Revisiting Biomarkers of Total-Body and Partial-Body Exposure in a Baboon Model of Irradiation.

In case of a mass casualty radiation event, there is a need to distinguish total-body irradiation (TBI) and partial-body irradiation (PBI) to concentrate overwhelmed medical resources to the individuals that would develop an acute radiation syndrome (ARS) and need hematologic support (i.e., mostly TBI victims). To improve the identification and medical care of TBI versus PBI individuals, reliable biomarkers of exposure could be very useful. To investigate this issue, pairs of baboons (n = 18) were exposed to different situations of TBI and PBI corresponding to an equivalent of either 5 Gy 60Co gamma irradiation (5 Gy TBI; 7.5 Gy left hemibody/2.5 right hemibody TBI; 5.55 Gy 90% PBI; 6.25 Gy 80% PBI; 10 Gy 50% PBI, 15 Gy 30% PBI) or 2.5 Gy (2.5 Gy TBI; 5 Gy 50% PBI). More than fifty parameters were evaluated before and after irradiation at several time points up to 200 days. A partial least square discriminant analysis showed a good distinction of TBI from PBI situations that were equivalent to 5 Gy. Furthermore, all the animals were pooled in two groups, TBI (n = 6) and PBI (n = 12), for comparison using a logistic regression and a non parametric statistical test. Nine plasmatic biochemical markers and most of hematological parameters turned out to discriminate between TBI and PBI animals during the prodromal phase and the manifest illness phase. The most significant biomarkers were aspartate aminotransferase, creatine kinase, lactico dehydrogenase, urea, Flt3-ligand, iron, C-reactive protein, absolute neutrophil count and neutrophil-to-lymphocyte ratio for the early period, and Flt3-ligand, iron, platelet count, hemoglobin, monocyte count, absolute neutrophil count and neutrophil-to-lymphocyte ratio for the ARS phase. These results suggest that heterogeneity could be distinguished within a range of 2.5 to 5 Gy TBI.

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.

Assessment of total- and partial-body irradiation in a baboon model: preliminary results of a kinetic study including clinical, physical, and biological parameters.

This biodosimetry study used irradiated baboons to investigate the efficacy of a kinetic multiparameter (clinical, physical, and biological) approach for discriminating partial-body irradiation (PBI) and total-body irradiation (TBI). Animals were unilaterally (front) exposed to 60Co gamma rays (8 to 32 cGy min) using either TBI or vertical left hemi-body irradiation (HBI), as follows: 2.5 Gy TBI (n = 2), 5 Gy TBI (n = 2), 5 Gy HBI (n = 2), and 10 Gy HBI (n = 2). Midline tissue doses were measured at the anterior iliac crest level with an ionization chamber, and body dosimetry was performed using thermoluminescent dosimeters. Blood samples were collected before exposure and from 1 h until 200 d after irradiation. Clinical status, complete blood cell count, biochemical parameters, and cytogenetic analysis were evaluated. The partial least square discriminant analysis chosen for statistical analysis showed that the four groups of irradiated baboons were clearly separated. However, the dicentric chromosome assay may not distinguish HBI from TBI in confounding situations where equivalent whole-body doses are similar and the time of exposure is sufficient for peripheral blood lymphocyte homogenization. Interestingly, as bone marrow shielding in HBI animals prevented aplasia from happening, hematologic parameters such as the platelet count and Flt-3 ligand level helped to distinguish HBI and TBI. Moreover, the ratio of neutrophil to lymphocyte counts, creatine kinase, and citrulline levels may be discriminating biomarkers of dose or injury. Both early and delayed clinical signs and bioindicators appear to be useful for assessment of heterogeneous irradiation.

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.

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.

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.

Revisiting therapeutic strategies in radiation casualties.

OBJECTIVE: Nuclear/radiological threats have evolved and scenarios for terrorist attacks involving radioactive material have been identified as complex situations. Mass casualty scenarios may happen, and individuals may be exposed to intentionally hidden sources of high activity, resulting in delayed diagnosis and treatment of acute radiation syndrome (ARS). Moreover, ARS must be considered as an emergency in order to better anticipate delayed radiation toxicity. In this context, therapeutic strategies in radiation casualties have to be revisited and new pharmacological approaches developed. METHODS: B6D2F1 mice were total-body irradiated (TBI) with a 9 Gy gamma dose and then received intraperitoneal doses of either early (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin-3 [SFT3] +/- keratinocyte growth factor (KGF); stem cell factor + erythropoietin + Peg-filgrastim [SEG]) or delayed treatments (SFT3 +/- KGF, erythropoietin, or hyaluronic acid). Survival was monitored and bone marrow hematopoiesis evaluated at 300 days following early treatments. RESULTS: SFT3 anti-apoptotic cytokine combination administered early (2 hours and 24 hours) after lethal TBI induced 60% survival versus 5% in controls. Early SEG treatment may be an alternative to SFT3 in terms of survival (55%), but SEG benefit might be obtained at the expense of long-term hematopoiesis. SFT3 + KGF induced 75% survival. No effectiveness was observed, over antimicrobial supportive care, when administration of SFT3 or its tested combinations was delayed at 48 hours. CONCLUSION: As a potentially multi-organ failure, ARS requires global therapy, beyond the hematopoietic syndrome, which may include pleiotropic cytokines such as KGF.

