) to stimulate granulopoiesis was assessed by evaluating key parameters for ANC recovery: the depth of nadir, duration of neutropenia (ANC < 500 cells μL(−1)) and recovery time to ANC ≥ 1,000 cells μL(−1). Following 10.0 Gy PBI/BM5, the mean duration of neutropenia was 11.6 days in the control cohort, versus 3.5 days and 4.6 days in the day 1 and day 3 Neupogen(®) cohorts, respectively. The respective ANC nadirs were 94 cells μL(−1), 220 cells μL(−1), and 243 cells μL(−1) for the control and day 1 and day 3 Neupogen(®) cohorts. Following 11.0 Gy PBI/BM5, the duration of neutropenia was 10.9 days in the control cohort, versus 2.8 days, 3.8 days, and 4.5 days in the day 1, day 3, and day 5 Neupogen(®) cohorts, respectively. The respective ANC nadirs for the control, and day 1, day 3, and day 5 Neupogen(®) cohorts were 131 cells μL(−1), 292 cells μL(−1), 236 cells μL(−1), and 217 cells μL(−1). Therefore, the acceleration of granulopoiesis by Neupogen(®) in this model is independent of the time interval between radiation exposure and treatment initiation up to 5 days post-exposure. The PBI/BM5 model can be used to assess medical countermeasure efficacy in the context of the concurrent GI- and H-ARS.

Radiation-induced lung injury is highly complex and characterized by multiple pathologies, which occur over time, and sporadically throughout the lung. This complexity makes biomarker investigations and medical countermeasure screenings challenging. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has the ability to spatially resolve differences in molecular profiles within the lung following radiation exposure and can aid in biomarker identification and pharmaceutical efficacy investigations. MALDI-MSI was applied to the investigation of a whole-thorax lung irradiation model in non-human primates (NHP) for lipidomic analysis and medical countermeasure distribution.

Long term or residual damage post-irradiation has been described for many tissues. In hematopoietic stem cells (HSC) this is only revealed when the HSC are stressed and required to regenerate and repopulate a myeloablated host. Such an assay cannot be used to assess the recovery potential of previously irradiated intestinal stem cells (ISC) due to their incompatibility with transplantation. The best approximation to the HSC assay is the crypt microcolony assay, also based on clonogen survival. In the current study the regenerative capacity of intestinal clonogenic cells in mice that had survived 13 Gy irradiation (with 5% bone marrow shielding to allow survival through the hematopoietic syndrome) and were then aged for 200 days, was compared to previously unirradiated age matched controls. Interestingly, at 200 days following 13 Gy there remained a statistically significant reduction in crypts present in the various small intestinal regions (illustrating that the gastrointestinal epithelium had not fully recovered despite the 200 day interval). However, upon re-irradiation on day 196, those mice previously irradiated had improved crypt survival and regeneration compared to the age matched controls. This was evident in all regions of the small intestine following 11–13 Gy re-exposure. Thus, there were either more clonogens per crypt within those previously irradiated and/or those that were present were more radioresistant (possibly because a subpopulation was more quiescent). This is contrary to the popular belief that previously irradiated animals may have an impaired/delayed regenerative response and be more radiosensitive.

The use of plasma citrulline as a biomarker for gastrointestinal acute radiation syndrome via exposure to total-body irradiation in a murine model was investigated. The radiation exposure covered lethal, mid-lethal, and sub-lethal gastrointestinal acute radiation syndrome. Plasma citrulline profiles were generated over the first 6 days following total-body irradiation exposure of 6–15 Gy. In addition, plasma citrulline was comprehensively evaluated in the context of matching small intestine citrulline and histopathology. Higher plasma citrulline was significantly associated with lower irradiation doses over the first 6 days following the irradiation insult. Furthermore, higher plasma citrulline was significantly associated with higher crypt survival. The correlation of the plasma citrulline to crypt survival was more robust for higher irradiation doses and for later time points. The data suggested plasma citrulline was most informative for reflecting gastrointestinal injury resulting from exposure to 9–15 Gy total-body irradiation covering time-points 2 to 5 days post the irradiation insult.

