The Natural History of Acute Radiation-induced H-ARS and Concomitant Multi-organ Injury in the Non-human Primate: The MCART Experience.

ABSTRACT: 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.

Acute Proteomic Changes in Non-human Primate Kidney after Partial-body Radiation with Minimal Bone Marrow Sparing.

ABSTRACT: Near total body exposure to high-dose ionizing radiation results in organ-specific sequelae, including acute radiation syndromes and delayed effects of acute radiation exposure. Among these sequelae are acute kidney injury and chronic kidney injury. Reports that neither oxidative stress nor inflammation are dominant mechanisms defining radiation nephropathy inspired an unbiased, discovery-based proteomic interrogation in order to identify mechanistic pathways of injury. We quantitatively profiled the proteome of kidney from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Kidney was analyzed by liquid chromatography-tandem mass spectrometry. Out of the 3,432 unique proteins that were identified, we found that 265 proteins showed significant and consistent responses across at least three time points post-irradiation, of which 230 proteins showed strong upregulation while 35 proteins showed downregulation. Bioinformatics analysis revealed significant pathway and upstream regulator perturbations post-high dose irradiation and shed light on underlying mechanisms of radiation damage. These data will be useful for a greater understanding of the molecular mechanisms of injury in well-characterized animal models of partial body irradiation with minimal bone marrow sparing. These data may be potentially useful in the future development of medical countermeasures.

AEOL 10150 Alleviates Radiation-induced Innate Immune Responses in Non-human Primate Lung Tissue.

ABSTRACT: To study the molecular and cellular mechanisms of radiation-induced lung injury (RILI) in a non-human primate model, 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. 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 ß6, ICAM-1) were further studied using real-time 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. Cytokine/chemokine-induced excessive innate immune response after thoracic irradiation plays an important role 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.

Acute Radiation Effects, the H-ARS in the Non-human Primate: A Review and New Data for the Cynomolgus Macaque with Reference to the Rhesus Macaque.

ABSTRACT: 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.

Multi-omic Analysis of Non-human Primate Heart after Partial-body Radiation with Minimal Bone Marrow Sparing.

ABSTRACT: 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.

Acute Proteomic Changes in Lung after Radiation: Toward Identifying Initiating Events of Delayed Effects of Acute Radiation Exposure in Non-human Primate after Partial Body Irradiation with Minimal Bone Marrow Sparing.

ABSTRACT: 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.

Metabolomics of Multiorgan Radiation Injury in Non-human Primate Model Reveals System-wide Metabolic Perturbations.

ABSTRACT: 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.

Complementary Lipidomic, Proteomic, and Mass Spectrometry Imaging Approach to the Characterization of the Acute Effects of Radiation in the Non-human Primate Mesenteric Lymph Node after Partial-body Irradiation with Minimal Bone Marrow Sparing.

ABSTRACT: 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.

Effect of Radiation on the Essential Nutrient Homeostasis and Signaling of Retinoids in a Non-human Primate Model with Minimal Bone Marrow Sparing.

ABSTRACT: 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.

Recent advances in medical countermeasure development against acute radiation exposure based on the US FDA animal rule.

Recent advances in medical countermeasures (MCMs) has been dependent on the Food and Drug Administration (FDA) animal rule (AR) and the final guidance document provided for industry on product development. The criteria outlined therein establish the path for approval under the AR. The guidance document, along with the funding and requirements from the federal agencies provided the basic considerations for animal model development in assessing radiation effects and efficacy against the potential lethal effects of acute radiation injury and the delayed effects of acute exposure. Animal models, essential for determining MCM efficacy, were developed and validated to assess organ-specific, potentially lethal, radiation effects against the gastrointestinal (GI) and hematopoietic acute radiation syndrome (H-ARS), and radiation-induced delayed effects to lung and associated comorbidities of prolonged immune suppression, GI, kidney and heart injury. Partial-body irradiation models where marginal bone marrow was spared resulted in the ability to evaluate the concomitant evolution of multiple organ injury in the acute and delayed effects in survivors of acute radiation exposure. There are no MCMs for prophylaxis against the major sequelae of the ARS or the delayed effects of acute exposure. Also lacking are MCMs that will mitigate the GI ARS consequent to potentially lethal exposure from a terrorist event or major radiation accident. Additionally, the gap in countermeasures for prophylaxis may extend to mixed neutron/gamma radiation if current modelling predicts prompt exposure from an improvised nuclear device. However, progress in the field of MCM development has been made due to federal and corporate funding, clarification of the critical criteria for efficacy within the FDA AR and the concomitant development and validation of additional animal models. These models provided for a strategic and tactical approach to determine radiation effects and MCM efficacy.

