Centers for Medical Countermeasures against Radiation Consortium: Past, Present, and Beyond

Abstract

The Centers for Medical Countermeasures against Radiation Consortium (CMCRC) has provided a strong research foundation for the radiobiology science that will follow. After 20 years, however, the CMCRC will continue to conduct research on preparedness in niche areas of radiobiology and advanced product development, many of which were initiated by the CMCRC. This manuscript offers a review of past and current strategies and advancements in medical countermeasures to address radiation injuries, carried out by the CMCRC and funded by the National Institute of Allergy and Infectious Diseases Radiation and Nuclear Countermeasures Program. It also explores the mechanisms of radiation-induced injuries, discusses existing medical countermeasures, and highlights emerging technologies and potential future directions for radiobiology researchers. This review aims to enhance our understanding of current medical countermeasures against radiation and contribute to the future development of more efficient and innovative approaches to mitigate and treat radiation-induced damage.

INTRODUCTION AND PROGRAMMATIC OVERVIEW

On September 11, 2001, the threat of terrorism underscored the significance of national security in relation to weapons of mass destruction, especially those involving ionizing radiation. These events revived concerns about U.S. vulnerability to radioactive contamination incidents, radiological dispersal devices, and potential attacks on or accidents at nuclear power plants—concerns that had waned after the Cold War concluded in 1991 (1). In response, Congress allocated funds in 2004 to direct the National Institute of Allergy and Infectious Diseases (NIAID) to develop the Radiation and Nuclear Countermeasures Program (RNCP) (2). In 2005, NIAID created the NIH Strategic Plan and Research Agenda for Medical Countermeasures Against Radiological and Nuclear Threats with input from a Blue Ribbon Panel of scientific experts (3). The strategic plan was subsequently updated, and a progress report was issued in 2012 (4).

Radiation exposure can lead to both acute radiation syndrome (ARS) and delayed effects of radiation exposure (DEARE). Figure 1 illustrates some major organ systems affected by radiation-related comorbidities studied by the Centers for Medical Countermeasures against Radiation Consortium (CMCRC), including gastrointestinal (GI-ARS), hematopoietic (H-ARS), bone marrow, cutaneous, and vascular injuries as part of ARS. In contrast, DEARE encompasses lung, kidney, liver, cardiovascular, endocrine, and neurological injuries.

FIG. 1.

Major organ systems studied through the CMCR program and possible radiation-associated comorbidities (created with BioRender).

For the past 20 years (2005–2025), the CMCRC, a collaborative network of national research centers, has built the basic research foundation to prepare the government for medical management of radiological or nuclear public health emergencies. At its peak, the CMCRC included eight centers, 36 scientific projects, and 42 cores, significantly advancing the field of radiobiology in line with NIAID’s Strategic Plan. Currently (2020–2025), the CMCRC includes three Centers, each with multiple research projects focused on multi-disciplinary, product-oriented development, an administrative core, and optional scientific cores. Additionally, four consortium-wide cores (Coordinating Center, Opportunities Fund Management, Radiation Physics and Nonhuman Primate Radiation Survivors) were historically managed by selected institutions. The organization is overseen by a steering committee consisting of the principal investigators from each center, the leads of the consortium-wide cores, the NIAID program officer, and an external advisory committee appointed by NIAID. The administrative cores manage coordination and compliance, while scientific cores provide essential support. Although there are currently only three CMCR, Fig. 2 shows that this consortium has published over 115 peer-reviewed publications in 48 journals with over 667 authors representing 30 countries since receipt of notice of grant award (2020) through 2024 (also see Supplementary Table S1;² https://doi.org/10.1667/RADE-24-00275.1.S1).

FIG. 2.

The country collaboration map in the CMCRC consortium 2020–2024. Depicted here are connections from 115 peer-reviewed publications in 48 journals with over 667 authors representing 30 countries. The Collaboration Network was generated using the R package Bibliometrix default parameters.³

The CMCRC’s primary objectives include: 1. Developing innovative biodosimetry techniques and devices for measuring radiation exposure in the human body; 2. Identifying biomarkers that indicate tissue damage and recovery; 3. Advancing novel medical countermeasures (MCMs) to minimize tissue damage, expedite recovery, restore normal functions, and improve survival rates. To date, this program has explored numerous radiation mitigators (Table 1) and enhanced diagnostic tools to assess radiation exposure at various maturity stages (Table 2).

TABLE 1.

CMCRC MCMs Development 2005–2025

Hematopoietica	Gastrointestinala	Skina	Lungb
Cord Blood	Clusterin	Bmil1	BCL-xL
Cordyceps Sinensis	Duke 9	Celecoxib	BIO300
Dinoprostone	DJ001	Curcumin Derivates	Lisinopril/Captopril/Fosinopril/Enalapril
dmPGE2	Extracellular Vesicles	TH-curcumin	EUK-189, 207, 423,451
Endothelial cells	EUK-207,451	EsA & h-EsA	FGF-PT
EUK-451	FGF-P	EUK-207, 423, 451	Genistein
Flt-3L	FGF-PT	FGrIL-12	Homspera
G-CSF	FSL-1	FGF-PT	Lovastatin
Human growth hormone	Hepoxilln A3 (HXA3)	JP4–039	MnTnHex-2-PyP5+
JP4–039	IL-22		MnTE-2-PyP5+
KGF	JP4–039		SecinH3
Liposomes	Kineret
		Renalb	Central Nervous Systemb
Mesenchymal stem cells	KGF
Meloxicam	Li2CO3	Atorvastatin	Atorvastatin
MnSOD-plasmid	Necrostatin	Captopril	EUK-189, 207,423,451
Nocloprost	N-methyl-D-aspartate	EUK-207	Minocycline
Rivenprost	PD0332991	Enalapril	Ramipril
Stem cell transplant	RP-1	Losartan	SecinH3
Sulprostone	OrbeShield	Lisinopril
Pleiotrophin	SOM-230
PUMA	STO-609
XJB-5–131	XJB-5–131
YeL1 & YeL2

Note. Medical countermeasures investigated by the CMCRC from 2005 to 2025 for acute

^aand delayed

^bsub-syndromes of irradiation.