The use of plasma citrulline as a biomarker for acute and prolonged gastrointestinal injury via exposure to total- and partial-body irradiation (6 MV LINAC-derived photons; 0.80 Gy min−1) in nonhuman primate models was investigated. The irradiation exposure covered gastrointestinal injuries spanning lethal, mid-lethal, and sub-lethal doses. The acute gastrointestinal injury was assessed via measurement of plasma citrulline and small intestinal histopathology over the first 15 days following radiation exposure and included total-body irradiation at 13.0 Gy, 10.5 Gy, and 7.5 Gy and partial-body irradiation at 11.0 Gy with 5% bone marrow sparing. The dosing schemes of 7.5 Gy total-body irradiation and 11.0 Gy partial-body irradiation included time points out to day 60 and day 180, respectively, which allowed for correlation of plasma citrulline to prolonged gastrointestinal injury and survival. Plasma citrulline values were radiation-dependent for all radiation doses under consideration with nadir values ranging from 63–80 % lower than radiation-naïve NHP plasma. The nadir values were observed at day 5 to 7 post irradiation. Longitudinal plasma citrulline profiles demonstrated prolonged gastrointestinal injury resulting from acute high-dose irradiation had long lasting effects on enterocyte function. Moreover, plasma citrulline did not discriminate between total-body or partial-body irradiation over the first 15 days following irradiation and was not predictive of survival based on the radiation models considered herein.

Recovery from severe immunosuppression requires hematopoietic stem cell reconstitution and effective thymopoiesis to restore a functional immune cell repertoire. Herein, a model of immune cell reconstitution consequent to potentially lethal doses of irradiation is described which may be valuable in evaluating potential medical countermeasures. Male rhesus macaques were total body irradiated by exposure to 6.00 Gy 250 kVp x-radiation (midline tissue dose, 0.13 Gy min-1) resulting in an approximate LD10/60 (n = 5/59). Animals received medical management and hematopoietic and immune cell recovery was assessed (n ≤ 14) through 370 days post exposure. A subset of animals (n ≤ 8) was examined through 700 days. Myeloid recovery was assessed by neutrophil and platelet-related parameters. Lymphoid recovery was assessed by the absolute lymphocyte count and FACS-based phenotyping of B- and T-cell subsets. Recent thymic emigrants were identified by T cell receptor excision circle quantification. Severe neutropenia, lymphopenia and thrombocytopenia resolved within 30 days. Total CD3+ cells μL-1 required 60 days to reach values 60% of normal, followed by subsequent slow recovery to approximately normal by 180 days post irradiation. Recovery of CD3+4+ and CD3+8+ cell memory and naïve subsets were markedly different. Memory populations were ≥ 100% of normal by day 60; whereas, naïve populations were only 57% normal at 180 days and never fully recovered to baseline post irradiation. Total (CD20+) B cells μL-1 were within normal levels by 77 days post exposure. This animal model elucidates the variable T- and B-cell subset recovery kinetics after a potentially lethal dose of total-body irradiation that are dependent on marrow-derived stem and progenitor cell recovery, peripheral homeostatic expansion and thymopoiesis.

Computed Tomography (CT) and Echocardiography (EC) are two imaging modalities that produce critical longitudinal data that can be analyzed for radiation-induced organ-specific injury to the lung and heart. The Medical Countermeasures Against Radiological Threats (MCART) consortium has a well established animal model research platform that includes nonhuman primate (NHP) models of the acute radiation syndrome and the delayed effects of acute radiation exposure. These models call for a definition of the latency, incidence, severity, duration, and resolution of different organ-specific radiation-induced subsyndromes. The pulmonary subsyndromes and cardiac effects are a pair of interdependent syndromes impacted by exposure to potentially lethal doses of radiation. Establishing a connection between these will reveal important information about their interaction and progression of injury and recovery. Herein, the authors demonstrate the use of CT and EC data in the rhesus macaque models to define delayed organ injury, thereby establishing: a) consistent and reliable methodology to assess radiation-induced damage to the lung and heart; b) an extensive database in normal age-matched NHP for key primary and secondary endpoints; c) identified problematic variables in imaging techniques and proposed solutions to maintain data integrity; and d) initiated longitudinal analysis of potentially lethal radiation-induced damage to the lung and heart.