Acute Radiation-induced Lung Injury in the Non-human Primate: A Review and Comparison of Mortality and Co-morbidities Using Models of Partial-body Irradiation with Marginal Bone Marrow Sparing and Whole Thorax Lung Irradiation.

The nonhuman primate, rhesus macaque, is a relevant animal model that has been used to determine the efficacy of medical countermeasures to mitigate major signs of morbidity and mortality of radiation-induced lung injury. Herein, a literature review of published studies showing the evolution of lethal lung injury characteristic of the delayed effects of acute radiation exposure between the two significantly different exposure protocols, whole thorax lung irradiation and partial-body irradiation with bone marrow sparing in the nonhuman primate, is provided. The selection of published data was made from the open literature. The primary studies conducted at two research sites benefitted from the similarity of major variables; namely, both sites used rhesus macaques of approximate age and body weight and radiation exposure by LINAC-derived 6 MV photons at dose rates of 0.80 Gy min and 1.00 Gy min delivered to the midline tissue via bilateral, anterior/posterior, posterior/anterior geometry. An advantage relative to sex difference resulted from the use of male and female macaques by the Maryland and the Washington sites, respectively. Subject-based medical management was used for all macaques. The primary studies (6) provided adequate data to establish dose response relationships within 180 d for the radiation-induced lung injury consequent to whole thorax lung irradiation (male vs. female) and partial-body irradiation with bone marrow sparing exposure protocols (male). The dose response relationships established by probit analyses vs. linear dose relationships were characterized by two main parameters or dependent variables, a slope and LD50/180. Respective LD50/180 values for the primary studies that used whole thorax lung irradiation for respective male and female nonhuman primates were 10.24 Gy [9.87, 10.52] (n = 76, male) and 10.28 Gy [9.68, 10.92] (n = 40, female) at two different research sites. The respective slopes were steep at 1.73 [0.841, 2.604] and 1.15 [0.65, 1.65] probits per linear dose. The LD50/180 value and slope derived from the dose response relationships for the partial-body irradiation with bone marrow sparing exposure was 9.94 Gy [9.35, 10.29] (n = 87) and 1.21 [0.70, 1.73] probits per linear dose. A secondary study (1) provided data on limited control cohort of nonhuman primates exposed to whole thorax lung irradiation. The data supported the incidence of clinical, radiographic, and histological indices of the dose-dependent lung injury in the nonhuman primates. Tertiary studies (6) provided data derived from collaboration with the noted primary and secondary studies on control cohorts of nonhuman primates exposed to whole thorax lung irradiation and partial-body irradiation with bone marrow sparing exposure. These studies provided a summary of histological evidence of fibrosis, inflammation and reactive/proliferative changes in pneumonocytes characteristic of lung injury and data on biomarkers for radiation-induced lung injury based on matrix-assisted laser desorption ionization-mass spectrometry imaging and gene expression approaches. The available database in young rhesus macaques exposed to whole thorax lung irradiation or partial-body irradiation with bone marrow sparing using 6 MV LINAC-derived radiation with medical management showed that the dose response relationships were equivalent relative to the primary endpoint all-cause mortality. Additionally, the latency, incidence, severity, and progression of the clinical, radiographic, and histological indices of lung injury were comparable. However, the differences between the exposure protocols are remarkable relative to the demonstrated time course between the multiple organ injury of the acute radiation syndrome and that of the delayed effects of acute radiation exposure, respectively.

A Systematic Review of the Hematopoietic Acute Radiation Syndrome (H-ARS) in Canines and Non-human Primates: Acute Mixed Neutron/Gamma vs. Reference Quality Radiations.