TABLE 2.

CMCRC BioDosimetry Tools 2006–2025

Blood/lymphocytes	Serum/plasma
γ-H2AX	Metabolomics
Microculture	Lipidomics
Automated	Targeted LCMS kits and assays
ImageStream	Targeted methods ± FPSE
CBMN	Proteomics
Microculture	Endothelial microparticles*
Automated	Exosomes*
Accelerated	miRNA*
Centrifuge-free	Cell-free DNA
ImageStream	Saliva
DCA	Metabolomics
Microculture	Targeted methods ± FPSE
Automated	qRT-PCR*
PCC	Skin
Microculture*	Micronuclei
Proteomics	Metabolites in sebum*
Peptide arrays*	γ-H2AX*
ATM phosphorylation*	Feces
Mass spectrometry	Metabolomics*
ELISA	Metagenomics*
ImageStream	Urine
Vertical flow POC*	Metabolomics
Aptamers (POC)*	Targeted methods ± FPSE
Transcriptomics	Exosomes*
Microarray	miRNA*
RNA-Seq	Proteome
qRT-PCR	Nails
CMOS (POC)*	EPR
Aptamers (POC)*	Hair
nCounter*	γ-H2AX
Vertical flow (POC)*	Teeth
Cheek Swab	EPR
miRNA mutations*
Blood / Lymphocytes	Serum/Plasma
γ-H2AX	Metabolomics
Microculture	Lipidomics
Automated	Targeted LCMS kits and assays
ImageStream	Targeted methods ± FPSE
CBMN	Proteomics
Microculture	Endothelial microparticles*
Automated	Exosomes*
Accelerated	miRNA*
Centrifuge-free	Cell-free DNA
ImageStream	Saliva
DCA	Metabolomics
Microculture	Targeted methods ± FPSE
Automated	qRT-PCR*
PCC	Skin
Microculture*	Micronuclei
Proteomics	Metabolites in sebum*
Peptide arrays*	γ-H2AX*
ATM phosphorylation*	Feces
Mass spectrometry	Metabolomics*
ELISA	Metagenomics*
ImageStream	Urine
Vertical flow POC*	Metabolomics
Aptamers (POC)*	Targeted methods ± FPSE
Transcriptomics	Exosomes*
Microarray	miRNA*
RNA-Seq	Proteome
qRT-PCR	Nails
CMOS (POC)*	EPR
Aptamers (POC)*	Hair
nCounter*	γ-H2AX
Vertical flow (POC)*	Teeth
Cheek Swab	EPR
miRNA mutations*

Abbreviations: γH2AX, phospho(S139) H2A.X variant histone; CBMN, cytokinesis block micronucleus assay; DCA, dicentric chromosome assay; PCC, premature chromatin condensation; PNA, peptide nucleic acid; ATM, ataxia telangiectasia mutated; ELISA, enzyme-linked immunosorbent assay; POC, point of care; RNA-Seq, RNA sequencing; qRT-PCR, quantitative real-time PCR; CMOS, complementary metal-oxide-semiconductor; LCMS, liquid chromatography mass spectrometry; FPSE, fabric phase sorptive extraction; miRNA, microRNA; EPR, electron para-magnetic resonance.

^*Denotes pilot project.

Under normal conditions, organ systems and tissues work together to maintain physiological homeostasis. However, exposure to elevated levels of radiation, such as may occur during a radiological or nuclear incident, can severely disrupt these mechanisms, leading to multi-organ failure and sometimes mortality due to ARS. Survivors of ARS face a risk of developing DEARE, characterized by systemic inflammation, immune aging, lung fibrosis, and cardiovascular and metabolic diseases. Understanding DEARE is crucial for informing public health officials about future medical needs following a radiation emergency.

The CMCRC has significantly contributed to radiation preparedness research, establishing a strong foundation for independent studies on how radiation impacts various systems, organs, tissues, and cells, including the hematological, gastrointestinal, lung, cutaneous, and cardiovascular systems. They have also investigated more complex scenarios such as combined injuries (i.e., radiation plus another trauma), radiation-induced immune dysfunction, and the role of the microbiome in reacting to radiation injury. Additionally, the CMCRC has played a pivotal role in studying biodosimetry, which involves developing methods to estimate the radiation dose a person has received and, to a lesser extent, radionuclide decorporation, which aids in eliminating radioactive substances from the body.

