Delayed renal injury in survivors of hematologic acute radiation syndrome

Abstract

Purpose

A mass casualty disaster involving radiological or nuclear agents continues to be a public health concern which requires consideration of both acute and late tissue toxicities in exposed victims. With the advent of advanced treatment options for the mitigation of hematological injuries, there are likely to be survivors of total body irradiation (TBI) exposures as high as 8–10 Gy. These survivors are at risk for a range of delayed multi-organ morbidities including progressive renal failure.

Material and methods

Here, we established the WAG/RijCmcr rat as an effective model for the evaluation of medical countermeasures (MCM) for acute hematologic radiation syndrome (H-ARS). The LD_50/30 dose for adult and pediatric WAG/RijCmcr rats was determined for both sexes. We then confirmed the FDA-approved MCM pegfilgrastim (peg-GCSF, Neulasta®) mitigates H-ARS in adult male and female rats. Finally, we evaluated survival and renal dysfunction up to 300 d post-TBI in male and female adult rats.

Results

In the WAG/RijCmcr rat model, 87.5% and 100% of adult rats succumb to lethal hematopoietic acute radiation syndrome (H-ARS) at TBI doses of 8 and 8.5 Gy, respectively. A single dose of the hematopoietic growth factor peg-GCSF administered at 24 h post-TBI improved survival during H-ARS. Peg-GCSF treatment improved 30 d survival from 12.5% to 83% at 8 Gy and from 0% to 63% at 8.5 Gy. We then followed survivors of H-ARS through day 300. Rats exposed to TBI doses greater than 8 Gy had a 26% reduction in survival over days 30–300 compared to rats exposed to 7.75 Gy TBI. Concurrent with the reduction in long-term survival, a dose-dependent impairment of renal function as assessed by blood urea nitrogen (BUN) and urine protein to urine creatinine ratio (UP:UC) was observed.

Conclusion

Together, these data show survivors of H-ARS are at risk for the development of delayed renal toxicity and emphasize the need for the development of medical countermeasures for delayed renal injury.

Keywords:

• Acute radiation syndrome (ARS)
• haematology – radiation
• growth factors
• multiple organ failure (MOF)
• radiation-induced tissue toxicity
• total body irradiation (TBI)

Introduction

In the event of a nuclear detonation in a major metropolitan area, there are likely to be several hundred thousand civilian victims of radiation exposure encompassing both sexes and all ages. For this reason, it is critical to evaluate medical countermeasures (MCM) for radiation injury in both adult and pediatric animal models. Among Dr. John Moulder’s many achievements in the field of radiation MCM development was the refinement and characterization of the WAG/RijCmcr rat strain for use as a highly reproducible model for radiation biology (Medhora et al. 2019). As part of this special IJRB issue in honor of Dr. Moulder, we have further characterized the WAG/RijCmcr rat strain as a model for H-ARS and delayed renal injury. Previously, we demonstrated the WAG/RijCmcr rat model of partial-body irradiation (PBI) with minimal bone marrow sparing (∼8%) is an effective model for the evaluation of mitigators to delayed radiation injury to the lung, heart, and kidneys. In this current study, we have defined the dose-response relationship (DRR) and natural history of hematopoietic acute radiation syndrome (H-ARS) in WAG/RijCmcr rats exposed to total body irradiation (TBI). To demonstrate the utility of this model for MCM evaluation, we evaluated pegfilgrastim (peg-GCSF, Neulasta®) which is FDA-approved for the treatment of ARS. Since victims exposed to potentially lethal hematopoietic doses (5–10 Gy) may be rescued from early mortality with hematopoietic growth factor treatment such as pegfilgrastim (Moulder 2014), we also evaluated long-term survival and the incidence of renal dysfunction in adult survivors of H-ARS. Here, we demonstrate survivors of doses above 8 Gy are at greater risk for the development of late renal dysfunction.

Methods

Animal care

All studies described were performed in accordance with an approved Institutional Animal Care and Use Committee protocol. WAG/RijCmcr rats were bred and maintained in a barrier facility at our institution. Rats, at weaning were provided Harlan Teklad Global Rodent Diet 8904 (Gamma sterilized 8604 global diet-high antioxidant diet, 350–650 mg/kg isoflavones, Envigo, Madison WI). In general, the feed is provided ad libitum from the feeder rack within the cage. Two weeks prior to irradiations rats are switched to Teklad 2018 diet (moderate antioxidant diet, 150–250 mg/kg isoflavones). All rats were provided reverse osmosis hyper-chlorinated water ad libitum.