Exposure to sufficiently high doses of ionizing radiation is known to cause fibrosis in many different organs and tissues. Connective tissue growth factor (CTGF/CCN2), a member of the CCN family of matricellular proteins, plays an important role in the development of fibrosis in multiple organs. The aim of the present study was to quantify the gene and protein expression of CTGF in a variety of organs from non-human primates (NHP) that were previously exposed to potentially lethal doses of radiation. Tissues from non-irradiated NHP and NHP exposed to whole thoracic lung irradiation (WTLI) or partial-body irradiation with 5% bone marrow sparing (PBI/BM5) were examined by real-time quantitative reverse transcription PCR, western blot, and immunohistochemistry. Expression of CTGF was elevated in the lung tissues of NHP exposed to WTLI relative to the lung tissues of the non-irradiated NHP. Increased expression of CTGF was also observed in multiple organs from NHP exposed to PBI/BM5 compared to non-irradiated NHP; these included the lung, kidney, spleen, thymus, and liver. These irradiated organs also exhibited histological evidence of increased collagen deposition compared to the control tissues. There was significant correlation of CTGF expression with collagen deposition in the lung and spleen of NHP exposed to PBI/BM5. Significant correlations were observed between spleen and multiple organs on CTGF expression and collagen deposition, respectively, suggesting possible crosstalk between spleen and other organs. These data suggest that CTGF levels are increased in multiple organs after radiation exposure and that inflammatory cell infiltration may contribute to the elevated levels of CTGF in multiple organs.

Purpose Radiation-induced pulmonary fibrosis (RIPF) is a severe and life-threatening complication of radiation therapy in patients with thoracic cancer; however, the exact molecular mechanisms remain unknown, and there is no effective treatment method in clinic. Here, we assessed the role of follistatin-like 1 (Fstl1) in RIPF. Methods and Materials Protein and messenger RNA levels of Fstl1 in lung tissues from symptomatic RIPF patients, Rhesus macaques, and mice were assessed. Fibrotic and inflammatory responses to radiation-induced lung injury and accumulation of myofibroblasts in Fstl1 haplodeficient (Fstl1+/–) mice were determined. Finally, radiation-induced differentiation and activation of fibroblasts in primary Fstl1+/– lung fibroblasts were evaluated. Results FSTL1 amounts were significantly increased in serum and/or radiation-injured lung specimens from symptomatic RIPF patients, Rhesus macaques, and mice. Haplodeletion of Fstl1 in Fstl1+/– mice was protective against x-ray–induced lung injury in mice in vivo, as well as myofibroblast activation in vitro. Conclusions These findings suggest that Fstl1 plays an important role in lung fibrosis and may offer a potential approach to attenuate RIPF in radiation therapy of patients with thoracic cancer.

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 24hr 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 effected 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 9mo 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.

