The NIAID/RNCP Biodosimetry Program: An Overview.

Established in 2004, the Radiation and Nuclear Countermeasures Program (RNCP), within the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health has the central mission to advance medical countermeasure mitigators/therapeutics, and biomarkers and technologies to assess, triage, and inform medical management of patients experiencing acute radiation syndrome and/or the delayed effects of acute radiation exposure. The RNCP biodosimetry mission space encompasses: (1) basic research to elucidate novel approaches for rapid and accurate assessment of radiation exposure, (2) studies to support advanced development for US Food and Drug Administration (FDA) clearance of promising triage or treatment devices/approaches, (3) characterization of biomarkers and/or assays to determine degree of tissue or organ dose that can predict outcome of radiation injuries (i.e., organ failure, morbidity, and/or mortality), and (4) outreach efforts to facilitate interactions with researchers developing cutting edge biodosimetry approaches. Thus far, no biodosimetry device has been FDA cleared for use during a radiological/nuclear incident. At NIAID, advancement of radiation biomarkers and biodosimetry approaches is facilitated by a variety of funding mechanisms (grants, contracts, cooperative and interagency agreements, and Small Business Innovation Research awards), with the objective of advancing devices and assays toward clearance, as outlined in the FDA's Radiation Biodosimetry Medical Countermeasure Devices Guidance. The ultimate goal of the RNCP biodosimetry program is to develop and establish accurate and reliable biodosimetry tools that will improve radiation preparedness and ultimately save lives during a radiological or nuclear incident.

Minimum reporting standards should be expected for preclinical radiobiology irradiators and dosimetry in the published literature.

The cornerstones of science advancement are rigor in performing scientific research, reproducibility of research findings and unbiased reporting of design and results of the experiments. For radiation research, this requires rigor in describing experimental details as well as the irradiation protocols for accurate, precise and reproducible dosimetry. Most institutions conducting radiation biology research in in vitro or animal models do not have describe experimental irradiation protocols in sufficient details to allow for balanced review of their publication nor for other investigators to replicate published experiments. The need to increase and improve dosimetry standards, traceability to National Institute of Standards and Technology (NIST) standard beamlines, and to provide dosimetry harmonization within the radiation biology community has been noted for over a decade both within the United States and France. To address this requirement subject matter experts have outlined minimum reporting standards that should be included in published literature for preclinical irradiators and dosimetry.

Radiation-induced multi-organ injury.

PURPOSE: Natural history studies have been informative in dissecting radiation injury, isolating its effects, and compartmentalizing injury based on the extent of exposure and the elapsed time post-irradiation. Although radiation injury models are useful for investigating the mechanism of action in isolated subsyndromes and development of medical countermeasures (MCMs), it is clear that ionizing radiation exposure leads to multi-organ injury (MOI). METHODS: The Radiation and Nuclear Countermeasures Program within the National Institute of Allergy and Infectious Diseases partnered with the Biomedical Advanced Research and Development Authority to convene a virtual two-day meeting titled 'Radiation-Induced Multi-Organ Injury' on June 7-8, 2022. Invited subject matter experts presented their research findings in MOI, including study of mechanisms and possible MCMs to address complex radiation-induced injuries. RESULTS: This workshop report summarizes key information from each presentation and discussion by the speakers and audience participants. CONCLUSIONS: Understanding the mechanisms that lead to radiation-induced MOI is critical to advancing candidate MCMs that could mitigate the injury and reduce associated morbidity and mortality. The observation that some of these mechanisms associated with MOI include systemic injuries, such as inflammation and vascular damage, suggests that MCMs that address systemic pathways could be effective against multiple organ systems.

Advanced Technologies in Radiation Research.

The U.S. Government is committed to maintaining a robust research program that supports a portfolio of scientific experts who are investigating the biological effects of radiation exposure. On August 17 and 18, 2023, the Radiation and Nuclear Countermeasures Program, within the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), partnered with the National Cancer Institute, NIH, the National Aeronautics and Space Administration, and the Radiation Injury Treatment Network to convene a workshop titled, Advanced Technologies in Radiation Research (ATRR), which focused on the use of advanced technologies under development or in current use to accelerate radiation research. This meeting report provides a comprehensive overview of the research presented at the workshop, which included an assembly of subject matter experts from government, industry, and academia. Topics discussed during the workshop included assessments of acute and delayed effects of radiation exposure using modalities such as clustered regularly interspaced short palindromic repeats (CRISPR) - based gene editing, tissue chips, advanced computing, artificial intelligence, and immersive imaging techniques. These approaches are being applied to develop products to diagnose and treat radiation injury to the bone marrow, skin, lung, and gastrointestinal tract, among other tissues. The overarching goal of the workshop was to provide an opportunity for the radiation research community to come together to assess the technological landscape through sharing of data, methodologies, and challenges, followed by a guided discussion with all participants. Ultimately, the organizers hope that the radiation research community will benefit from the workshop and seek solutions to scientific questions that remain unaddressed. Understanding existing research gaps and harnessing new or re-imagined tools and methods will allow for the design of studies to advance medical products along the critical path to U.S. Food and Drug Administration approval.

