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

ABSTRACT: Medical countermeasure development under the US Food and Drug Administration animal rule requires validated animal models of acute radiation effects. The key large animal model is the non-human primate, rhesus macaque. To date, only the rhesus macaque has been used for both critical supportive data and pivotal efficacy trials seeking US Food and Drug Administration approval. The potential for use of the rhesus for other high priority studies such as vaccine development underscores the need to identify another non-human primate model to account for the current lack of rhesus for medical countermeasure development. The cynomolgus macaque, Macaca fascicularis, has an existing database of medical countermeasure development against the hematopoietic acute radiation syndrome, as well as the use of radiation exposure protocols that mimic the likely nonuniform and heterogenous exposure consequent to a nuclear terrorist event. The review herein describes published studies of adult male cynomolgus macaques that used two exposure protocols-unilateral, nonuniform total-body irradiation and partial-body irradiation with bone marrow sparing-with the administration of subject-based medical management to assess mitigation against the hematopoietic acute radiation syndrome. These studies assessed the efficacy of cytokine combinations and cell-based therapy to mitigate acute radiation-induced myelosuppression. Both therapeutics were shown to mitigate the myelosuppression of the hematopoietic acute radiation syndrome. Additional studies being presented herein further defined the dose-dependent hematopoietic acute radiation syndrome of cynomolgus and rhesus macaques and a differential dose-dependent effect with young male and female cynomolgus macaques. The database supports the investigation of the cynomolgus macaque as a comparable non-human primate for efficacy testing under the US Food and Drug Administration animal rule. Critical gaps in knowledge required to validate the models and exposure protocols are also identified.

BABOON RADIATION QUALITY (MIXED-FIELD NEUTRON AND GAMMA, GAMMA ALONE) DOSE-RESPONSE MODEL SYSTEMS: ASSESSMENT OF H-ARS SEVERITY USING HAEMATOLOGIC BIOMARKERS.

Results from archived (1986 and 1996) experiments were used to establish a baboon radiation-quality dose-response database with haematology biomarker time-course data following exposure to mixed-fields (i.e. neutron to gamma ratio: 5.5; dose: 0-8 Gy) and 60Co gamma-ray exposures (0-15 Gy). Time-course (i.e. 0-40 d) haematology changes for relevant blood-cell types for both mixed-field (neutron to gamma ratio = 5.5) and gamma ray alone were compared and models developed that showed significant differences using the maximum likehood ratio test. A consensus METREPOL-like haematology ARS (H-ARS) severity scoring system for baboons was established using these results. The data for mixed-field and the gamma only cohorts appeared similar, and so the cohorts were pooled into a single consensus H-ARS severity scoring system. These findings provide proof-of-concept for the use of a METREPOL H-ARS severity scoring system following mixed-field and gamma exposures.

Use of Proteomic and Hematology Biomarkers for Prediction of Hematopoietic Acute Radiation Syndrome Severity in Baboon Radiation Models.

Use of plasma proteomic and hematological biomarkers represents a promising approach to provide useful diagnostic information for assessment of the severity of hematopoietic acute radiation syndrome. Eighteen baboons were evaluated in a radiation model that underwent total-body and partial-body irradiations at doses of Co gamma rays from 2.5 to 15 Gy at dose rates of 6.25 cGy min and 32 cGy min. Hematopoietic acute radiation syndrome severity levels determined by an analysis of blood count changes measured up to 60 d after irradiation were used to gauge overall hematopoietic acute radiation syndrome severity classifications. A panel of protein biomarkers was measured on plasma samples collected at 0 to 28 d after exposure using electrochemiluminescence-detection technology. The database was split into two distinct groups (i.e., "calibration," n = 11; "validation," n = 7). The calibration database was used in an initial stepwise regression multivariate model-fitting approach followed by down selection of biomarkers for identification of subpanels of hematopoietic acute radiation syndrome-responsive biomarkers for three time windows (i.e., 0-2 d, 2-7 d, 7-28 d). Model 1 (0-2 d) includes log C-reactive protein (p < 0.0001), log interleukin-13 (p < 0.0054), and procalcitonin (p < 0.0316) biomarkers; model 2 (2-7 d) includes log CD27 (p < 0.0001), log FMS-related tyrosine kinase 3 ligand (p < 0.0001), log serum amyloid A (p < 0.0007), and log interleukin-6 (p < 0.0002); and model 3 (7-28 d) includes log CD27 (p < 0.0012), log serum amyloid A (p < 0.0002), log erythropoietin (p < 0.0001), and log CD177 (p < 0.0001). The predicted risk of radiation injury categorization values, representing the hematopoietic acute radiation syndrome severity outcome for the three models, produced least squares multiple regression fit confidences of R = 0.73, 0.82, and 0.75, respectively. The resultant algorithms support the proof of concept that plasma proteomic biomarkers can supplement clinical signs and symptoms to assess hematopoietic acute radiation syndrome risk severity.