The dose response relationship for the acute gastrointestinal syndrome following total-body irradiation prevents analysis of the full recovery and damage to the gastrointestinal system, since all animals succumb to the subsequent 100% lethal hematopoietic syndrome. A partial-body irradiation model with 5% bone marrow sparing was established to investigate the prolonged effects of high-dose radiation on the gastrointestinal system, as well as the concomitant hematopoietic syndrome and other multi-organ injury including the lung. Herein, cellular and clinical parameters link acute and delayed coincident sequelae to radiation dose and time course post-exposure. Male rhesus Macaca mulatta were exposed to partial-body irradiation with 5% bone marrow (tibiae, ankles, feet) sparing using 6 MV linear accelerator photons at a dose rate of 0.80 Gy min(-1) to midline tissue (thorax) doses in the exposure range of 9.0 to 12.5 Gy. Following irradiation, all animals were monitored for multiple organ-specific parameters for 180 d. Animals were administered medical management including administration of intravenous fluids, antiemetics, prophylactic antibiotics, blood transfusions, antidiarrheals, supplemental nutrition, and analgesics. The primary endpoint was survival at 15, 60, or 180 d post-exposure. Secondary endpoints included evaluation of dehydration, diarrhea, hematologic parameters, respiratory distress, histology of small and large intestine, lung radiographs, and mean survival time of decedents. Dose- and time-dependent mortality defined several organ-specific sequelae, with LD50/15 of 11.95 Gy, LD50/60 of 11.01 Gy, and LD50/180 of 9.73 Gy for respective acute gastrointestinal, combined hematopoietic and gastrointestinal, and multi-organ delayed injury to include the lung. This model allows analysis of concomitant multi-organ sequelae, thus providing a link between acute and delayed radiation effects. Specific and multi-organ medical countermeasures can be assessed for efficacy and interaction during the concomitant evolution of acute and delayed key organ-specific subsyndromes.

The acute effects of irradiation on the gastrointestinal (GI) system are well documented but the longer-term effects are less well known. Increased incidence of adenocarcinoma has been noted but apart from descriptions of fibrosis, the development of other pathologies specific to survivors of acute radiation is poorly understood. Samples were taken from C57BL/6 mice irradiated with partial-body irradiation where the thorax, head and forelimbs were shielded (i.e. sparing 40% of the bone marrow). Tissue from age matched controls was also collected. There were clear pathological changes in the intestine associated with DEARE (Delayed Effects of Acute Radiation Exposure) at doses greater than 12 Gy, with a dose related increase in observed pathologies. Mice maintained on the synthetic antibiotic ciprofloxacin during the acute phase (days four to twenty), however, had a lower or delayed incidence of symptoms. After twenty days mice developed structures similar to early adenomas. Abnormally high levels of apoptotic and mitotic cells were present in some crypts, along with the early adenomas, suggesting tissue regeneration and areas of deregulated cell turnover. Over time, in animals with advanced symptoms there was inhibited crypt cell proliferation, a blunting of the crypts and villi and an enlargement of villus girth, with an increasingly acellular and fibrotic extracellular matrix (a characteristic that has been previously demonstrated in aging mice). Together these changes may lead to a reduced functional surface area and less motile intestine. These observations are similar to those seen in geriatric animals, suggesting a premature aging of the GI tract.

The development of medical countermeasures against the acute gastrointestinal subsyndrome of the acute radiation syndrome in humans requires well characterized and validated animal models. These models must adhere to the criteria of the U.S. Food and Drug Administration's Animal Rule and consider the natural history and clinical context of the human radiation response and treatment in the nuclear terrorist scenario. The models must define the radiation dose- and time-dependent relationships for mortality and major signs of morbidity, including concurrent damage in other organs, such as the bone marrow, that may contribute to the overall mortality and morbidity. There are no such models of the gastrointestinal syndrome in response to total-body irradiation in the nonhuman primate. Herein, these parameters are defined for the rhesus macaque exposed to potentially lethal doses of radiation and administered medical management. Rhesus macaques (n = 69) were exposed bilaterally to 6 MV linear accelerator-derived photon total body irradiation to midline tissue (thorax) doses ranging from 10.0 to 14.0 Gy at 0.80 Gy min(-1). Following irradiation, all animals were administered supportive care consisting of fluids, anti-emetics, anti-diarrheal medication, antibiotics, blood transfusions, analgesics, and nutrition. The primary endpoint was survival at 15 d post-irradiation. Secondary endpoints included indices of dehydration, diarrhea, weight loss, hematological parameters, cellular histology of the small and large intestine, and mean survival time of decedents. Mortality within the 15-d in vivo study defined the acute gastrointestinal syndrome and provided an LD30/15 of 10.76 Gy, LD50/15 of 11.33 Gy, and an LD70/15 of 11.90 Gy. Intestinal crypt and villus loss were dose- and time-dependent with an apparent nadir 7 d post-irradiation and recovery noted thereafter. Severe myelosuppression and thrombocytopenia were noted in all animals, requiring the administration of antibiotics and blood transfusions. The model defines the dose response relationship and time course of acute gastrointestinal syndrome-induced morbidity and mortality in the rhesus macaque.