The CMCRC established small and large animal models and in vitro models to evaluate radiation MCMs and biodosimetry approaches aimed at achieving these goals. Additionally, understanding the cellular and molecular mechanisms of radiation injuries, such as DNA damage, oxidative stress, inflammation, and tissue impairment, is crucial for developing effective treatments. Furthermore, stem cell-based therapies may aid in tissue regeneration and repair. The knowledge gained from CMCRC research has advanced the development of MCMs and biodosimetry tools, bringing them closer to potential U.S. Food and Drug Administration (FDA) approval and inclusion in the Strategic National Stockpile for emergency use. After two decades of pioneering research, the CMCRC will conclude its operations. Nevertheless, the NIAID is committed to continuing funding for specific and advanced areas of radiobiological research, many of which originated under the CMCRC’s auspices.

ANIMAL MODELS

Due to ethical reasons and the infeasibility of human studies to determine the effectiveness of MCMs against ARS and/or DEARE, the FDA employs the Animal Rule to regulate, approve, and license MCMs. This non-traditional regulatory pathway relies on demonstrating therapeutic efficacy in “adequate and well-controlled” studies conducted in one or more well-characterized animal models as a surrogate for human clinical trials. Relevant animal models that mimic the human response to acute, high-dose, and potentially lethal radiation exposure have been established and validated to test potential MCMs. The rationale for defining the requirements for these models was based on the current understanding of radiation effects on human survivors from the Hiroshima and Nagasaki atomic bombings, as well as Belarus and Ukraine survivors of the Chernobyl Nuclear Power Plant accident.

In a radiological or nuclear incident, exposed individuals may receive non-homogeneous radiation doses that affect multiple organs. The degree and severity of radiation-induced injuries depend not only on the type and dose of radiation but also on the affected tissue. Animal models were designed to evaluate and understand key organ-specific and multi-organ sequelae for survivable nuclear radiation exposures. Additionally, animal models are needed to define the radiation dose, incidence, severity, and time-dependent relationship for ARS to progress into DEARE and to identify longitudinal biomarkers to guide MCM development.

Early approaches to developing potential mitigators of radiation injury focused on specific organ toxicities (e.g., hematopoietic, gastrointestinal, cutaneous, neurologic, etc.) or “syndromes.” This reductive method facilitated investigation into the effects of various potential mitigators on specific syndromes. It also tracked the clinical manifestations of these syndromes, noting that lower doses of radiation resulted in damage to the bone marrow and the development of H-ARS, while higher doses caused damage to the GI and central nervous system, accompanied by associated symptomology. Importantly, concentrating on the single syndrome of H-ARS led to four FDA-approved products as radiation MCMs, as well as several biosimilars.

While informative, organ-based radiation exposures like whole thoracic lung irradiation (WTLI) do not accurately simulate exposures likely to occur during mass casualty incidents, where multiple tissues are exposed to high radiation doses. In such an incident, total-body irradiation (TBI) doses significantly above conventional clinical practice would likely occur, with some shielding of the bone marrow, resulting in the development of H-ARS and GI-ARS, along with survivors subsequently experiencing late multi-organ injuries. Consequently, multi-organ injury models are being developed to compare critical radiation endpoints across sexes, strains, and acute versus delayed radiation-related morbidity and mortality. This strategy also aligns with the FDA’s preference for a multi-organ injury model for MCM development.

Several animal models have been used to study radiation-induced injury and for the development of MCMs (6). Various species, including small rodents and larger animals such as swine and non-human primates (NHPs), have greatly contributed to the current understanding of ARS; however, none of these models can perfectly reproduce every aspect of radiation-induced injuries in humans. It is understood that although a single animal model cannot completely replicate all radiation-induced injuries, using more than one model to cover different aspects of the syndrome often provides sufficient information to grant FDA approval under the Animal Rule. Here, we summarize the advantages and disadvantages of the animal models commonly used at the CMCRC institutions.

Mouse

Small animals such as rodents (mice and rats) are useful for investigating the effects of H-ARS, GI-ARS, and DEARE and screening efficacy of potential MCMs. Radiation mouse models have been utilized for decades in radiation oncology, including fractionated, focal, electron, and proton FLASH radiation exposures. A multi-organ injury mouse model was developed using partial-body irradiation (PBI) with 2.5% bone marrow shielding (PBI/BM2.5) (7). The TBI/BM2.5 model recapitulates the breathing dysfunction and microscopic abnormalities consistent with radiation pneumonitis and fibrosis.

Early studies on radiation-induced lung pathology and MCM development focused on focal irradiation of the upper thorax using the WTLI model. Several MCMs have been shown to enhance pathology and reduce mortality in the WTLI model (8, 9). However, the MCMs that showed efficacy in WTLI models did not always demonstrate the same effectiveness in the PBI/BM2.5 model, likely due to the multi-organ damage induced. Nonetheless, this model offers a more relevant radiation exposure, consistent with a mass casualty scenario, which is needed for the continued development of MCMs that target mediators of DEARE, such as monocyte/macrophage polarization, fibrosis, and senescence.

Advantages.

Several mouse strains are available; they are easy to handle, and they have a short gestation period and lifespan. Housing can conveniently be accommodated at most institutions, and the mice are economical. Reagents for murine genomic manipulation are readily available, and easily accessible databases already exist.

Disadvantages.

