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Summary Sanchita P. Ghosh1, Gregory Holmes-Hampton1, George Cox2, and Vidya P. Kumar1. Background: The threat of nuclear exposure is heightened and there is unmet need to identify potential countermeasures for acute radiation syndrome (ARS). Currently no countermeasures have been approved for prophylactic administration. Effective countermeasures should function to increase survival in the short term as well as to increase the overall prognosis of an exposed individual long term. Here we report BBT-059 (PEGylated IL-11 analog developed by Bolder Biotechnology), a promising prophylactic radiation countermeasure in mice, protect multi-organ systems exposed to total body radiation (TBI) or partial body irradiation (PBI), as well as delayed effects of acute radiation exposure (DEARE). Method: A single dose (0.3 mg/kg) of BBT-059 was tested in mice, injected at several time points from 24 h pre- to 24 h post-TBI.CBC, cytokines, bone marrow progenitor cells and gastrointestinal crypt cells were evaluated in irradiated mice. Mice were exposed to either total body Co-60 gamma radiation or partial body irradiation using the clinical linear accelerator (4MV X-rays).Animal were monitored up to one year following exposure and radiation-induced damage was evaluated in major organs. Results: BBT-059 significantly increased survival in mice exposed to supralethal doses of TBI, protected hematopoietic system(recovery of CBC, bone marrow CFU, markers of bone marrow aplasia EPO, TPO, FLt3L) as well as gastrointestinal (GI) system (recovery of viable crypts, reduction of bacterial translocation and sepsis markers SAA, PCT). A dose reduction factor of 1.28 was calculated for BBT-059 administered pre-TBI compared to saline. BBT-059 also demonstrated significantly higher survival in GI-ARS PBI model, protected crypts. In the long term study in surviving (6 and 12 months post-TBI) animals showed accelerated recovery of CBC, bone marrow CFU, sternal cellularity and megakaryocyte numbers in, RITM0040047, Background: The threat of nuclear exposure is heightened and there is unmet need to identify potential countermeasures for acute radiation syndrome (ARS). Currently no countermeasures have been approved for prophylactic administration. Effective countermeasures should function to increase survival in the short term as well as to increase the overall prognosis of an exposed individual long term. Here we report BBT-059 (PEGylated IL-11 analog developed by Bolder Biotechnology), a promising prophylactic radiation countermeasure in mice, protect multi-organ systems exposed to total body radiation (TBI) or partial body irradiation (PBI), as well as delayed effects of acute radiation exposure (DEARE). Method: A single dose (0.3 mg/kg) of BBT-059 was tested in mice, injected at several time points from 24 h pre- to 24 h post-TBI.CBC, cytokines, bone marrow progenitor cells and gastrointestinal crypt cells were evaluated in irradiated mice. Mice were exposed to either total body Co-60 gamma radiation or partial body irradiation using the clinical linear accelerator (4MV X-rays).Animal were monitored up to one year following exposure and radiation-induced damage was evaluated in major organs. Results: BBT-059 significantly increased survival in mice exposed to supralethal doses of TBI, protected hematopoietic system(recovery of CBC, bone marrow CFU, markers of bone marrow aplasia EPO, TPO, FLt3L) as well as gastrointestinal (GI) system (recovery of viable crypts, reduction of bacterial translocation and sepsis markers SAA, PCT). A dose reduction factor of 1.28 was calculated for BBT-059 administered pre-TBI compared to saline. BBT-059 also demonstrated significantly higher survival in GI-ARS PBI model, protected crypts. In the long term study in surviving (6 and 12 months post-TBI) animals showed accelerated recovery of CBC, bone marrow CFU, sternal cellularity and megakaryocyte numbers in drug treated mice compared to control. In addition, increased senescence was observed

Dose-Dependent Effects of Radiation on Mitochondrial Morphology and Function, and Clonogenic Cell Survival in Human Microvascular Endothelial Cells Li Wang, Rafael Rivas, Angelo Wilson, Yu Min Park, Shannon Walls, Tianzheng Yu, and Alexandra C. Miller Uniformed Services University of the Health Sciences, Bethesda, MD 20814, U.S.A • Radiation exposure significantly reduced clonogenic growth in HMEC-1 cells at moderate-to-high doses (1 Gy and above). However, low doses of radiation (less than 1 Gy), specifically 0.25 Gy, resulted in a 15% increase in colony number compared to control cells. • Moderate-to-high doses, but not low doses, increased rH2AX & p53 and induced premature senescence & oxidative stress in irradiated HMEC-1. • Mitochondrial dysfunction induced by radiation is dependent on the radiation dose and may contribute to oxidative stress and cellular damage. • Upon exposure to radiation, all doses, except for 0.1 Gy, significantly increased the percentage of elongated mitochondria, with low doses having a greater effect than high doses. However, only moderate-to-high doses (1 Gy and greater) caused a notable increase in mitochondrial fragmentation/swelling. • Low doses-induced mitochondrial elongation may be due to upregulation of Mfn1 expression, while moderate-to-high doses-triggered mitochondrial fragmentation may be caused by downregulation of Opa1 expression. • In conclusion, our findings suggest that in response to varying levels of radiation exposure, mitochondria undergo changes in morphology to regulate radiation-induced protection or damage to endothelial function, thus contributing to a better