Gastrointestinal Acute Radiation Syndrome: Mechanisms, Models, Markers, and Medical Countermeasures.

There have been a number of reported human exposures to high dose radiation, resulting from accidents at nuclear power plants (e.g., Chernobyl), atomic bombings (Hiroshima and Nagasaki), and mishaps in industrial and medical settings. If absorbed radiation doses are high enough, evolution of acute radiation syndromes (ARS) will likely impact both the bone marrow as well as the gastrointestinal (GI) tract. Damage incurred in the latter can lead to nutrient malabsorption, dehydration, electrolyte imbalance, altered microbiome and metabolites, and impaired barrier function, which can lead to septicemia and death. To prepare for a medical response should such an incident arise, the National Institute of Allergy and Infectious Diseases (NIAID) funds basic and translational research to address radiation-induced GI-ARS, which remains a critical and prioritized unmet need. Areas of interest include identification of targets for damage and mitigation, animal model development, and testing of medical countermeasures (MCMs) to address GI complications resulting from radiation exposure. To appropriately model expected human responses, it is helpful to study analogous disease states in the clinic that resemble GI-ARS, to inform on best practices for diagnosis and treatment, and translate them back to inform nonclinical drug efficacy models. For these reasons, the NIAID partnered with two other U.S. government agencies (the Biomedical Advanced Research and Development Authority, and the Food and Drug Administration), to explore models, biomarkers, and diagnostics to improve understanding of the complexities of GI-ARS and investigate promising treatment approaches. A two-day workshop was convened in August 2022 that comprised presentations from academia, industry, healthcare, and government, and highlighted talks from 26 subject matter experts across five scientific sessions. This report provides an overview of information that was presented during the conference, and important discussions surrounding a broad range of topics that are critical for the research, development, licensure, and use of MCMs for GI-ARS.

Immune Dysfunction from Radiation Exposure.

Exposure to ionizing radiation causes acute damage and loss of bone marrow and peripheral immune cells that can result in high mortality due to reduced resistance to infections and hemorrhage. Besides these acute effects, tissue damage from radiation can trigger inflammatory responses, leading to progressive and chronic tissue damage by radiation-induced loss of immune cell types that are required for resolving tissue injuries. Understanding the mechanisms involved in radiation-induced immune system injury and repair will provide new insights for developing medical countermeasures that help restore immune homeostasis. For these reasons, The Radiation and Nuclear Countermeasures Program (RNCP) and the Basic Immunology Branch (BIB) under the Division of Allergy, Immunology, and Transplantation (DAIT) within the National Institute of Allergy and Infectious Diseases (NIAID) convened a two-day workshop, along with partners from the Biomedical Advanced Research and Development Authority (BARDA), and the Radiation Injury Treatment Network (RITN). This workshop, titled "Immune Dysfunction from Radiation Exposure," was held virtually on September 9-10, 2020; this Commentary provides a high-level overview of what was discussed at the meeting.

Considerations of Medical Preparedness to Assess and Treat Various Populations During a Radiation Public Health Emergency.

During a radiological or nuclear public health emergency, given the heterogeneity of civilian populations, it is incumbent on medical response planners to understand and prepare for a potentially high degree of interindividual variability in the biological effects of radiation exposure. A part of advanced planning should include a comprehensive approach, in which the range of possible human responses in relation to the type of radiation expected from an incident has been thoughtfully considered. Although there are several reports addressing the radiation response for special populations (as compared to the standard 18-45-year-old male), the current review surveys published literature to assess the level of consideration given to differences in acute radiation responses in certain sub-groups. The authors attempt to bring clarity to the complex nature of human biology in the context of radiation to facilitate a path forward for radiation medical countermeasure (MCM) development that may be appropriate and effective in special populations. Consequently, the focus is on the medical (as opposed to logistical) aspects of preparedness and response. Populations identified for consideration include obstetric, pediatric, geriatric, males, females, individuals of different race/ethnicity, and people with comorbidities. Relevant animal models, biomarkers of radiation injury, and MCMs are highlighted, in addition to underscoring gaps in knowledge and the need for consistent and early inclusion of these populations in research. The inclusion of special populations in preclinical and clinical studies is essential to address shortcomings and is an important consideration for radiation public health emergency response planning. Pursuing this goal will benefit the population at large by considering those at greatest risk of health consequences after a radiological or nuclear mass casualty incident.