Murine physiology and organ structures differ from those of humans. Small body size confounds the variable of radiation dose distribution and makes it difficult to follow the effects of radiation exposure longitudinally. Two additional challenges arise with the use of mice in radiation studies. First, radiosensitivity varies widely between strains and should be considered when attempting to reproduce a particular type of radiation-induced injury in humans (10). For example, mouse strains differ in their development of DEARE, with some strains being more prone to pneumonitis or fibrosis. The C57L/J mouse strain is believed to best mimic the dose-response relationship (DRR) and lung injury pathology observed in NHP (11). The C57BL/6 strain is more radioresistant than the C57L/J in the development of DEARE, with an extended timeline for the development of lung injury that does not fully recapitulate the injuries anticipated in humans. Given the differences in radiosensitivity, it is recommended that MCMs be tested in more than one mouse strain (e.g., one resistant and one sensitive). Second, chronoradiosensitivity has been recognized since the 1960s as an important consideration in rodent models used for radiation studies. Studies by the CMCRC have shown that rodents are more radiosensitive during the morning hours (12). Investigators must consider this effect when designing experiments involving the irradiation of models.

Rabbits

Several ethical issues and a dwindling supply of NHPs for MCM studies have raised awareness of the need to develop larger non-clinical models that recapitulate aspects of human sequelae following acute radiation exposure. Rabbits have shown usefulness in understanding the physiological and pathological processes following radiation-induced injuries. These animals are easily managed, readily available, and compatible with imaging technologies such as computed tomography (CT) and magnetic resonance imaging (MRI).

Rabbits are considered the largest small animal model by the FDA, providing sufficient blood volume to allow repetitive sampling or monitor disease onset, dynamics, and progression during a longitudinal study. In addition, the coagulation profile in New Zealand White (NZW) rabbits, unlike rodents, is similar to that of humans and, therefore, more suitable for MCM development. In a model of TBI-induced ARS, the clinical manifestations of NZW rabbits exposed to different radiation doses recapitulated several human symptoms (13).

With moderate levels of supportive care, severe hemorrhage and bone marrow failure in rabbits were reported to be similar to those in other species, including humans. For each radiation dose tested (6.5–9.5 Gy), the damage to the small intestine was greater than that to the colon, as previously reported in NHPs (14). In addition, thrombocytopenia always preceded the onset of severe anemia, and the fall in hematocrit and red blood cell counts indicated an impending mortality (13). Gross findings of vital organs (stomach, small and large intestine, kidneys) at necropsy indicated architectural damage, inflammation, and hemorrhage consistent with a multiorgan injury model. The NZW rabbit model thus recapitulates several pathological symptoms associated with ARS in humans and may provide an alternative to the mouse model for MCM efficacy screening, dose finding, and schedule optimization studies.

Advantages.

Rabbits are docile, efficiently handled animals that are small enough to easily accommodate while having sufficient blood volume to allow multiple blood samplings for longitudinal studies. They are readily available, cost-effective, have a longer lifespan than rodents, and are immunologically more similar to humans than rodents. Further, their size facilitates more reliable imaging assessments (e.g., CT, MRI).

Disadvantages.

Although rabbits have previously been used to support radiobiology research and radiation MCM development, the model has yet to be developed to the same extent as other species, such as rodents and NHPs. In addition, substantial physiological differences from humans, including heart size, heart rate, and body weight, may complicate interpretation and extrapolation of findings to human response to ARS and MCMs. Finally, compared to reagents for mice and NHPs, few rabbit-specific reagents are available. This reality places limitations on analyses of pharmacodynamics, biomarkers, and mechanisms of action.

Swine

Swine have been used in medical research for quite some time. Similarities in size, anatomy, pathophysiology, metabolism, and pathology to humans make swine suitable models in multiple areas of medicine. In addition, the pig genome has 66% sequence identity to the human genome, and epigenetics are highly conserved (15–18). In the context of radiation-induced injuries, swine have been the preferred models for the evaluation of cutaneous injuries due to the comparability of swine skin to human skin (6). They are also useful models for studying the natural history of ARS progression as well as potential MCM evaluation (19–21).

Minipig radiation models have gained traction as suitable animal models for radiation exposure because their size is more appropriate than that of larger swine for laboratory housing, husbandry, and radiation studies. Minipigs serve as models for studying GI-ARS since they exhibit physiological signs, such as vomiting, which are not present in rodent and rabbit models. Currently, Sinclair and Göttingen minipig models are under development in the current CMCRC. Both strains exhibit similar hematological profiles to those observed in other species, including humans, with neutrophils, lymphocytes, platelets, and hematocrit decreasing after acute radiation exposure. In addition, gross observation at necropsy shows hemorrhage in the lungs, skin, heart, and GI in both strains, as could be expected in multiorgan injury models. Moreover, radiation absorption patterns in minipigs are expected to be comparable to those of young adult humans, given their similar body thickness (22). The minipig thus provides an additional large animal model to screen potential radiation mitigators and study H-ARS and GI-ARS.

Advantages.

The pathophysiology of H-ARS in minipigs is similar to the human response. Minipigs are also relevant models for studying GI-ARS with endpoints of vomiting and diarrhea. Minipig skin and body thicknesses are comparable to humans, thus facilitating the extrapolation of radiation absorption data to human exposure. Minipigs have a longer lifespan compared to mice and rabbits, and their larger size supports their use as an additional large non-clinical model to evaluate MCM against acute radiation exposure.

Disadvantages.

Minipigs exhibit increased radiosensitivity compared to humans and other large animal species and may need supportive care to extend survival long enough to assess crypt-epithelial cell transit time and countermeasure efficacy. Radiosensitivity also varies widely between minipig strains. For example, after 2.3 Gy TBI, Göttingen minipigs had 25% survival at day 45 postirradiation, compared to 100% survival in Sinclair minipigs (17). The difference in radiation dose tolerance is unclear but may be related to the inbred nature of the Göttingen compared to the outbred nature of the Sinclair minipig. Furthermore, while a PBI model has been developed in Göttingen minipigs, only the abdomen and lower extremities were exposed to radiation, minimizing the contribution of multi-organ injury in the development of GI-ARS and DEARE (20).