Age determination for pediatric cohorts

The WAG/RijCmcr strain reaches sexual maturity at 65 d (Medhora et al. 2019). Based on the lifespan of the laboratory rat and the time to reach sexual maturity, it is estimated that rats aged 5–6 weeks are at an equivalent age of 7–9 years in a human (Sengupta 2013).

Total body irradiation (TBI) model

Adult female (n = 116) and male (n = 125) WAG/RijCmcr rats (11–12 weeks old) were exposed to doses ranging from 6.5 to 8.5 Gy (TBI). Pediatric female (n = 125) and male (n = 170) WAG/RijCmcr rats (5–6 weeks old) were exposed to doses ranging from 5.5 to 7.5 Gy total body TBI. All rats were irradiated without the use of anesthetics by being placed in a plastic jig, and the entire body was exposed posterior to anterior using an XRAD 320 kV orthovoltage x-ray system (Precision X-Ray, Madison, Connecticut). The x-ray system was operated at 320 kVp and 13 mAs with a half-value layer of 1.4 mm copper and a dose rate of 169 cGy/min for adult females, 173 cGy/min for adult males and 173 cGy/min for juvenile rats. The jigs were placed on a plane perpendicular to the direction of the beam, with the distance from the source to the midline of rats set at 61.0 cm for adult females, 60.8 cm for adult males, and 60.5 for juvenile rats. Collimator jaws and dosimetry were used as previously described (Medhora et al. 2014). The irradiation field at the midline was large enough to cover both chambers with adequate (at least 2 cm) margins. This geometry resulted in a uniform dose gradient across the body. Non-irradiated control rats were included for body weight and CBC comparisons (10 adult females, 9 adult males, 5 pediatric females, and 5 pediatric male rats).

Pegfilgrastim (neulasta®)

Pegylated GCSF (pegfilgrastim, Neulasta®) was administered subcutaneously 24 h after TBI at a dose of 0.55 mg kg⁻¹. Peg-GCSF was given to 36 WAG/RijCmcr (18 male and 18 female) adult rats exposed to X-ray doses of 8–9 Gy. The dose for Neulasta® was selected based on prior studies in our lab using PEGylated G-CSF (Gasperetti et al. 2021).

Determination of morbidity

In accordance with our IACUC protocol, morbidity was used as a surrogate marker for death as death is not a humane study endpoint. Animals were humanely euthanized if any of the following single criteria for euthanasia were met: a comatose state or minimal response to tactile stimulation, open-mouthed breathing, or a blood urea nitrogen (BUN) level >120 mg/dl. Additionally, animals that met two or more of the following criteria were humanely euthanized: severe emaciation characterized by extremely prominent skeletal structure, respiratory distress, severe ataxia, and severe morbidity characterized by marked depression of activity, reluctance to move upon prodding, or piloerection.

Body weight change

Body weight was measured twice per week through day 30 and weekly thereafter. Percent body weight change was calculated from the rat’s weight on the day of irradiation.

Blood collection

Blood was collected from anesthetized rats via the jugular vein. Briefly, rats were restrained with one hand by positioning the forelimbs in the caudo-dorsal direction with the thumb and middle finger. The head of the rat was secured with the index finger, creating a straight line along the ventral side of the rat. A 23-gauge needle attached to a syringe was carefully inserted into either the right or left external jugular vein and blood was collected. Syringes were coated with EDTA to prevent clotting for all CBC blood draws.

Urine collection

Rats were singly housed in metabolic cages for 24 h to collect urine samples. At collection, each sample was tested with an Albustix reagent strip (Siemens 2191) for a general protein level.

Peripheral blood analysis

Complete blood counts (CBC) were determined using a Heska Element HT5 Veterinary Hematology Analyzer on a subset of rats. CBC were measured at day 3, 7, 10, 13, 17, 21, and 30 d after irradiation.

Blood urea nitrogen (BUN) determination

Starting at 90 d post-irradiation, BUN levels were monitored every 30 d until study termination using a urease-nitroprusside colorimetric assay (Pointe Scientific #B7551-120 kit).