Immune Dysfunction from Radiation Exposure.

The hematopoietic system is highly sensitive to ionizing radiation. Damage to the immune system may result in opportunistic infections and hemorrhage, which could lead to mortality. Inflammation triggered by tissue damage can also lead to additional local or widespread tissue damage. The immune system is responsible for tissue repair and restoration, which is made more challenging when it is in the process of self-recovery. Because of these challenges, the Radiation and Nuclear Countermeasures Program (RNCP) and the Basic Immunology Branch (BIB) under the Division of Allergy, Immunology, and Transplantation (DAIT) within the National Institute of Allergy and Infectious Diseases (NIAID), along with partners from the Biomedical Advanced Research and Development Authority (BARDA), and the Radiation Injury Treatment Network (RITN) sponsored a two-day meeting titled Immune Dysfunction from Radiation Exposure held on September 9-10, 2020. The intent was to discuss the manifestations and mechanisms of radiation-induced immune dysfunction in people and animals, identify knowledge gaps, and discuss possible treatments to restore immune function and enhance tissue repair after irradiation.

Development of Biomarkers for Radiation Biodosimetry and Medical Countermeasures Research: Current Status, Utility, and Regulatory Pathways.

Biomarkers are important indicators of biological processes in health or disease. For this reason, they play a critical role in advanced development of radiation biodosimetry tools and medical countermeasures (MCMs). They can aid in the assessment of radiation exposure level, extent of radiation-induced injury, and/or efficacy of a MCM. This meeting report summarizes the presentations and discussions from the 2020 workshop titled, "Biomarkers in Radiation Biodosimetry and Medical Countermeasures" sponsored by the Radiation and Nuclear Countermeasures Program (RNCP) within the National Institute of Allergy and Infectious Diseases (NIAID). The main goals of this meeting were to: 1. Provide an overview on biomarkers and to focus on the state of science with regards to biomarkers specific to radiation biodosimetry and MCMs; 2. Understand developmental challenges unique to the role of biomarkers in the fields of radiation biodosimetry and MCM development; and 3. Identify existing gaps and needs for translational application.

Animal Care in Radiation Medical Countermeasures Studies.

Animal models are necessary to demonstrate the efficacy of medical countermeasures (MCM) to mitigate/treat acute radiation syndrome and the delayed effects of acute radiation exposure and develop biodosimetry signatures for use in triage and to guide medical management. The use of animal models in radiation research allows for the simulation of the biological effects of exposure in humans. Robust and well-controlled animal studies provide a platform to address basic mechanistic and safety questions that cannot be conducted in humans. The U.S. Department of Health and Human Services has tasked the National Institute of Allergy and Infectious Diseases (NIAID) with identifying and funding early- through advanced-stage MCM development for radiation-induced injuries; and advancement of biodosimetry platforms and exploration of biomarkers for triage, definitive dose, and predictive purposes. Some of these NIAID-funded projects may transition to the Biomedical Advanced Research and Development Authority (BARDA), a component of the Office of the Assistant Secretary for Preparedness and Response in the U.S. Department of Health and Human Services, which is tasked with the advanced development of MCMs to include pharmacokinetic, exposure, and safety assessments in humans. Guided by the U.S. Food and Drug Administration's (FDA) Animal Rule, both NIAID and BARDA work closely with researchers to advance product and device development, setting them on a course for eventual licensure/approval/clearance of their approaches by the FDA. In August 2020, NIAID partnered with BARDA to conduct a workshop to discuss currently accepted animal care protocols and examine aspects of animal models that can influence outcomes of studies to explore MCM efficacy for potential harmonization. This report provides an overview of the two-day workshop, which includes a series of special topic presentations followed by panel discussions with subject-matter experts from academia, industry partners, and select governmental agencies.

Acute Radiation Syndrome and the Microbiome: Impact and Review.