understanding of the mechanisms underlying the dose-dependent relationship between radiation and cellular effects. Summary and Conclusion The impact of irradiation (IR) on endothelial cells (ECs) varies depending on the dose administered. Low doses of radiation (<1 Gy) have the potential to enhance EC function, while moderate-to-high doses can lead to EC i, RITM0040077, The impact of irradiation (IR) on endothelial cells (ECs) varies depending on the dose administered. Low doses of radiation (<1 Gy) have the potential to enhance EC function, while moderate-to-high doses can lead to EC impairment. Mitochondria are the primary cellular components affected by IR, and this study explored their role in the protection or damage of ECs at low and high gamma doses. Human dermal microvascular ECs (HMEC-1) were exposed to varying IR doses (0.1 Gy - 8 Gy, ~ 0.4 Gy/min) in the AFRRI 60-Cobalt facility. Results indicated that moderate-to-high doses (? 1 Gy) led to a dose-dependent reduction in clonogenic growth, increased H2AX phosphorylation, elevated p53 levels, and enhanced senescence-associated ?-galactosidase (SA-?-Gal) activity, p21 expression, and overproduction of ROS. However, low radiation doses (0.25 Gy) significantly increased clonogenic growth by 15% without detectable changes in H2AX phosphorylation, p53 level, SA-?-Gal activity, p21 expression, or ROS production. Mitochondrial potential (??m) and morphology were also assessed, revealing that radiation doses ? 1 Gy decreased ??m, while low doses had no effect. Furthermore, all doses, except for 0.1 Gy, increased the percentage of elongated mitochondria, with low doses having a greater effect than high doses. However, only moderate-to-high doses caused an increase in mitochondrial fragmentation/swelling. The study further revealed that low doses induced mitochondrial elongation via an increase in mitochondrial fusion protein 1 (Mfn- 1), while moderate-to-high doses caused mitochondrial fragmentation via a decrease in optic atrophy protein 1 (Opa-1). In conclusion, the study demonstrated, for the first time, that the effects of radiation on ECs are dosedependent, with moderate-to-high doses leading to EC damage and low doses enhancing EC function, by changing mitochondrial morphology.

6.5 7.0 7.5 8.0 8.5 9.0 9.5 Probit analysis of Male C57BL/6 TBI Survival Dose (Gy) %Mortality 1 10 30 50 70 90 99 LD50/30 7.9 Gy LD70/30 8.1 Gy LD90/30 8.4 Gy 6 7 8 9 10 Probit analysis of Female C57BL/6 TBI Survival Dose (Gy) % Mortality 1 10 30 50 70 90 99 LD50/30 8.1 Gy LD70/30 8.4 Gy LD90/30 8.7 Gy 6 7 8 9 10 Sex as a Radiation Dose Modifier TBI Model Dose (Gy) % Mortality 1 10 30 50 70 90 99 Female Data Male Data LD50/30 Males 7.9Gy LD50/30 Females 8.1Gy Females vs. Males DRF 1.03 (95% CI 1.01-1.053) A B C Abstract Comparing animal survival with Countermeasure X in Co60 and TRIGA (65% neutron 35% gamma) Disclaimer: The opinions and assertions expressed herein are those of the author(s) and do not necessarily reflect the official policy or position of the Uniformed Services University or the Department of Defense. None of the authors or their family members have a financial interest in any commercial product, service, or organization providing financial support for this research. The work was supported by NIAID IAA, JPC-7, and AFRRI Intramural Research. In the current geopolitical climate the threat of exposure to ionizing radiation is heightened both as a result of intentional means such as weapon detonation and unintentional means such as accidents. It is imperative that medical countermeasure be developed and available for use in such instances. The detonation of a nuclear weapon will result in the production of various qualities of radiation; initially producing neutrons with relatively high linear energy transfer (LET), which will result in the activation of environmental materials and the sustained presence of relatively lower LET gamma irradiation in the wake of the nuclear fallout. Development of radiation countermeasures should be framed in the context of radiation of different qualities such that they are effective regardless the LET associated with the exposure. At the Armed Forces Radiobiology Research Institute we have established murine models of t, RITM0041851, In the current geopolitical climate the threat of exposure to ionizing radiation is heightened both as a result of intentional means such as weapon detonation and unintentional means such as accidents. It is imperative that medical countermeasure be developed and available for use in such instances. The detonation of a nuclear weapon will result in the production of various qualities of radiation; initially producing neutrons with relatively high linear energy transfer (LET), which will result in the activation of environmental materials and the sustained presence of relatively lower LET gamma irradiation in the wake of the nuclear fallout. Development of radiation countermeasures should be framed in the context of radiation of different qualities such that they are effective regardless the LET associated with the exposure. At the Armed Forces Radiobiology Research Institute we have established murine models of total body irradiation (TBI) using both our Training, Research, Isotopes, General Atomics (TRIGA) reactor and our 60Co facilities. The former is capable of producing a mixed field of neutron and gamma irradiation and the latter produces gamma irradiation. Here we describe the observed differences between these two exposure environments including lethality and extent of hematopoietic injury and progression of recovery, as well as the efficacy of promising countermeasures in both of these environments.