Urine protein urine creatinine ratio (up:UC) determination

Urine protein and creatinine levels were determined using commercial kits (protein, Bradford reagent Sigma #B6916, and creatinine, Teco Diagnostics #C515-480). Urine protein excretion was expressed as the ratio of urine protein to urine creatinine (UP:UC) which accounts for the known urine-concentrating defect that occurs in renal injury and to normalize for animal size differences (Moulder et al. 2011).

Renal histopathology

Kidneys were harvested, fixed, and embedded in paraffin as described previously (Gao et al. 2021). Whole mount kidney sections (4 μm thick) were stained with Masson’s trichrome. Embedding, cutting, and staining were carried out by the histology core of Children’s Hospital of Wisconsin. Slides were viewed and photographed on EVOS M5000 Microscope (Invitrogen).

Statistical analyses

Statistical analysis was performed using GraphPad Prism version 9 (GraphPad Software, Inc.). p-values for comparison of survival curves were determined using Log-rank (Mantel-Cox) test. Two-way ANOVA with multiple comparisons test was used for the assessment of UP:UC and BUN. Dose-response curves were fit by probit regression in IBM SPSS and used to determine 50% lethal doses (LD₅₀).

Results

Adult and pediatric survival through H-ARS

Although there are well-established mouse models for hematological injury during ARS, it is necessary under the FDA Animal Efficacy rule to demonstrate efficacy in more than one species. To establish the WAG/RijCmcr rat as an alternate species for MCM evaluation, here we have rigorously defined the dose-response relationship (DRR) in the WAG/RijCmcr rat in both sexes as well as adult and pediatric rats. We evaluated survival during H-ARS, after a single dose of TBI ranging from 6.5 to 8.5 Gy in 11–12 week-old adults and 5.5 to 7.5 Gy in 5–6 week-old pediatric rats. Figure 1 depicts the percent morbidity in adult females (Figure 1(A)), adult males (Figure 1(C)), pediatric females (Figure 1(E)), and pediatric males (Figure 1(G)) rats. Percent body weight changes for the respective cohorts are shown adjacent to the survival curves (Figure 1(B,D,F,H)). Both adult sexes show a dose-dependent increase in morbidity across the range of doses tested (Figure 1(A,C)). However, in pediatric male and female rats (Figure 1(E,G)), doses lower than 6.25 Gy result in similar morbidity levels (15%–25% morbidity). At doses above 6.25 Gy in pediatric rats, both sexes exhibit a dose-dependent increase in morbidity.

Figure 1. Dose-dependent lethality observed after total body irradiation in female and male WAG/RijCmcr rats. (A) Survival and (B) percent body weight change in adult (11–12-week-old) female rats which received TBI at doses ranging from 6.5 to 8.5 Gy. (C) Survival and (D) percent body weight change in adult (11–12-week-old) male rats which received TBI at doses ranging from 6.5 to 8.5 Gy. (E) Survival and (F) percent body weight change in pediatric (5–6-week-old) female rats which received TBI at doses ranging from 5.5 to 7.0 Gy. (G) Survival and (H) percent body weight change in pediatric (5–6-week-old) male rats which received TBI at doses ranging from 5.5 to 7.5 Gy.

Age differences in a dose-response relationship

Mortality through 30 d (ARS) was plotted as a function of dose to determine the respective LD_50/30 dose (with 95% confidence limit) using probit analysis for females (Figure 2(A)) and males (Figure 2(B)) rats. For pediatric rats, doses above 6 Gy were used for probit analysis. The LD_50/30 for adult female rats is 7.71 (CI: 7.61–7.85) Gy versus an LD_50/30 of 6.47 (CI: 6.14–8.12) Gy for pediatric female rats. Comparing these probit curves for adult and pediatric female rats the dose modifying factor (DMF) was 1.19. The LD_50/30 for adult male rats is 7.71 Gy (CI: 7.62–7.81) Gy versus an LD_50/30 of 6.45 (CI: 6.01–7.11) Gy for pediatric male rats. Similar to female rats, the dose modifying factor (DMF) for adult versus juvenile male rats was 1.19. No differences were seen when comparing the same-aged female to male rats.