Non-human Primates

The NHP model is the closest model related to humans, with 95% DNA sequence identity and a high degree of physiological receptor and pathway similarity. The NHP model most closely reproduces the clinical, histopathological, and pathophysiological aspects of radiation injury in humans; therefore, it is considered the gold standard by most investigators. Based on convenience, cost, and availability, the genus Macaca (rhesus and cynomolgus) has historically been the most commonly used NHP for biomedical research. Rhesus macaques are known as either Chinese or Indian, depending on their country of origin.

The rhesus macaque has anatomic, tissue composition, and tissue complexity similar to humans. Their response to radiation and treatments is analogous to the human radiation response, which facilitates the application of derived information to clinical situations. The model also allows for dose-effect correlation with humans due to similar requirements for supportive care, body size/thickness, and a longer lifespan than other animal models (6). The LD_50/60 of humans that received TBI is proposed to be approximately 4.5 Gy without supportive care and 6.0 Gy with supportive care, in line with the 7.2 Gy LD_50/60 reported for rhesus macaques with supportive care (23).

The pharmacokinetic/pharmacodynamic parameters for MCMs in NHPs approximate the human response. These criteria adhere to the FDA Animal Rule guidance for MCM approval (5). For these reasons, the rhesus macaques and, more specifically those of Chinese origin, had historically been the model of choice in biomedical research for the development of radiation MCMs. However, recent restrictions on the export of these animals have led to the increased use of other species, including rhesus macaques of Indian origin, cynomolgus (Macaca fasicularis) macaques, and squirrel monkeys. Although all these animals significantly contribute to advanced biomedical research, comparison between historical and current studies will be limited to animals of similar species and genetic backgrounds.

A particular advantage of NHP models is the opportunity to study DEARE in surviving irradiated NHPs. A multi-morbidity pattern has emerged, which in some ways resembles aging but also has a distinctive pattern of fibrosis, vascular pathology, metabolic derangement, gonadal injury, cataractogenesis, and neuroinflammation reflecting mechanisms characteristic of radiation injury (Fig. 3) (24). The Wake Forest Radiation Late Effects Cohort (RLEC) of rhesus macaque (Macaca mulatta) NHPs are a unique colony of long-term radiation survivors, consisting of animals exposed TBI, PBI, and WTLI. The cohort is composed of 290 NHPs (215 males/75 females), with 239 irradiated NHPs (168 males/71 females) and 51 non-irradiated controls (47 males/4 females). There are 217 animals under current observation, and 153 are deceased. Each irradiated animal received 1.14–8.5 Gy TBI (mean exposure was 6.13 Gy). The NHPs were irradiated at 2.3–15.5 years of age (mean age was = 4.6 years). This cohort has been under observation for nearly 18 years. All NHPs undergo clinical screening on an annual or semi-annual basis, including physical exams, MRI, CT, body composition analysis, echocardiograms, abdominal ultrasound, complete blood counts, and blood chemistries. Clinical diagnoses, including type II diabetes, heart disease, hypertension, renal disease, neoplasia, underweight, MRI brain lesions, and cataracts, are made using rigorously defined criteria (Fig. 3). Euthanized or dying animals receive a comprehensive postmortem and histopathologic exam performed by veterinary pathologists, and over 80 unique tissue samples are archived.

FIG. 3.

Chord diagram of the co-occurrence of major morbidities by organ or disease process in (N = 270) irradiated rhesus macaques. The category “no other” represents the low proportion of animals with only one morbidity.

NHPs receiving any dose of radiation had a mean accumulation of 4.3 morbidities (SD = 2.6), whereas the mean morbidity accumulation for unirradiated NHPs was 2.7 (SD = 1.9; P < 0.0003). Additionally, we have observed that certain morbidities, such as heart disease, tend to manifest earlier, with a mean age of onset of 9.3 years (SD = 4.4) after irradiation, than others, like type II diabetes which has a mean age of onset of 11.3 years (SD = 3.0 years; P < 0.05). This finding suggests unique organ responses to systemic injury, such as inflammation, vascular injury, and resultant fibrosis, routinely observed in the cohort. The ability to intensively monitor these NHPs over decades represents an unparalleled resource for modeling DEARE in a well-established large animal model.

Advantages.

The NHP model is as close as possible to humans, sharing more than 95% of identical DNA sequences, resulting in similar organ structure, metabolism, and a long life span. The model also allows easy sequential sampling and is suitable for monitoring GI symptoms, including vomiting and diarrhea. A comparable body size, thickness, and supportive care treatment facilitate the correlation of dose-effect relationships to human symptoms and treatment response.

Disadvantages.

The social and cognitive similarity between humans and NHPs raises ethical considerations unique to NHPs. A longer breeding period increases housing, feeding, and handling costs. Acquisition due to recent export restriction seriously limits their availability. Finally, cutaneous studies are complicated by the hirsute nature of NHP skin.