High-LET radiation induces less injury in the bone marrow of female mice than in male mice due to increasing IL-2 and SAA, maintaining CD27, and lowering the miR-34a-regulated pyroptosisin female mice RESULTS MATERIALS & METHDS INTRODUCTION ExposurePreviousreportshaveindicatedthatmixed-field(neutron+gamma)irradiationcausessignificantlyincreasedmortality,decreasedsurvivaltimeandlatencyinacuteradiationsyndrome(ARS),andevendelayedhealingofinjuries(woundand/orburn)sufferedincombinationwithradiation[LedneyandElliott2010]comparedwithpurephotonirradiation.Asmechanismsofinjurybylow-LETradiationaredifferentfromthoseofhigh-LETradiation,thismayaffectdoseassessmentandcountermeasureefficacy[MacVittieetal.1991;Ledneyetal.1991and2000;Caryetal.2012].High-LETincreasesC-reactiveprotein(CRP),complementcomponent3(C3),IgMandPGE2incirculation[KiangandLedney,2013].Moreover,reductionofbloodcellcountsandincreasesincirculatingIL-18,G-CSFandGM-CSFafterhigh-LET[Ossetrovaetal.,2018a]aregreaterthanthatobservedafterlow-LET[Ossetrovaetal.,2018b;Kiangetal.,2018].In2018,SusannaRosiandhercolleagues[Krukowskietal.,2018]investigatedforthefirsttimehowcombinedgalacticcosmicradiation(GCR)exposureimpactslong-termbehavioralandcellularresponsesinmaleandfemalemousebrains.TheyfoundthatasingleexposuretosimulatedGCRinducedlong-termcognitiveandbehavioraldeficitsinmalecohorts,includingdiminishedsocialinteraction,increasedanxiety-likephenotypeandimpairedrecognitionmemory.Remarkably,femalecohortsdidnotdisplayanycognitiveorbehavioraldeficitsafterGCRexposure.Theirresultsidentifiedsex-dependentdifferencesinbehavioralandcognitivedomains.Inconcordancewiththesefindings,ourpreviousreports[Kiangetal.,2022]observedthathigh-LETradiationinducedhigherlevelsofmiR-34ainmaleileumcomparedtofemaleileum.Moreover,histologicalexaminationsofsternumsandileumsalongwithassessmentsoftheirtissuecitrullinelevels(biomarkerforentero-epithelialcellproliferation)suggestedorganinjurytobemoresevereinmalesthanfemales.Inthisreport,weinvestigatedbiomo, RITM0041736, Overexposure to ionizing radiation (IR) is life-threatening in nuclear and radiological incidents. IR depletes blood cell sand increases circulating cytokine/chemokine concentrations as well as mortality. It is reported that microglia cells of female mice are less damaged than those in male mice by IR. We have reported that the GI of female mice is more resistant to IR than the GI of male mice mediated by increases in G-CSF which down-regulate the IR-induced elevation of miR-34a and Bax, leading to decreases in both caspase 3-dependent apoptosis and caspase 1-dependent pyroptosis. It is unclear whether sex varied physiopathological responses in bone marrow (BM) and/or how it happened. We exposed B6D2F1 male and female mice to 0,1.5,3,or 6 Gy with radiation containing 67% neutrons and 33% gamma at 0.6 Gy/min. Blood, sternums and femurs were collected on days 1, 4,and 7. On day 7, IR increased adipocytes and depleted BM cells and megakaryocytes in a radiation dose-dependent manner. However, females exhibited less damage in sternum BM histology than males, which was also confirmed with Flt-3 lig and measurements. IR increased cytokines/chemokines in femurs of females and males in a dose-dependent manner, Cytokines and chemokines generally peaked on day 4 and declined on day 7 in femoral BM cells of both males and females. However, IL-2 (a promoter of T reg cells) increased on days 1 and 4 and returned to the baseline on day 7 in females but decreased on days 4 and 7 in males. IR persistently increased serum amyloid A (SAA) and maintained CD 27 (for Ig production) from day 1 to day 7 in females, where as IR decreased both on day 7 in males. MiR-34a is known to trigger gasderm in D-mediated pyroptosis viacaspase-1 activation. IR significantly increased mi R-34a, caspase-1 activation and gasderm in D activation in males but did little in females, suggesting the presence of pyroptosis in males but not females. No apoptosis was found with BM. The results indicate that BM in