Figure 2. Age-Related Differences in LD_50/30. (A) Percent morbidity in pediatric and adult female rats (individual data points) was fit with a probit regression analysis (solid line) to determine the expected percent mortality as a function of radiation dose. The calculated LD_50/30 (red dashed line) with 95% confidence interval (gray region) for adult and pediatric female rats are 7.71 (CI: 7.61–7.85) Gy and 6.47 (CI: 6.14–8.12), respectively. The dose modifying factor (DMF) for female rats is 1.19. (B) Percent morbidity in pediatric and adult male rats (individual data points) was fit with a probit regression analysis (solid line) to determine the expected percent mortality as a function of radiation dose. The calculated LD_50/30 (red dashed line) with a 95% confidence interval (gray region) for adult and pediatric male rats are 7.71 Gy (CI: 7.62–7.81) Gy and LD_50/30 of 6.45 (CI: 6.01–7.11) Gy, respectively. The dose modifying factor (DMF) for male rats is 1.19.

Peripheral hematology at the LD50/30

Complete blood cell counts (CBCs) were measured at days 3, 7, 10, 13, 17, 21, and 30 after TBI at the respective ∼ LD_50/30 for adult and pediatric rats (7.75 and 6.5 Gy, Figure 3). The time course and nadir for CBCs was similar in pediatric animals exposed to 6.5 Gy as adult animals exposed to 7.75 Gy for all CBC measures. The nadir in lymphocyte counts (Figure 3(C,D)) occurred first at day 7 post-TBI. Total white blood cell counts (WBCs, Figure 3(A,B)), neutrophils (Figure 3(E,F)), and platelets (Figure 3(G,H)) have a similar nadir around day 12–13 post-TBI.

Figure 3. Time courses for white blood cell, lymphocyte, neutrophil, and platelet counts at the LD_50/30 for female and male, adult (7.75 Gy), and pediatric (6.5 Gy) rats are shown in panels (A–H). Complete blood counts were assessed from day 3 to 30 after TBI. Data are presented as means and 95% CI for non-irradiated rats (open symbols) and TBI rats (solid symbols).

Pegfilgrastim for mitigation of ARS

We next evaluated whether a single dose of pegylated G-CSF (peg-G-CSF, pegfilgrastim) administered at 24 h following TBI mitigates lethality at TBI doses above the LD_50/30 for adult male and female rats (8.0, 8.5, and 8.75 Gy). Survival in female and male rats are shown respectively in Figure 4(A,B). For comparison, the survival following 8 and 8.5 Gy TBI in untreated adult rats (Figure 1(A,C)) is included. Peg-GCSF administered at 24 h post-TBI improved 30 d survival from 12.5% to 83% at 8 Gy and from 0% to 63% at 8.5 Gy.

Figure 4. Kaplan–Meier (KM) survival plots after TBI (A) in adult female and (B) male WAG/RijCmcr rats treated with peg-GCSF (dashed lines) subcutaneously at 24 h after TBI. For comparison, survival curves from untreated female and male rats exposed to 8 and 8.5 Gy are included for comparison (solid lines). Peg-G-CSF significantly improves survival at the 8 and 8.5 Gy doses. Log-rank p-values for 8 Gy + peg-G-CSF versus 8 Gy alone for female and male rats are 0.0389 and 0.0009, respectively. Log-rank p-values for 8.5 Gy + peg-G-CSF versus 8.5 Gy alone for female and male rats are 0.05 and 0.0016, respectively. (B) Percent morbidity in pooled male and female rats (solid points) was fit with a probit regression analysis (solid line) to determine the expected percent mortality as a function of radiation dose. The calculated LD_50/30 (red dashed line) with a 95% confidence interval (gray region) for untreated adult rats was 7.71 (7.6–7.8) Gy and for peg-G-CSF treated rats was 8.41 (8.2–8.6) Gy. The dose modifying factor (DMF) for Neulasta treatment is 1.09.

No benefit of peg-GCSF was observed at 8.75 Gy or above. Male and female rats were pooled for probit analysis (Figure 4(C)) and the LD_50/30 for peg-GCSF treatment was determined to be 8.41 (8.2–8.6) Gy. This is increased from 7.71 (7.6–7.8) Gy in untreated pooled male and female rats (Figure 4(C)). Thus, the dose modifying factor (DMF) for peg-GCSF treatment in adult rats is 1.09.