In this study, nonhuman primates (NHPs) exposed to lethal doses of total body irradiation (TBI) within the gastrointestinal (GI) acute radiation syndrome range, sparing ∼5% of bone marrow (TBI-BM5), were used to evaluate the mechanisms involved in development of the chronic GI syndrome. TBI increased mucosal permeability in the jejunum (12-14 Gy) and proximal colon (13-14 Gy). TBI-BM5 also impaired mucosal barrier function at doses ranging from 10-12.5 Gy in both small intestine and colon. Timed necropsies of NHPs at 6-180 days after 10 Gy TBI-BM5 showed that changes in small intestine preceded those in the colon. Chronic GI syndrome in NHPs is characterized by continued weight loss and intermittent GI syndrome symptoms. There was a long-lasting decrease in jejunal glucose absorption coincident with reduced expression of the sodium-linked glucose transporter. The small intestine and colon showed a modest upregulation of several different pro-inflammatory mediators such as NOS-2. The persistent inflammation in the post-TBI-BM5 period was associated with a long-lasting impairment of mucosal restitution and a reduced expression of intestinal and serum levels of alkaline phosphatase (ALP). Mucosal healing in the postirradiation period is dependent on sparing of stem cell crypts and maturation of crypt cells into appropriate phenotypes. At 30 days after 10 Gy TBI-BM5, there was a significant downregulation in the gene and protein expression of the stem cell marker Lgr5 but no change in the gene expression of enterocyte or enteroendocrine lineage markers. These data indicate that even a threshold dose of 10 Gy TBI-BM5 induces a persistent impairment of both mucosal barrier function and restitution in the GI tract and that ALP may serve as a biomarker for these events. These findings have important therapeutic implications for the design of medical countermeasures.

Cachexia, or muscle wasting, is a serious health threat to victims of radiological accidents or patients receiving radiotherapy. Here, we propose a non-human primate (NHP) radiation-induced cachexia model based on clinical and molecular pathology findings. NHP exposed to potentially lethal partial-body irradiation developed symptoms of cachexia such as body weight loss in a time- and dose-dependent manner. Severe body weight loss as high as 20–25% was observed which was refractory to nutritional intervention. Radiographic imaging indicated that cachectic NHP lost as much as 50% of skeletal muscle. Histological analysis of muscle tissues showed abnormalities such as presence of central nuclei, inflammation, fatty replacement of skeletal muscle and muscle fiber degeneration. Biochemical parameters such as hemoglobin and albumin levels decreased after radiation exposure. Levels of FBXO32 (Atrogin-1), ActRIIB and myostatin were significantly changed in the irradiated cachectic NHP compared to the non-irradiated NHP. Our data suggest NHP that have been exposed to high dose radiation manifest cachexia-like symptoms in a time- and dose-dependent manner. This model provides a unique opportunity to study the mechanism of radiation-induced cachexia and will aid in efficacy studies of mitigators of this disease.

Multi-organ dose evaluations and the effects of heterogeneous tissue dose calculations have been retrospectively evaluated following irradiation to the whole thorax and lung in non-human primates (NHP). A clinical-based approach was established to evaluate actual doses received in the heart and lungs during whole thorax lung irradiation. Anatomical structure and organ densities have been introduced in the calculations to show the effects of dose distribution through heterogeneous tissue. Mean organ doses received by non-human primates undergoing whole thorax lung irradiations were calculated using a treatment planning system that is routinely used in clinical radiation oncology. The doses received by non-human primates irradiated following conventional dose calculations have been retrospectively reconstructed using computerized tomography-based, heterogeneity-corrected dose calculations. The use of dose volume descriptors for irradiation to organs at risk and tissue exposed to radiation is introduced. Mean and partial-volume doses to lung and heart are presented and contrasted. The importance of exact dose definitions is highlighted, and the relevance of precise dosimetry to establish organ-specific dose response relationships in NHP models of acute and delayed effects of acute radiation exposure is emphasized.