CBC recovery in male and female mice Disclaimer LA-GM-CSF by Calibr The opinions and assertions expressed herein are those of the author(s) and do not necessarily reflect the official policy or position of the Uniformed Services University or the Department of Defense. None of the AFRRI/USUHS authors or their family members have a financial interest in any commercial product, service, or organization providing financial support for this research. AW and SJ are an employees at Calibr and therefore have a financial interest in the development of the product. The work was supported by NIAID AFRRI Interagency Agreement. Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that stimulates bone marrow to divide and differentiate into neutrophils and macrophages. Calibr, a division of Scripps Research has developed a product mPDM608 which is a long-acting granulocyte-macrophage colony stimulating factor (LA GM-CSF) construct, which is a mouse specific construct, a fusion of the FDA approved antibody synagis with murine GM-CSF. The major drawback with the FDA approved medical countermeasure, rhuGM-CSF is its short half-life, currently daily dosing is required for recovery from exposure to myelosuppressive doses of radiation. To this end, a push to develop a long acting formulation has emerged. LA GM-CSF is designed for less frequent dosing. In this study, we demonstrate, a single dose of LA-GM-CSF protected mice from lethal dose of radiation, accelerated recovery from radiation-induced peripheral blood cytopenia, as well as attenuated protein biomarkers of hematopoietic acute radiation syndrome (H-ARS). We found that a dose of 10 mg/kg was well tolerated in non-irradiated mice. The compound was tested for its ability to increase survival efficacy in animals, 3.0 mg/kg of LA-GM-CSF was administered once subcutaneously 24 h post-exposure. Efficacy was validated in female cohorts. There was significantly accelerated recovery from radiation-induced p, RITM0041791, Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that stimulates bone marrow to divide and differentiate into neutrophils and macrophages. Calibr, a division of Scripps Research has developed a product mPDM608 which is a long-acting granulocyte-macrophage colony stimulating factor (LA GM-CSF) construct, which is a mouse specific construct, a fusion of the FDA approved antibody synagis with murine GM-CSF. The major drawback with the FDA approved medical countermeasure, rhuGM-CSF is its short half-life, currently daily dosing is required for recovery from exposure to myelosuppressive doses of radiation. To this end, a push to develop a long acting formulation has emerged. LA GM-CSF is designed for less frequent dosing. In this study, we demonstrate, a single dose of LA-GM-CSF protected mice from lethal dose of radiation, accelerated recovery from radiation-induced peripheral blood cytopenia, as well as attenuated protein biomarkers of hematopoietic acute radiation syndrome (H-ARS). We found that a dose of 10 mg/kg was well tolerated in non-irradiated mice. The compound was tested for its ability to increase survival efficacy in animals, 3.0 mg/kg of LA-GM-CSF was administered once subcutaneously 24 h post-exposure. Efficacy was validated in female cohorts. There was significantly accelerated recovery from radiation-induced peripheral blood cytopenia in males treated with LA-GM-CSF up to day 15 post-TBI, no difference was observed in the CBC counts in females during this period. The drug also increased bone marrow cellularity and megakaryocytes and accelerated multi-lineage hematopoietic recovery. In addition, LA-GM-CSF inhibited radiation-induced hematopoietic biomarkers and inflammatory cytokines. Significant survival benefit with LA-GM-CSF along with hematopoietic recovery suggests that the drug could be developed as a novel radiation countermeasure for personnel who might exposed to lethal dose of irradiation aftermath of a radiation event.