Long-term survival in H-ARS survivors

To determine if acute radiation dose affects long-term survival, we compared survival to day 300 in adult rats exposed to 7.75 Gy (9 male and 8 female) to rats treated with pegfilgrastim exposed to doses of 8–8.5 Gy (12 male and 8 female). By day 300, no deaths were observed in rats exposed to 7.75 Gy (Figure 5(A)). However, five rats (26%) required euthanasia due to severe morbidity (emaciation, marked depression of activity) by day 300 in the cohort exposed to doses greater than 8 Gy which received peg-GCSF treatment. Morbidities in the 8 Gy and above rats treated with peg-GCSF were primarily attributed to renal failure based on gross necropsy and pathological evidence of kidney fibrosis (Figure 5(B)). Renal function was assessed non-invasively in survivors at day 300 by measurement of urine protein creatinine ratio (UP:UC; Figure 5(C)) and blood urea nitrogen levels (BUN; Figure 5(D)). Here, female rats were more sensitive to radiation-induced increases in UP:UC ratio than male rats (Figure 5(C)). A positive association with radiation dose was observed in both UP:UC and BUN levels (Figure 5(C,D)). At 8.5 Gy, both renal function measures were significantly elevated in male and female rats compared to 0 Gy age-matched controls (Figure 5(C,D)).

Figure 5. (A) Kaplan–Meier (KM) survival plots days 30–300 in pooled male and female adult WAG/RijCmcr rats exposed to either 7.75 Gy (black line) or 8–8.5 Gy (peg-GCSF treated, red line). Survival at doses above 8 Gy is significantly reduced by about 26% with a log-rank p-value of 0.025. (B) Representative Masson’s trichrome staining of kidneys from a non-irradiated aged rat (left) and a rat euthanized at day 265 following 8.5 Gy TBI (right). A dashed white region indicates fibrotic Trichrome-stained glomeruli. Scale bar = 150 µm. (C) Urine protein/creatinine ratio measured at day 300 in female and male rats. (8 and 8.5 Gy doses received peg-GCSF). (D) BUN levels measured at day 300 in female and male rats. (8 and 8.5 Gy doses received peg-GCSF).

Discussion

If an act of radiological terrorism or nuclear power plant accident were to occur in the USA, approximately 22% of the victims would be children under age 18. Children are likely to absorb higher doses due to their smaller body and organ size (Linet et al. 2018). The current FDA-recommended treatment regimen for H-ARS in both exposed adults and children is granulocyte-colony stimulating factors in addition to supportive care and antibiotics (Harrold et al. 2020). However, there are currently no FDA-approved MCMs for the late effects of radiation injury. This is a particular concern for pediatric populations who are likely to survive for many decades following exposure. Here, we have established both pediatric and adult rat models for the evaluation of MCMs of ARS.

In the acute response to radiation exposure, pediatric rats aged 5–6 weeks with a human equivalent age of 7–9 years were found to have a greater susceptibility to hematopoietic death than adult rats as evidenced by a DMF of 1.19. This is consistent with Patterson et al., who showed mice are most radiosensitive at age 3 weeks with a human equivalency of 8.7 years (Patterson et al. 2021). However, while sex differences in hematopoietic syndrome have been reported in the murine model (Patterson et al. 2021), we did not observe a sex difference in this study. In the adult rat model, we have confirmed that peg-G-CSF (pegfilgrastim, Neulasta) provides a survival benefit as has been reported in the mouse (Chua et al. 2014), non-human primate (Hankey et al. 2015), and mini-pig models (Legesse et al. 2019). Here we were able to evaluate peg-G-CSF at a range of TBI doses and determine a DMF of 1.09 for peg-G-CSF treatment. To our knowledge, this study is the first to report a DMF for peg-G-CSF in the mitigation of H-ARS.

In survivors of H-ARS at doses of 8–8.5 Gy, we saw a significant reduction in survival to day 300 as compared to 7.75 Gy. Concurrent with the reduction in survival, we observed dose-dependent increases in endogenous measures of renal function: BUN and UP:UC. These findings are consistent with work from the Orschell lab documenting progressive renal dysfunction and histological evidence of renal fibrosis in the C57Bl/6 following TBI doses higher than 8.5 Gy (Unthank et al. 2015; Unthank et al. 2019; Miller et al. 2020). In the current study, female rats were more sensitive to radiation-induced renal injury than male rats, as evidenced by significantly increased urine P/C ratio at all doses above 7.75 Gy. Conversely, UP:UC was only significantly increased at the 8.5 Gy dose in male rats. Moulder et al. previously demonstrated WAG/RijCmcr rats receiving TBI doses above 7 Gy with a rescue bone marrow transplant (Moulder et al. 2011) exhibit elevated BUN levels and renal dysfunction by 180 d. In the current study, at a TBI dose of 7.75 Gy, we observed only a slight non-significant increase in BUN by day 300. The explanation for the difference in renal progression between the current study and Dr. Moulder’s work may have to do with the transplanted bone marrow as transplant-specific toxicities distinct from myeloablative conditioning regimens are known to increase the risk for renal dysfunction in bone marrow transplant recipients (Cohen et al. 2010; Kemmner et al. 2017). Additionally, differences in diet may also contribute to the progression of renal dysfunction in this strain.