Study of the human microbiota has been a centuries-long endeavor, but since the inception of the National Institutes of Health (NIH) Human Microbiome Project in 2007, research has greatly expanded, including the space involving radiation injury. As acute radiation syndrome (ARS) is multisystemic, the microbiome niches across all areas of the body may be affected. This review highlights advances in radiation research examining the effect of irradiation on the microbiome and its potential use as a target for medical countermeasures or biodosimetry approaches, or as a medical countermeasure itself. The authors also address animal model considerations for designing studies, and the potential to use the microbiome as a biomarker to assess radiation exposure and predict outcome. Recent research has shown that the microbiome holds enormous potential for mitigation of radiation injury, in the context of both radiotherapy and radiological/nuclear public health emergencies. Gaps still exist, but the field is moving forward with much promise.

Commonalities Between COVID-19 and Radiation Injury.

As the multi-systemic components of COVID-19 emerge, parallel etiologies can be drawn between SARS-CoV-2 infection and radiation injuries. While some SARS-CoV-2-infected individuals present as asymptomatic, others exhibit mild symptoms that may include fever, cough, chills, and unusual symptoms like loss of taste and smell and reddening in the extremities (e.g., "COVID toes," suggestive of microvessel damage). Still others alarm healthcare providers with extreme and rapid onset of high-risk indicators of mortality that include acute respiratory distress syndrome (ARDS), multi-organ hypercoagulation, hypoxia and cardiovascular damage. Researchers are quickly refocusing their science to address this enigmatic virus that seems to unveil itself in new ways without discrimination. As investigators begin to identify early markers of disease, identification of common threads with other pathologies may provide some clues. Interestingly, years of research in the field of radiation biology documents the complex multiorgan nature of another disease state that occurs after exposure to high doses of radiation: the acute radiation syndrome (ARS). Inflammation is a key common player in COVID-19 and ARS, and drives the multi-system damage that dramatically alters biological homeostasis. Both conditions initiate a cytokine storm, with similar pro-inflammatory molecules increased and other anti-inflammatory molecules decreased. These changes manifest in a variety of ways, with a demonstrably higher health impact in patients having underlying medical conditions. The potentially dramatic human impact of ARS has guided the science that has identified many biomarkers of radiation exposure, established medical management strategies for ARS, and led to the development of medical countermeasures for use in the event of a radiation public health emergency. These efforts can now be leveraged to help elucidate mechanisms of action of COVID-19 injuries. Furthermore, this intersection between COVID-19 and ARS may point to approaches that could accelerate the discovery of treatments for both.

Metabolomics in Radiation Biodosimetry: Current Approaches and Advances.

Triage and medical intervention strategies for unanticipated exposure during a radiation incident benefit from the early, rapid and accurate assessment of dose level. Radiation exposure results in complex and persistent molecular and cellular responses that ultimately alter the levels of many biological markers, including the metabolomic phenotype. Metabolomics is an emerging field that promises the determination of radiation exposure by the qualitative and quantitative measurements of small molecules in a biological sample. This review highlights the current role of metabolomics in assessing radiation injury, as well as considerations for the diverse range of bioanalytical and sampling technologies that are being used to detect these changes. The authors also address the influence of the physiological status of an individual, the animal models studied, the technology and analysis employed in interrogating response to the radiation insult, and variables that factor into discovery and development of robust biomarker signatures. Furthermore, available databases for these studies have been reviewed, and existing regulatory guidance for metabolomics are discussed, with the ultimate goal of providing both context for this area of radiation research and the consideration of pathways for continued development.

Determination and analysis of site-specific 125I decay-induced DNA double-strand break end-group structures.

End groups contribute to the structural complexity of radiation-induced DNA double-strand breaks (DSBs). As such, end-group structures may affect a cell's ability to repair DSBs. The 3'-end groups of strand breaks caused by gamma radiation, or oxidative processes, under oxygenated aqueous conditions have been shown to be distributed primarily between 3'-phosphoglycolate and 3'-phosphate, with 5'-phosphate ends in both cases. In this study, end groups of the high-LET-like DSBs caused by 125I decay were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by 125I-labeled triplex-forming oligonucleotide targeting. End-group structure was assessed enzymatically as a function of the DSB end to serve as a substrate for ligation and various forms of end labeling. Using this approach, we have demonstrated 3'-hydroxyl (3'-OH) and 3'-phosphate (3'-P) end groups and 5'-ends (> or = 42%) terminated by phosphate. A 32P postlabeling assay failed to detect 3'-phosphoglycolate in a restriction fragment terminated by the 125I-induced DNA double-strand break, and this is likely due to restricted oxygen diffusion during irradiation as a frozen aqueous solution. Even so, end-group structure and relative distribution varied as a function of the free radical scavenging capacity of the irradiation buffer.