MEDICAL COUNTERMEAURES

The Strategic National Stockpile Radiation Working Group noted that animal models are key to MCM development and crucial for U.S. preparedness for a radiological or nuclear event (25). Table 1 shows the numerous MCMs studied by the CMCRC, which span many organ systems, given the expected multi-organ insults after radiation exposure. Early MCMs repurposed for radiation exposure include leukocyte growth factors such as granulocyte colony-stimulating factor (G-CSF), studied by the CMCRC, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In 2015, collaborations between NIAID, Amgen, and the FDA led to the approval of Filgrastim (Neupogen^®) (26) and Pegfilgrastim (Neulasta^®) (27) for H-ARS based on studies conducted under the FDA Animal Rule (28). Preclinical studies demonstrated Neupogen’s efficacy in mice (29) and NHPs (29) when administered 24 h postirradiation; Neulasta showed similar efficacy, leading to its approval. The Biomedical Advanced Research and Development Authority (BARDA), in partnership with Partner Therapeutics and the FDA, spearheaded the approval of Leukine^® (Sargramostim) in 2018, showing that delayed treatment up to 48 h postirradiation significantly reduced mortality and enhanced recovery of hemopoietic cell counts (30). In 2021, Nplate^® (Romiplostim) (31), an anti-thrombocytopenic that stimulates platelet recovery, was approved through collaborations with NIAID, Amgen, and the FDA. These public-private partnerships also enabled the approval of several growth factor biosimilars, including Nypozi™ (filgrastim-txid), Udenyca (pegfilgrastim-cbqv), Stimufend (pegfilgrastim-FPGA), Zarxio (filgrastim-sndz), and Ziextenzo (pegfilgrastim-bmez). The development and approval of these MCMs underscore the importance of collaborative efforts in enhancing U.S. radiation medical preparedness. These advancements ensure that effective treatments are available to mitigate the adverse effects of radiation exposure on hematopoiesis.

Other MCMs tested under the CMCRC continue to advance through academic and industry collaborations. For example, a genetically engineered probiotic Limosilactobacillus reuteri that releases IL-22 is being developed for GI-ARS under an NIAID contract with ChromoLogic, LLC, and the University of Pittsburgh (32). Lisinopril is also being developed for lung DEARE under an NIAID contract with the Medical College of Wisconsin (33). BIO300 was being developed under the CMCRC and a NIAID contract as a mitigator (7, 8). Humanetics Corporation was awarded funding to pursue a Phase 2 clinical trial to study BIO300 as a therapeutic to mitigate lung injury in discharged COVID-19 patients.⁴ The Department of Defense has also funded BIO 300 studies for prophylactic radioprotection (34).

BIODOSIMETRY

Biodosimetry tools will be needed to triage a large population and guide medical management after a mass casualty radiation incident. Predicting the severity and specific manifestations of radiation injuries is complex due to the variability in symptoms, which can take days to years to evolve, and the influence of individual factors such as age, immune status, and genetic background. The CMCRC has been at the forefront of biodosimetry diagnostic tools and biomarker discovery, aiming to predict early and delayed damage to specific organs and tissues. This research enables precise and timely medical interventions, reducing morbidity and mortality.

Cytogenetic approaches have historically been the cornerstone of biodosimetry, using lymphocyte DNA damage to assess radiation exposure. The dicentric chromosome assay (DCA) is considered the “gold standard” in the field, where lymphocyte division is induced but halted at the metaphase stage, allowing the identification of dicentric chromosomes specific to radiation exposure (35, 36). The formation of dicentric chromosomes requires at least two double-strand breaks formed on adjacent chromosomes and is highly specific for radiation. The average number of dicentric chromosomes per metaphase has a linear-quadratic relationship with dose and can be used for dose reconstruction.

Another useful assay is the cytokinesis blocked micronucleus (CBMN) assay, where lymphocyte division is blocked in cytokinesis, forming a binucleate cell. Irradiated lymphocytes may contain micronuclei, representing chromosome fragments not incorporated into the daughter nuclei during division (36, 37). Micronuclei are small, generally round objects in the cytoplasm of the cells outside the main nucleus. They represent chromosome fragments not incorporated into the daughter nuclei during division. The average number of micronuclei per binucleate cell also has a linear-quadratic relationship with dose and can be used for dose reconstruction. The main disadvantage of these assays is that 2–3 days are required for culture, but this has been accelerated and automated within the CMCRC for faster and more reliable results (38–40).

Radiation exposure causes protein modifications, expression, and localization, as well as rapid transcriptional and metabolic cellular reprogramming. These changes can be detected in easily sampled biofluids such as blood, serum, and urine. From cell samples, the γ-H2AX assay can be used to measure DNA damage by immunolabeling phosphorylated histone H2AX, which localizes to double-strand breaks (41). This assay can provide a dose estimate within a few hours and has the potential for automation (42, 43). Scoring is done by counting the number of foci per cell or the total fluorescence within the nucleus. While focus counting is more precise at low doses, it requires higher resolution (ideally confocal) imaging and saturates at low doses. Total fluorescence is easier but precision is not as good at low doses (44, 45). The major limitation of γ-H2AX analysis is its low persistence. The peak signal is achieved at 2–4 h, followed by a decrease in yields until 24 h postirradiation, when levels are close to the background.

The CMCRC has been instrumental in developing effective clinical tools for detecting radiation exposure using multiple approaches to biodosimetry (Table 2). Although γ-H2AX (46) and cytogenetic markers such as DCA (47), CBMN (37), and premature chromosome condensation (PCC) (48) have been used for biodosimetry, the assays are not high throughput, so CMCRC efforts focused on increasing throughput. Assays were developed using microculture in 96-well plates (38, 39, 42, 49) on robotic platforms with different configurations [e.g., custom robotics (50), high-throughput screening platforms (38, 39, 51–53), and imaging flow cytometry (45, 54)].