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.

Dendritic cells (DC) orchestrate immune responses under direction of cytokines/chemokines in their microenvironment. To investigate the influence of that generated during T cell activation, we stimulated peripheral blood mononuclear cells (PBMC) with anti-CD3 and anti-CD28 coated beads and tested cell-free culture supernatants (lymphocyte conditioned medium, LCM) for cytokine/chemokine composition and biologic activity. LCM contained a battery of mediators important in the biology of myeloid (mDC) and plasmacytoid (pDC) DC. LCM differentiated monocytes into functional immature mDC, and induced maturation of immature mDC. LCM also augmented maturation and IFNalpha-production of CpG-treated pDC. Functional activity of LCM-derived DC was confirmed by their ability to enhance in vitro recall T cell responses and substantially augment in vivo cellular and humoral immune responses to various vaccines in non-human primates. These results demonstrate that products of anti-CD3/anti-CD28 stimulated PBMC generate biologically active DC in vitro and function as a vaccine adjuvant in vivo.

The objective of this study was to re-evaluate the previously published hematopoietic stem cell (HSC) expansion work using human brain endothelial cells (HUBEC). The expansion effect of contact and non-contact conditions was reported to be equivalent by others. However, we report here different results that the expansion can be achieved only with direct contact. We co-cultured human CD34+ cells with and without HUBEC contact for seven days with cytokines and the readouts were CD34+ / CD38 - phenotype and SCID repopulating cell (SRC) frequency. Also tested was the inhibitory effect of Wnt receptor inhibitor Dkk-1 on HUBEC contact ex vivo expansion; whether an increased expression of Wnt3 occurs on the HUBEC surface; and detection of an increased nuclear localization of beta-catenin in CD34+ / CD38- cells in HUBEC contact culture condition. We conclude that the successful expansion by HUBEC contact culture is a candidate explanation based on the Wnt family protein, possibly Wnt3, expression on HUBEC.

We engineered a 21-mer peptide representing amino acids 170–190 of the respiratory syncytial virus (RSV) G protein as a fusion with the Alfalfa mosaic virus (AlMV) coat protein (CP), produced recombinant AlMV particles presenting this peptide (VMR-RSV) on their surfaces and tested the immunogenicity in vitro in human dendritic cells and in vivo in non-human primates. Significant pathogen-specific immune responses were generated in both systems: (i) human dendritic cells armed with VMR-RSV generated vigorous CD4+ and CD8+ T cell responses; (ii) non-human primates that received these particles responded by mounting strong cellular and humoral immune responses. This approach may validate the use of a novel RSV vaccine delivery vehicle in humans.

Mathematical modeling of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) pharmacokinetics (PK) and pharmacodynamics (PD) suggest that variations in the PEG-rHuMGDF treatment schedule could reduce the severity and duration of thrombocytopenia following myeloablation and bone marrow transplant (BMT). We tested this hypothesis in a rhesus monkey model of autologous (Au) bone marrow-derived mononuclear cell (BM-MNC) transplantation following lethal myeloablation. On day 0, animals were myeloablated by total body exposure to 920 cGy, 250 kVp x-irradiation (TBI). Four cohorts of animals were infused with 1 x 10(8) AuBM-MNC/kg body weight within 2 hours of TBI. The AuBMT-alone cohort received no cytokine, the daily dosage cohort received PEG-rHuMGDF (2.5 micro g/kg/day, s.c.) post TBI and AuBMT, and the pre/post-transplant cohort received PEG-rHuMGDF (2.5 micro g/kg/day, s.c.) pre (day -9 to day -5) and post TBI and AuBMT. The post-transplant PEG-rHuMGDF administration in the above cohorts was begun on day 1 post TBI and continued until platelet counts reached 200,000 micro l (range, 15-31 days). Another group received PEG-rHuMGDF (300 micro g/kg/day, s.c.) on days 1 and 3 only following TBI and AuBMT. The TBI controls received neither AuBMT nor cytokine therapy. In this model of AuBMT, with regard to the PEG-rHuMGDF administration schedule, the daily dosage of the post-transplant cohort did not significantly improve platelet recovery; the pre/post-transplant schedule and an abbreviated high-dosage, post-transplant schedule (days 1 and 3) significantly improved the duration and nadir of thrombocytopenia and platelet recovery. These data confirm predictions from PK/PD modeling of PEG-rHuMGDF that thrombocytopenia is preventable following AuBMT.