Pre-Exposure Gene Expression in Baboons with and without Pancytopenia after Radiation Exposure.

Radiosensitivity differs in humans and likely among primates. The reasons are not well known. We examined pre-exposure gene expression in baboons (n = 17) who developed haematologic acute radiation syndrome (HARS) without pancytopenia or a more aggravated HARS with pancytopenia after irradiation. We evaluated gene expression in a two stage study design where stage I comprised a whole genome screen for messenger RNAs (mRNA) (microarray) and detection of 667 microRNAs (miRNA) (real-time quantitative polymerase chain reaction (qRT-PCR) platform). Twenty candidate mRNAs and nine miRNAs were selected for validation in stage II (qRT-PCR). None of the mRNA species could be confirmed during the validation step, but six of the nine selected candidate miRNA remained significantly different during validation. In particular, miR-425-5p (receiver operating characteristic = 0.98; p = 0.0003) showed nearly complete discrimination between HARS groups with and without pancytopenia. Target gene searches of miR-425-5p identified new potential mRNAs and associated biological processes linked with radiosensitivity. We found that one miRNA species examined in pre-exposure blood samples was associated with HARS characterized by pancytopenia and identified new target mRNAs that might reflect differences in radiosensitivity of irradiated normal tissue.

MicroRNA Expression for Early Prediction of Late Occurring Hematologic Acute Radiation Syndrome in Baboons.

For effective medical management of radiation-exposed persons after a radiological/nuclear event, blood-based screening measures in the first few days that could predict hematologic acute radiation syndrome (HARS) are needed. For HARS severity prediction, we used microRNA (miRNA) expression changes measured on days one and two after irradiation in a baboon model. Eighteen baboons underwent different patterns of partial or total body irradiation, corresponding to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts (BCC) the surviving baboons (n = 17) exhibited mild (H1-2, n = 4) or more severe (H2-3, n = 13) HARS. In a two Stage study design we screened 667 miRNAs using a quantitative real-time polymerase chain reaction (qRT-PCR) platform. In Stage II we validated candidates where miRNAs had to show a similar regulation (up- or down-regulated) and a significant 2-fold miRNA expression difference over H0. Seventy-two candidate miRNAs (42 for H1-2 and 30 for H2-3) were forwarded for validation. Forty-two of the H1-2 miRNA candidates from the screening phase entered the validation step and 20 of them showed a statistically significant 2-4 fold up-regulation relative to the unexposed reference (H0). Fifteen of the 30 H2-3 miRNAs were validated in Stage II. All miRNAs appeared 2-3 fold down-regulated over H0 and allowed an almost complete separation of HARS categories; the strongest candidate, miR-342-3p, showed a sustained and 10-fold down-regulation on both days 1 and 2. In summary, our data support the medical decision making of the HARS even within the first two days after exposure where diagnostic tools for early medical decision are required but so far missing. The miRNA species identified and in particular miR-342-3p add to the previously identified mRNAs and complete the portfolio of identified mRNA and miRNA transcripts for HARS prediction and medical management.