Historically, individual gene and protein markers have been explored as tools for biodosimetry. The CMCRC enabled extensive efforts in discovery using platforms like microarrays, RNA-Seq, antibody screening, and mass spectrometry (MS) (55–58), followed by signature development (59, 60) and point of care (POC) assays that would identify irradiated individuals in 30 min or less (61–63). The CMCRC also pioneered analysis of small molecule metabolites obtainable from other easily accessible biofluids (e.g., serum/plasma, urine, saliva) (64, 65), mainly using non-targeted MS approaches in discovery and later stages to increase speed and throughput (66, 67). In the past, other CMCRC studies addressed different approaches, such as exploration of the fecal microbiome and metabolome (68), gene expression signatures in saliva (69), cell-free DNA in serum (70), and γH2AX in skin (71) and hair (72). Physical dosimetry approaches using biological samples (e.g., tooth- and fingernail-based electron paramagnetic resonance) were also refined (73, 74). These studies have relied heavily on in vivo mouse and ex vivo human samples for their development. Studies have been conducted in NHPs both in vivo and ex vivo (75–79), genetically engineered mouse models representing alterations in DNA repair and immune pathways (80, 81), and in mice reconstituted with a human immune system (82–84).

Several biodosimetry tools have progressed from basic research under the CMCRC to advanced development funded by NIAID and BARDA. For example, the Columbia University Center launched technologies such as ASELL CytoRADx™ (85) and a MRIGlobal ARad Biodosimetry assay in partnership with Thermo Fisher Scientific and Arizona State University (86). The Duke University Center contributed to the discovery phase of DxTerity Diagnostics’ demonstration of the utility of gene signatures in animal models to predict radiation exposure in humans (87). Other biodosimetry technologies studied under the CMCRC, such as tooth EPR and micro DNA, also transitioned to BARDA but were not commercialized.

Rapid individual radiation dose estimates are essential for optimal medical management of mass radiation casualties and for determining the appropriate administration of counter-measures. In a mass casualty incident, quick identification of those not exposed to radiation can prevent medical facilities from being overwhelmed, allowing resources to focus on those with radiation exposures. After a large-scale radiological or nuclear incident, rapid radiation dose reconstruction can estimate the affected organ systems and identify individuals most likely to benefit from specific MCMs (88). Significant progress has been made in this area since the launch of the CMCRC, with a recent focus on developing biomarkers for radiological injury to particular organs or organ systems (e.g., lung, GI, etc.) and monitoring treatment responses. International interlaboratory intercomparison exercises have also been conducted to standardize methods and compare results from multiple biodosimetry approaches (89–92).

Methods based on signatures of responsive biomolecules are also under development. The earliest to emerge were signatures of genes measured in peripheral blood cells (93–96) and proteins in serum (97, 98). More recently, non-coding RNAs in blood or serum (99) and metabolomic profiles in serum or urine (65, 100, 101) have also shown great promise. These methods all rely on highly dynamic changes over time after irradiation, but most have shown markers that persist for at least a week. The use of multiple biomolecules in signatures is thought to militate against confounding by other exposures, disease states, or individual variation, but these factors are not yet completely understood. Measuring biomolecules in fluids such as urine is particularly attractive for large-scale incidents because it is a non-invasive approach for biodosimetry, avoiding the major potential bottleneck presented by the need to draw blood for most assays. These assays also have a great potential for both automation and integration into POC devices for rapid in-field testing (61, 102–105), a growing area of biodosimetry research.

EMERGING DIRECTIONS

Advancements in science and technology offer promising avenues for the development of novel MCMs against radiation injury and biodosimetry development. With TBI and PBI, there is a systemic effect rather than a local organ effect, providing a better understanding of tissue injury and actions of countermeasures. There have been several fruitful areas of research.

Endothelial/Vascular Injury

One scientific premise is that endothelial cell (EC) injury is the sine qua nonincident for radiation injury, initiating both downstream events and the perpetuation of injury (106). Processes that regulate whether ECs repair/regenerate or die are fundamental to the outcomes of patients. Endothelial cellS elaborate cytokines to modulate other cells in an autocrine, paracrine, and endocrine fashion. After irradiation, ECs will elaborate cytokines that incite tissue damage or facilitate cellular repair (107). In addition, ECs have a role in innate and adaptive immune responses. For example, CCL5 is secreted by ECs and other cells, facilitating tissue injury repair, and also acts as a potent chemoattractant for T cells (108). Thus, ECs not only participate in T cell recruitment to areas of injury but also direct T cells to produce inflammatory cytokines (e.g., IFNg) and modulate T cell function. Endothelial cells also mediate innate immune responses through enhanced neutrophil trafficking, activation of toll-like receptors (109), and increased secretion of other inflammatory cytokines like IL-1 beta, IL-6, and IL-8 in response to pathogens (110). Targeting inflammatory proteins could reset the inflammatory pathways and ameliorate the bone marrow microenvironment.