A systematic review of relevant studies that determined the dose response relationship (DRR) for the hematopoietic (H) acute radiation syndrome (ARS) in the canine relative to radiation quality of mixed neutron:gamma radiations, dose rate, and exposure uniformity relative to selected reference radiation exposure has not been performed. The datasets for rhesus macaques exposure to mixed neutron:gamma radiation are used herein as a species comparative reference to the canine database. The selection of data cohorts was made from the following sources: Ovid Medline (1957-present), PubMed (1954-present), AGRICOLA (1976-present), Web of Science (1954-present), and US HHS RePORT (2002-present). The total number of hits across all search sites was 3,077. Several referenced, unpublished, non-peer reviewed government reports were unavailable for review. Primary published studies using canines, beagles, and mongrels were evaluated to provide an informative and consistent review of mixed neutron:gamma radiation effects to establish the DRRs for the H-ARS. Secondary and tertiary studies provided additional information on the hematologic response or the effects on hematopoietic progenitor cells, radiation dosimetry, absorbed dose, and organ dose. The LD50/30 values varied with neutron quality, exposure aspect, and mixed neutron:gamma ratio. The reference radiation quality varied from 250 kVp or 1-2 MeV x radiation and Co gamma radiation. A summary of a published review of a data set describing the DRR in rhesus macaques for mixed neutron:gamma radiation exposure in the H-ARS is included for a comparative reference to the canine dataset. The available evidence provided a reliable and extensive database that characterized the DRR for the H-ARS in canines and young rhesus macaques exposed to mixed neutron:gamma radiations of variable energy relative to 250 kVp, 1-2 MeV x radiation and Co gamma, and uniform and non-uniform total-body irradiation without the benefit of medical management. The mixed neutron:gamma radiation showed an energy-dependent RBE of ~ 1.0 to 2.0 relative to reference radiation exposure within both species. A marginal database described the DRR for the gastrointestinal (GI)-ARS. Medical management showed benefit in both species relative to the mixed neutron:gamma as well as exposure to reference radiation. The DRR for the H-ARS was characterized by steep slopes and relative LD50/30 values that reflected the radiation quality, exposure aspect, and dose rate over a range in time from 1956-2012.

Evaluation of Plasma Biomarker Utility for the Gastrointestinal Acute Radiation Syndrome in Non-human Primates after Partial Body Irradiation with Minimal Bone Marrow Sparing through Correlation with Tissue and Histological Analyses.

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 primates 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 d 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.

Proteomics of Non-human Primate Plasma after Partial-body Radiation with Minimal Bone Marrow Sparing.

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 over a time period of 3 wk. 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.

Proteomic Evaluation of the Natural History of the Acute Radiation Syndrome of the Gastrointestinal Tract in a Non-human Primate Model of Partial-body Irradiation with Minimal Bone Marrow Sparing Includes Dysregulation of the Retinoid Pathway.

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.

Lack of Cellular Inflammation in a Non-human Primate Model of Radiation Nephropathy.

Inflammation is commonly cited as a mechanism of delayed effects of acute radiation exposure (DEARE). Confirmation of its presence could provide significant insight to targeted use of treatments or mitigators of DEARE. We sought to quantify the presence of cellular inflammation in kidneys of non-human primates that developed acute and chronic kidney injury after a partial body irradiation exposure. We show herein that cellular inflammation is not found as a component of either acute or chronic kidney injury. Other mechanistic pathways of injury must be sought.

MALDI-MSI spatially maps N-glycan alterations to histologically distinct pulmonary pathologies following irradiation.

Radiation-induced lung injury is a highly complex combination of pathological alterations that develop over time and severity of disease development is dose-dependent. Following exposures to lethal doses of irradiation, morbidity and mortality can occur due to a combination of edema, pneumonitis and fibrosis. Protein glycosylation has essential roles in a plethora of biological and immunological processes. Alterations in glycosylation profiles have been detected in diseases ranging from infection, inflammation and cancer. We utilized mass spectrometry imaging to spatially map N-glycans to distinct pathological alterations during the clinically latent period and at 180 days post-exposure to irradiation. Results identified alterations in a number of high mannose, hybrid and complex N-glycans that were localized to regions of mucus and alveolar-bronchiolar hyperplasia, proliferations of type 2 epithelial cells, accumulations of macrophages, edema and fibrosis. The glycosylation profiles indicate most alterations occur prior to the onset of clinical symptoms as a result of pathological manifestations. Alterations in five N-glycans were identified as a function of time post-exposure. Understanding the functional roles N-glycans play in the development of these pathologies, particularly in the accumulation of macrophages and their phenotype, may lead to new therapeutic avenues for the treatment of radiation-induced lung injury.

Characterizing the Natural History of Acute Radiation Syndrome of the Gastrointestinal Tract: Combining High Mass and Spatial Resolution Using MALDI-FTICR-MSI.

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.

A Comparative Dose-response Relationship Between Sexes for Mortality and Morbidity of Radiation-induced Lung Injury in the Rhesus Macaque.

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.