Soluble Vascular Endothelial Cadherin as a New Biomarker of Irradiation in Highly Irradiated Baboons with Bone Marrow Protection.

Vascular endothelial cadherin is the main component of adherens junctions enabling cohesion of the endothelial monolayer in vessels. The extracellular part of vascular endothelial cadherin (VE-cadherin) can be cleaved, releasing soluble fragments in blood (sVE-cadherin). In some diseases with endothelial dysfunction, a correlation between increased blood sVE-cadherin levels and disease state has been proposed. Irradiation is known to induce endothelial damage, but new serum biomarkers are needed to evaluate endothelial damage after irradiation. Here, the authors investigated whether sVE-cadherin may be an interesting biomarker of irradiation in highly irradiated baboons with bone marrow protection. sVE-cadherin was detected in the plasma of young as well as old baboons. Plasma sVE-cadherin levels significantly decrease a few days after irradiation but recover in the late time after irradiation. Kinetic analysis of plasma sVE-cadherin levels suggests a correlation with white blood cell counts in both the acute phase of irradiation and during hematopoietic recovery, suggesting that plasma sVE-cadherin levels may be partly linked to the disappearance and recovery of white blood cells. Interestingly, after hematopoietic recovery was completed, sVE-cadherin levels were found to exceed control values, suggesting that plasma sVE-cadherin may represent a new biomarker of endothelial damage or neovascularization in the late time after irradiation.

Experimental Quantification of Delayed Radiation-Induced Organ Damage in Highly Irradiated Rats With Bone Marrow Protection: Effect of Radiation Dose and Organ Sensitivity.

The evolution of organ damage following extensive high-dose irradiation remains largely unexplored and needs further investigation. Wistar rats [with or without partial bone marrow protection (∼20%)] were irradiated at lethal gamma-ray doses (12, 14, and 16 Gy) and received antibiotic support. While total-body-irradiated rats did not survive, bone marrow protection (achieved by protecting hind limbs behind a lead wall) combined with antibiotic support allowed survival of 12-Gy and 14-Gy irradiated rats for more than 3 mo, with a late phase of body weight loss and altered clinical status. Histological analysis of radiation-induced damages in visceral organs (liver, kidney, and ileum), performed 64 and 104 d after high-dose body irradiation, indicates that the extent and the evolution of damage depend on both the irradiation dose and organ. A dose-related aggravation of lesions was observed in the liver and kidney but not in the ileum. In contrast to the liver, alterations in the kidney and ileum aggravate with time, emphasizing the need to develop new efficient countermeasures to protect both the gastrointestinal tract and kidney from late-occurring radiation effects. Specifically, the complex evolution of organ damage presented in this paper offers the possibility to explore and then validate specific therapeutic windows using candidate drugs targeted to each injured visceral organ.

The extent of irradiation-induced long-term visceral organ damage depends on cranial/brain exposure.

In case of high-dose radiation exposure, mechanisms controlling late visceral organ damage are still not completely understood and may involve the central nervous system. To investigate the influence of cranial/brain irradiation on late visceral organ damage in case of high-dose exposure, Wistar rats were irradiated at 12 Gy, with either the head and fore limbs or the two hind limbs protected behind a lead wall (head- and hind limbs-protected respectively), which allows long-term survival thanks to bone marrow protection. Although hind limbs- and head-protected irradiated rats exhibited similar hematopoietic and spleen reconstitution, a late body weight loss was observed in hind limbs-protected rats only. Histological analysis performed at this time revealed that late damages to liver, kidney and ileum were attenuated in rats with head exposed when compared to animals whose head was protected. Plasma measurements of inflammation biomarkers (haptoglobin and the chemokine CXCL1) suggest that the attenuated organ damage in hind limbs-protected rats may be in part related to reduced acute and chronic inflammation. Altogether our results demonstrate the influence of cranial/brain exposure in the onset of organ damage.