Molecular analysis of base damage clustering associated with a site-specific radiation-induced DNA double-strand break.

Base damage flanking a radiation-induced DNA double-strand break (DSB) may contribute to DSB complexity and affect break repair. However, to date, an isolated radiation-induced DSB has not been assessed for such structures at the molecular level. In this study, an authentic site-specific radiation-induced DSB was produced in plasmid DNA by triplex forming oligonucleotide-targeted (125)I decay. A restriction fragment terminated by the DSB was isolated and probed for base damage with the E. coli DNA repair enzymes endonuclease III and formamidopyrimidine-DNA glycosylase. Our results demonstrate base damage clustering within 8 bases of the (125)I-targeted base in the DNA duplex. An increased yield of base damage (purine > pyrimidine) was observed for DSBs formed by irradiation in the absence of DMSO. An internal control fragment 1354 bp upstream from the targeted base was insensitive to enzymatic probing, indicating that the damage detected proximal to the DSB was produced by the (125)I decay that formed the DSB. Gas chromatography-mass spectrometry identified three types of damaged bases in the approximately 32-bp region proximal to the DSB. These base lesions were 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine. Finally, evidence is presented for base damage >24 bp upstream from the (125)I-decay site that may form via a charge migration mechanism.

Characterization of a complex 125I-induced DNA double-strand break: implications for repair.

PURPOSE: To examine the role of radiation-induced DNA double-strand break (DSB) structural organization in DSB repair, and characterize the structural features of 125I-induced DSBs that may impact the repair process. METHODS: Plasmid DNA was linearized by sequence-specific targeting using an 125I-labeled triplex-forming oligonucleotide (TFO). Following isolation from agarose gels, base damage structures associated with the DSB ends in plasmids linearized by the 125I-TFO were characterized by probing with the E. coli DNA damage-specific endonuclease and DNA-glycosylases, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-glycosylase (Fpg). RESULTS: Plasmid DNA containing DSBs produced by the high-LET-like effects of 125I-TFO has been shown to support at least 2-fold lower end joining than gamma-ray linearized plasmid, and this may be a consequence of the highly complex structure expected near an 125I-induced DSB end. Therefore, to determine if a high density of base damage exists proximal to the DSBs produced by 125I-TFOs, short fragments of DNA recovered from the DSB end of 125I-TFO-linearized plasmid were enzymatically probed. Base damage and AP site clustering was demonstrated within 3 bases downstream and 7 bases upstream of the targeted base. Furthermore, the pattern and extent of base damage varied depending upon the presence or absence of 2 M DMSO during irradiation. CONCLUSIONS: 125I-TFO-induced DSBs exhibit a high degree of base damage clustering proximal to the DSB end. At least 60% of the nucleotides within 10 bp of the 125I decay site are sensitive to cleavage by endo IV, endo III, or Fpg following damage accumulation in the presence of DMSO, whereas > or = 80% are sensitive in the absence of DMSO. The high degree of base damage clustering associated with the 125I-TFO-induced DSB end may be a major factor leading to its negligible in vitro repair by the non-homologous end-joining pathway (NHEJ).

Repair of radiation-induced DNA double-strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks.

Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co gamma rays, and the effects of 125I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by gamma rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of gamma-ray-induced DSBs. In addition, DSB ends produced by 125I were shown to be blocked by 3'-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.

Therapeutic and space radiation exposure of mouse brain causes impaired DNA repair response and premature senescence by chronic oxidant production.

Despite recent epidemiological evidences linking radiation exposure and a number of human ailments including cancer, mechanistic understanding of how radiation inflicts long-term changes in cerebral cortex, which regulates important neuronal functions, remains obscure. The current study dissects molecular events relevant to pathology in cerebral cortex of 6 to 8 weeks old female C57BL/6J mice two and twelve months after exposure to a γ radiation dose (2 Gy) commonly employed in fractionated radiotherapy. For a comparative study, effects of 1.6 Gy heavy ion 56Fe radiation on cerebral cortex were also investigated, which has implications for space exploration. Radiation exposure was associated with increased chronic oxidative stress, oxidative DNA damage, lipid peroxidation, and apoptosis. These results when considered with decreased cortical thickness, activation of cell-cycle arrest pathway, and inhibition of DNA double strand break repair factors led us to conclude to our knowledge for the first time that radiation caused aging-like pathology in cerebral cortical cells and changes after heavy ion radiation were more pronounced than γ radiation.