Mesenchymal stem cells support expansion of in vitro irradiated CD34(+) cells in the presence of SCF, FLT3 ligand, TPO and IL3: potential application to autologous cell therapy in accidentally irradiated victims.

Ex vivo expansion of residual autologous hematopoietic stem and progenitor cells collected from victims soon after accidental irradiation (autologous cell therapy) may represent an additional or alternative approach to cytokine therapy or allogeneic transplantation. Peripheral blood CD34+ cells could be a useful source of cells for this process provided that collection and ex vivo expansion of hematopoietic stem and progenitor cells could be optimized. Here we investigated whether mesenchymal stem cells could sustain culture of irradiated peripheral blood CD34+ cells. In vitro irradiated (4 Gy 60Co gamma rays) or nonirradiated mobilized peripheral blood CD34+ cells from baboons were cultured for 7 days in a serum-free medium supplemented with stem cell factor+thrombopoietin+interleukin 3+FLT3 ligand (50 ng/ml each) in the presence or absence of mesenchymal stem cells. In contrast to cultures without mesenchymal stem cells, irradiated CD34+ cells cultured with mesenchymal stem cells displayed cell amplification, i.e. CD34+ (4.9-fold), CD34++ (3.8-fold), CD34++/Thy-1+ (8.1-fold), CD41+ (12.4-fold) and MPO+ (50.6-fold), although at lower levels than in nonirradiated CD34+ cells. Fourteen times more clonogenic cells, especially BFU-E, were preserved when irradiated cells were cultured on mesenchymal stem cells. Moreover, we showed that the effect of mesenchymal stem cells is related mainly to the reduction of apoptosis and involves cell-cell contact rather than production of soluble factor(s). This experimental model suggests that mesenchymal stem cells could provide a crucial tool for autologous cell therapy applied to accidentally irradiated victims.

Effect of soman poisoning on populations of bone marrow and peripheral blood cells in mice.

According to recent reports, brain lesions resulting from ischemia, mechanical injury or neurodegenerative diseases can be partially treated using bone marrow-derived stromal cell (BMSC) engraftment approaches. Nevertheless, for brain lesions resulting from organophosphate poisoning, nerve agents such as soman (pinacolyl methylphosphono-fluoridate) could affect blood and bone marrow (BM) micro-environments, thus preventing efficient BMSC migration and engraftment. It is therefore necessary to verify the hematologic response to soman exposure. To assess this issue, the survival of BM cells, in particular hematopoietic progenitor and precursor cells (HPC), as well as distribution of the different populations of peripheral blood cells, were investigated in soman-intoxicated mice. Nine-week-old adult male B6D2F1 mice were treated with 110 microg/kg soman and 5.0 mg/kg methyl nitrate atropine. BM and peripheral blood (PB) samples were collected 1, 4, 8 and 22 days after poisoning. Various parameters were determined such as PB cell counting or, for BM samples, myelogram, in vitro colony-forming cells and phenotypic flow cytometry analysis. On post-soman day 1, a significant decrease in numbers of white blood cells and an increase in erythrocyte and platelet counts were noted. On post-soman day 4, the number of HPC decreased significantly, probably due to reduction of the replication rate of these immature cells. However, the number of more immature cells (Sca1+/Lin- phenotype) remained unchanged. On post-soman day 8 and day 22, the number of monocytes and granulocytes in the blood had considerably increased, probably due to a strong inflammatory reaction in response to soman poisoning. In conclusion, PB cell and BM-derived HPC populations are affected by acute soman poisoning, suggesting particular care, mainly for graft kinetic aspects, during future development of autologous BM stem cell therapy strategy to treat nerve agent-induced brain damage.

Single administration of stem cell factor, FLT-3 ligand, megakaryocyte growth and development factor, and interleukin-3 in combination soon after irradiation prevents nonhuman primates from myelosuppression: long-term follow-up of hematopoiesis.

Preservation of hematopoietic stem and progenitor cell survival is required for recovery from radiation-induced myelosuppression. We recently showed that short-term injection of antiapoptotic cytokine combinations into mice soon after lethal gamma irradiation promoted survival. The present study investigated the hematopoietic response of cynomolgus monkeys to a single dose of stem cell factor, FLT-3 ligand, megakaryocyte growth and development factor, and interleukin-3 in combination (4F, each factor given intravenously at 50 microg/kg) administered 2 hours after 5-Gy gamma irradiation. Treated monkeys (n = 4) experienced no thrombocytopenia. Only 1 in 4 displayed a transient period of neutropenia (neutrophil [ANC] count < 0.5 x 10(9)/L), whereas all irradiated controls (n = 4) experienced neutropenia (5-12 days) and thrombocytopenia (platelet [PLT] count < 20 x 10(9)/L, 5-31 days). Treated animals exhibited an impressive 2-wave PLT response that peaked at days 8 and 22 after total body irradiation (TBI). Areas under the curve (AUC) of PLTs, ANCs, white blood cells (WBCs), and red blood cells (RBCs) between days 0 and 90 were significantly higher in treated animals than in controls. Humeral bone marrow-derived clonogenic activity was significantly spared at 24 hours and 4 days after TBI in treated monkeys. No apparent impairment of the hematopoietic status and stem cell pool, in terms of long-term culture-initiating cells (LTC-ICs) and side population (SP) cells, was observed after 15 months. These results strongly suggest that the 4F cytokine combination, as a single dose regimen, could act as an emergency treatment for nuclear accident or terrorism victims.