Mitigating radiation-induced toxicity: an overview of new approaches developed at the French Military Biomedical Research Institute.

Acute radiation syndrome represents the clinical response of radiation-sensitive key tissues (i.e., hematopoietic, gastrointestinal and neurovascular) following exposure to high doses of ionizing radiation. In this context, the hematopoietic syndrome remains the first therapeutic challenge. Today, identifying new drugs and developing new strategies to cope with acute radiation syndrome remain a priority, especially to prevent/cure the radiation-induced multiple organ dysfunction syndrome then failure. Cytokine, non-cytokine drugs, and cell/gene therapy represent a pattern of sophisticated approaches developed by this group and others to achieve this goal.

Useful radiation dose biomarkers for early identification of partial-body exposures.

Although less urgent than the physical injuries caused by an unexpected nuclear and radiological (NR) event, radiation damages can be treated more effectively if the intensity and extent of the exposure are evaluated rapidly. In this work, the authors followed within the first 7 d after exposure a few early biomarkers that could be easily deployable (simple and fast sampling and analysis) to determine their potential in distinguishing a total body irradiation (TBI) from a dose-equivalent partial exposure. A plausible accident scenario was created for this study by exposing unilaterally nonhuman primates of body mass comparable to humans to 5 Gy, to either the whole body or to part of it. Using logistic regression analysis, it was shown that both a combination of three parameters together [absolute neutrophil count; monocyte count on the first day; and C-reactive protein (CRP) 5 d after exposure] or CRP 7 d after irradiation completely separated the partial exposures from the TBIs. A quasi-complete separation using receiver-operator characteristic (ROC) was observed for neutrophil count to lymphocyte count ratio during the first day after exposure.

Gene therapy to mitigate radiation-induced bone marrow aplasia: preliminary study in highly irradiated monkeys.

The hematopoietic syndrome represents the first therapeutic challenge following exposure to high doses of ionizing radiation. Today there is a crucial need to identify/develop new treatments in order to reach the transplantation threshold. The authors propose the concept of a global niche therapy strategy based on local and short-term secretion of selected morphogenes to favor a vascular niche in order to raise the transplantation threshold regeneration and to stimulate residual hematopoietic stem and progenitor cells. The present study was aimed at setting up a monkey model of gene therapy using Sonic hedgehog (Shh) as a first candidate. Multipotent mesenchymal stem cells from adipocyte tissues were nucleofected with mock and Sonic hedgehog pIRES2 plasmids using Amaxa technology. 8-Gy gamma irradiated monkeys were given a single intraosseous injection of manipulated or unmanipulated adipocyte stem cells 48 h following total body irradiation. Mock and Shh-grafts were well tolerated. This preliminary study establishes the feasibility of transient gene therapy in highly irradiated monkeys. Ongoing studies will determine the putative efficacy of this therapeutic strategy.

Revisiting emergency anti-apoptotic cytokinotherapy: erythropoietin synergizes with stem cell factor, FLT-3 ligand, trombopoietin and interleukin-3 to rescue lethally-irradiated mice.

We have re-evaluated the benefit of using erythropoietin (Epo) as a pleiotropic cytokine to counteract hematological and extra-hematological toxicity following lethal irradiation. B6D2F1 mice were exposed to a dose of 9 Gy gamma radiation resulting in 90% mortality at 30 days, and then injected with stem cell factor, FLT-3 ligand, thrombopoietin and interleukin-3 [i.e. SFT3] at two and 24 hours with or without Epo (1,000 IU/kg) at 2 hours and day 8. As controls, two groups of irradiated mice were given only Epo or Phosphate-buffered saline. Epo synergized with SFT3 to rescue lethally-irradiated mice from radiation-induced death (survival: 60%, 95% and 5% respectively for SFT3, SFT3+Epo and controls at 30 days, p<0.05), whereas Epo alone exhibited no protective effect. Hematopoietic parameters did not differ significantly between SFT3 and SFT3+Epo groups during the animal death period. Some beneficial effects on gastro-intestinal toxicity were noticed following administration of Epo, although lung, liver and kidney were not protected. Further studies are necessary to understand fully the mechanisms involved in these effects of Epo in order to optimize treatment with cytokines following high-dose irradiation.