Promegapoietin-1a (PMP-1a), a multifunctional agonist for the human interleukin 3 and Mpl receptors, was evaluated for its ability to stimulate hematopoietic reconstitution in nonhuman primates following severe radiation-induced myelosuppression. Animals were total body x-irradiated (250 kVp) to 600 cGy total midline tissue dose. PMP-1a was administered s.c. in several protocols: A) daily (50 microg/kg) for 18 days; B) nine doses (5 microg/kg) every other day for 3 weeks; C) a single high dose (100 microg/kg) at 20 hours, or D) a single high dose (100 microg/kg) at 1 hour following TBI. The irradiation controls received 0.1% autologous serum for 18 consecutive days. Hematopoietic recovery was assessed by bone marrow clonogenic activity, peripheral blood cell nadirs, duration of cytopenias, time to recovery to cellular thresholds, and requirements for clinical support. PMP-1a, irrespective of administration schedule, significantly improved all platelet-related parameters: thrombocytopenia was eliminated, the severity of platelet nadirs was significantly improved, and recovery of platelet counts toor =20,000/miccrol was significantly reduced in all PMP-1a-treated cohorts. As a consequence, all PMP-1a-treated cohorts were transfusion-independent. Neutrophil regeneration was augmented in all treatment schedules. Additionally, all PMP-1a-treated cohorts showed an improvement in red blood cell nadir and recovery. PMP-1a in conventional or abbreviated schedules induced significant thrombopoietic regeneration relative to the control cohort, whereas significant improvement in neutrophil recovery was schedule-dependent in radiation-myelosuppressed nonhuman primates.

SB-251353 is an N-terminal truncated form of the human CXC chemokine GROβ. Recombinant SB-251353 was profiled in murine and rhesus monkey peripheral blood stem cell mobilization and transplantation models. SB-251353 rapidly and transiently mobilized hematopoietic stem cells and neutrophils into the peripheral blood after a single subcutaneous injection. Transplantation of equivalent numbers of hematopoietic stem cells mobilized by SB-251353 into lethally irradiated mice resulted in faster neutrophil and platelet recovery than stem cells mobilized by granulocyte colony-stimulating factor (G-CSF). A single injection of SB-251353 in combination with 4 days of G-CSF administration resulted in augmented stem and progenitor cell mobilization 5-fold greater than G-CSF alone. Augmented stem cell mobilization could also be demonstrated in mice when a single injection of SB-251353 was administered with only one-day treatment with G-CSF. In addition, SB-251353, when used as a single agent or in combination with G-CSF, mobilized long-term repopulating stem cells capable of hematopoietic reconstitution of lethally irradiated mice. In rhesus monkeys, a single injection of SB-251353 induced rapid increases in peripheral blood hematopoietic progenitor cells at a 50-fold lower dose than in mice, which indicates a shift in potency. These studies provide evidence that the use of SB-251353 alone or in combination with G-CSF mobilizes hematopoietic stem cells with long-term repopulating ability. In addition, this treatment may (1) reduce the number of apheresis sessions and/or amount of G-CSF required to collect adequate numbers of hematopoietic stem cells for successful peripheral blood cell transplantation and (2) improve hematopoietic recovery after transplantation.