Immunity

The immune response, discussed at a high level above, is a key system that encompasses the most important mechanisms for healing. For example, monocytes, which constitute 5–10% of circulating white blood cells in humans and NHPs, respond to tissue injury and may transform into tissue-resident macrophages upon exiting the vasculature (111, 112). Classical monocytes represent the majority of monocytes (~85%), with intermediate and non-classical monocytes comprising the remainder (~15% total). These monocyte subsets have disparate phenotypes (113), where classical monocytes are associated with inflammatory responses and anaerobic metabolism, intermediate monocytes are associated with antigen presentation and processing, and non-classical monocytes are associated with oxidative phosphorylation and protein metabolism. Depending on the signals received from the local and systemic environment, monocytes may adopt a spectrum of phenotypes, developing into pro-inflammatory (M1-like) or anti-inflammatory-tissue remodeling/profibrotic (M2-like) macrophages (114). These definitions are too simplistic, and the role monocytes and macrophages play as master regulators of inflammation and fibrosis needs to be explored in more detail.

Microbiome

As epithelial organs, the gut, lungs, and skin are targets of radiation injury and are also hosts to extensive communities of microorganisms. Hundreds of studies have demonstrated associations between the microbiome and human health and disease, including associations between the gut microbiota and radiation-induced damage (115, 116). The outcome from radiation may be affected by the microbiome, microorganisms that colonize epithelial niches and contribute to health and disease. For example, gut bacteria may attenuate radiation responses by interacting with host cells to promote or inhibit apoptosis. Thus, targeting the microbiome can help mitigate radiation injury to these organs and improve survival. Previously published murine studies have shown that while most C57BL/6 mice receiving 9.2 Gy TBI will die from radiation injury, a small population of “elite survivors” can live relatively normal life spans (117). These mice harbored a distinct gut microbiota with protection against radiation-induced damage and death.

Moreover, the microbiota transfer from elite survivors to specific pathogen-free mice conferred these protective benefits, improving survival after irradiation. These microbiomes were characterized by elevated levels of Lachnospiraceae and Enterococcaceae, which were also found to be more abundant in humans who had fewer radiation-related toxicities after receiving TBI as part of hematopoietic stem cell transplantation. In addition, elite survivor mice had increased levels of microbially derived propionate and other short-chain fatty acids, and administration of these specific lipids protected against radiation injury, including mitigating H-ARS and reducing inflammatory responses.

Fibrosis (Long-term Effect)

A major DEARE manifestation is radiation-induced fibrosis (RIF), a debilitating condition occurring 4–12 months postirradiation in humans that has been referred to as a “wound that fails to heal.” RIF has been documented in many tissues, including the heart, lungs, liver, and skin (118–120). The initiating signals and cells that lead to progression from quiescence to RIF are largely unknown but likely involve an aberrant healing response mediated by monocytes, macrophages, fibroblasts, myofibroblasts, endothelial cells, and platelets (121). Ultimately, this response results in excessive extracellular matrix deposition, which reduces tissue compliance, impairs functionality, and increases morbidity. Unfortunately, there are currently no therapies that can halt or reverse RIF.

Nonetheless, monocytes and macrophages are known to be master regulators of inflammation and fibrosis, playing key roles in initiating, producing, and resolving fibrosis (122). Increased intermediate monocyte polarization and subsequent tissue infiltration have been implicated in the pro-fibrotic activity in pathologic fibrotic disorders, including systemic sclerosis, renal fibrosis, idiopathic pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis, and skeletal muscle fibrosis (123–125). Early monocyte and macrophage depletion by clodronate in a bleomycin-induced pulmonary fibrosis model reduces fibrosis (126). Depletion of circulating monocytes during the early stages after infarction impaired healing and increased necrotic debris accumulation, whereas depletion during the later stages decreased collagen deposition and endothelial cells (127). These findings suggest monocyte-macrophages may be a target for intervention to inhibit radiation-induced fibrosis.

CONCLUSIONS

The NIAID CMCRC program has contributed significantly over the past 20 years to basic radiobiology, novel MCMs development, dose reconstruction, and development of biodosimetry tools for triage and medical management of patients. The goal is to continue to develop and mature these technologies for use after a radiation mass casualty scenario. Many products were matured under NIAID funding and some were transferred to BARDA for advanced development.

A robust early product development pipeline was established by funding high-risk, high-reward projects through the CMCRC and associated pilot projects program. Pilot projects were initially selected and administered by the individual centers, and for the last ten years through the Opportunities Funds Management Core (OFMC). The scientists and projects brought into the CMCRC portfolio have been extremely productive. In the nearly ten years of the OFMC, pilot awardees have published at least 83 related peer-reviewed publications. They have reported that data generated in their pilot projects contributed to the submission of 52 grant proposals (11 of which were reported as funded), nine patent filings, one invention report, and one patent award.

The CMCRC has successfully met its original objectives, resulting in a comprehensive radiation medical preparedness research portfolio. As the CMCRC concludes its operations, the NIAID/RNCP views this as a transition to a stronger and more advanced research preparedness portfolio, thanks to the consortium’s pioneering efforts. Radiation MCMs play a crucial role in mitigating the adverse effects of radiation exposure, and the biodosimetry tools developed will help in the allocation of essential medical supplies, protective equipment, and MCMs. This review provides an overview of current strategies, challenges, and future directions in this field, underscoring the CMCRC’s lasting impact and the continued evolution of research and medical preparedness in radiobiology.

Supplementary Material

SUPPLEMENTARY MATERIALS

Supplementary Table S1. Detailed information on 115 publications during the period of notice of awards for the three centers for medical countermeasures against radiation, starting in 2020.