Autologous adipocyte derived stem cells favour healing in a minipig model of cutaneous radiation syndrome.

Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Here we examined for the first time in a large animal model the therapeutic potential of autologous adipose tissue-derived stroma cells (ASCs). For experiments, Göttingen minipigs were locally gamma irradiated using a (60)Co source at the dose of 50 Gy and grafted (n = 5) or not (n = 8). ASCs were cultured in MEM-alpha with 10% fetal calf serum and basic fibroblast growth factor (2 ng.mL(-1)) and post irradiation were intradermally injected on days 25, 46, 67 and finally between days 95 and 115 (50 × 10(6) ASCs each time) into the exposed area. All controls exhibited a clinical evolution with final necrosis (day 91). In grafted pigs an ultimate wound healing was observed in four out of five grafted animals (day 130 +/- 28). Immunohistological analysis of cytokeratin expression showed a complete epidermis recovery. Grafted ASCs accumulated at the dermis/subcutis barrier in which they attracted numerous immune cells, and even an increased vasculature in one pig. Globally this study suggests that local injection of ASCs may represent a useful strategy to mitigate CRS.

Radiation victim management and the haematologist in the future: time to revisit therapeutic guidelines?

PURPOSE: The use of nuclear/radiation devices against the civilian population is now a realistic scenario. Haematopoietic syndrome is the primary therapeutic challenge in the case of whole body acute exposure over 2 Grays (Gy) whereas burns and combined injuries would be frequently observed in myelo-suppressed patients. Optimisation of scoring and treatments are important goals to achieve. CONCLUSION: The European Response Category (RC) concept represents an attempt to integratively assess haematological/extrahematological radiation-induced lesions. Based on the frequently observed heterogeneity of bone marrow damage in accidental/intentional irradiations, the stimulation of residual stem cells using granulocyte Colony-stimulating factor remains the therapeutic standard after exposure to less than the lethal dose 50 % (Haematopoietic[H] score 3-H3). Allogeneic stem cell transplantation is indicated in case of medullary eradication (Haematopoietic score 4-H4) whereas extramedullary toxicity may determine the outcome. Especially in case of numerous casualties exhibiting acute radiation syndrome, the administration of survival factor combinations remains questionable, at least as a palliative treatment. In addition pleiotropic cytokines injection such as erythropoietin and keratinocyte growth factor and grafting multipotent mesenchymal stem cells - from underexposed bone marrow areas or fat tissues - could be proposed to prevent multiple organ failure syndrome development. Multi-disciplinary teams should be prepared to manage such patients.

Multipotent mesenchymal stem cell grafting to treat cutaneous radiation syndrome: development of a new minipig model.

OBJECTIVE: Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Recent grafting of three ionizing radiation-burned patients has suggested the benefit of local bone marrow mesenchymal stem cell (MSC) injection in favor of wound healing and pain control. Here, we have developed a new minipig model of severe CRS to study underlying mechanisms of this cell therapy approach. MATERIALS AND METHODS: Göttingen minipigs were locally irradiated using a (60)Co gamma source as follows: ungrafted 50 and 60 Gy (n = 4) and grafted 50 and 60 Gy (n = 3). Bone marrow MSCs were cultured in minimum essential medium with 10% fetal calf serum and basic fibroblast growth factor (2 ng.mL(-1)). Autologous MSCs were intradermally injected twice or three times from days 27 to 96 (range, 99-128.5 × 10(6) MSCs per injection). RESULTS: All animals exhibited a clinical evolution similar to humans after a latency phase of several weeks, including early erythema, hair loss, and dry/moist desquamation followed by necrosis during 81 to 222 days post-ionizing radiation. Skin damage in higher exposed animals appeared slightly earlier. Immunohistology revealed severe skin damage in all animals and rhabdomyolysis in the muscle tissue below the entry area, with the latter being more severe in controls. In grafted animals, MSCs led to local accumulation of lymphocytes at the dermis/subcutis border and improved vascularization. CONCLUSIONS: This study establishes a new minipig model that is close to human and allows development of stem cell therapy strategies that can be applied in treatment of human radiation burns.