While the threshold dose for renal dysfunction is higher in the current study, we do observe the degree of renal dysfunction increases in a dose-dependent manner. This is consistent with both Dr. Moulder’s prior findings (Moulder et al. 2011) and observations in human populations exposed to high-dose radiation (Sera et al. 2013). In a cohort analysis of Japanese atomic bomb survivors, the radiation dose received was significantly associated with chronic kidney disease (Sera et al. 2013). Additionally, declining renal function following radiation exposure is observed in adult and pediatric bone marrow transplant (BMT) recipients who received myeloablative total body radiation conditioning prior to transplant (Lawton et al. 1991; Kal and van Kempen-Harteveld 2006; Watanabe Nemoto et al. 2014). In bone marrow transplant (BMT) recipients, increasing radiation dose to the kidneys is associated with a greater risk of renal dysfunction (Kal and van Kempen-Harteveld 2006) and renal shielding reduces both decline in renal function (Lawton et al. 1991; Juckett et al. 2001) and the incidence of late renal failure (Igaki et al. 2005).

Conclusion

In his 2013 Dade W. Moeller Lecture, Dr. Moulder noted that human survivors of dose above 8 Gy would likely exceed renal tolerance and for this reason survivors of H-ARS would require medical management of renal impairment (Moulder 2014). Here, we have demonstrated this is true as rat survivors of lethal H-ARS are indeed at greater risk for progressive renal failure. The current study establishes the WAG/RijCmcr rat strain as a model for both acute H-ARS and evaluating MCMs against the delayed onset of renal injury in survivors of H-ARS.

Disclosure statement

The authors have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Additional information

Funding

This work was supported by funding from the National Institute of Allergy and Infectious Diseases; NIH/NIAID U01AI133594 (HH), U01AI138331 (HH), NIH/NIAID Contract 75N93020C00005 (DP), the MCW Department of Radiation Oncology, and the MCW Cancer Center.

Notes on contributors

Tracy Gasperetti

Tracy Gasperetti is a lab manager in the Department of Radiation Oncology at the Medical College of Wisconsin, Milwaukee, WI, USA.

Anne Frei

Anne Frei is a research program coordinator in the Department of Radiation Oncology at the Medical College of Wisconsin, Milwaukee, WI, USA.

Guru Prasad Sharma

Guru Prasad Sharma, PhD, is Postdoctoral fellow in the laboratory of Dr. Himburg in the Department of Radiation Oncology at the Medical College of Wisconsin, Milwaukee, WI, USA.

Lauren Pierce

Lauren Pierce is a research technician in the Department of Radiation Oncology at the Medical College of Wisconsin, Milwaukee, WI, USA.

Dana Veley

Dana Veley is a research technician in the Department of Radiation Oncology at the Medical College of Wisconsin, Milwaukee, WI, USA.

Nathan Szalewski

Nathan Szalewski is a research technician in the Department of Radiation Oncology at the Medical College of Wisconsin, Milwaukee, WI, USA.

Srishti Munjal Mehta

Srishti Munjal Mehta, PhD, is a scientist in research and development at Myelo Therapeutics Gmbh, Berlin, Germany.

Brian L. Fish

Brian L. Fish is Program Director of Radiation Biology in the Department of Radiation Oncology at the Medical College of Wisconsin, Milwaukee, WI, USA.

Dirk Pleimes

Dirk D. Pleimes, MD, is a physician-scientist in research and drug development and Chief Executive Officer at Myelo Therapeutics Gmbh, Berlin, Germany.

Heather A. Himburg

Heather A. Himburg, PhD, is an Associate Professor of Radiation Oncology and Biomedical Engineering at the Medical College of Wisconsin, Milwaukee, WI, USA.