Characterisation of normal and cancer stem cells: one experimental paradigm for two kinds of stem cells.

The characterisation of normal stem cells and cancer stem cells uses the same paradigm. These cells are isolated by a fluorescence-activated cell sorting step and their stemness is assayed following implantation into animals. However, differences exist between these two kinds of stem cells. Therefore, the translation of the experimental procedures used for normal stem cell isolation into the research field of cancer stem cells is a potential source of artefacts. In addition, normal stem cell therapy has the objective of regenerating a tissue, while cancer stem cell-centred therapy seeks the destruction of the cancer tissue. Taking these differences into account is critical for anticipating problems that might arise in cancer stem cell-centred therapy and for upgrading the cancer stem cell paradigm accordingly.

Cytokines in combination to treat radiation-induced myelosuppresssion: evaluation of SCF + glycosylated EPO + pegylated G-CSF as an emergency treatment in highly irradiated monkeys.

Multicytokine therapy may be useful to counteract radiation-induced myelosuppression. We assessed the stem cell factor + glycosylated erythropoietin + pegylated granulocyte colony-stimulating factor combination (SEG) as an emergency treatment. SEG in highly irradiated monkeys efficacy appeared to be restricted to granulopoiesis. Early administration of Erythropoietin did not prevent radiation-induced anemia.

Antiapoptotic cytokines in combination with pegfilgrastim soon after irradiation mitigates myelosuppression in nonhuman primates exposed to high irradiation dose.

OBJECTIVE: Preservation of hematopoietic stem and progenitor cells from early radiation-induced apoptosis is the rationale for emergency antiapoptotic cytokine therapy (EACK) after radiation accidents. This strategy is based on the combination of stem cell factor + Flt3-ligand + thrombopoietin + interleukin 3 (SFT3). The long-term safety and efficacy of EACK in managing severe radiation exposure were evaluated. MATERIAL AND METHODS: Early administration of SFT3 + pegfilgrastim was assessed in 7-Gy gamma total body-irradiated (TBI) monkeys. Efficiency of delayed administration was also addressed after 5-Gy TBI. RESULTS: Here we showed that a single, intravenous injection of SFT3 2 hours after 7-Gy TBI reduced the period of thrombocytopenia (platelet count <20 x 10(9)/L: 0.8 +/- 1.5 day vs 23.8 +/- 15.9 days in controls; p < 0.05) and blood transfusion needs. Moreover, addition of pegfilgrastim to SFT3 treatment shortened the period of neutropenia compared with SFT3 and control groups (neutrophil count <0.5 x 10(9)/L: 7 +/- 1.4 days vs 13 +/- 3.2 days and 15.2 +/- 1.5 days; p < 0.05). In both SFT3 groups, bone marrow activity recovered earlier and, in contrast with controls, platelet count returned to baseline values from 250 days after irradiation. Furthermore, delayed (48 hours) single SFT3 administration in 5-Gy irradiated monkeys significantly reduced thrombocytopenia compared to controls. Finally, SFT3 did not increase frequency of total chromosome translocations observed in the blood lymphocytes of controls 1 year after 5 Gy TBI. CONCLUSION: These results suggest the safety and efficacy of EACK in managing severe radiation exposure.

Effects of Hoechst 33342 on C2C12 and PC12 cell differentiation.

Accumulative evidence demonstrates that normal as well as cancer stem cells can be identified as a side population following Hoechst 33342 staining and flow cytometric analysis. This popular method is based on the ability of stem cells to efflux this fluorescent vital dye. We demonstrate that Hoechst 33342 can affect cell differentiation, suggesting potential complications in the interpretation of data.