Comparison of established and emerging biodosimetry assays

Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3-0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5-4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools

Characterization of lethal inhalational infection with Francisella tularensis in the common marmoset (Callithrix jacchus)

The intracellular Gram-negative pathogen Francisella tularensis is the causative agent of tularaemia and is prevalent in many countries in the northern hemisphere. To determine whether the common marmoset (Callithrix jacchus) would be a suitable non-human primate model of inhalational tularaemia, a pathophysiology study was undertaken. Ten animals were challenged with ∼102 c.f.u. F. tularensis strain SCHU S4 (F. tularensis subsp. tularensis). To look for trends in the infection, pairs of animals were sacrificed at 24 h intervals between 0 and 96 h post-challenge and blood and organs were assessed for bacteriology, pathology and haematological and immunological parameters. The first indication of infection was a raised core temperature at 3 days post-challenge. This coincided with a number of other factors: a rapid increase in the number of bacteria isolated from all organs, more pronounced gross pathology and histopathology, and an increase in the immunological response. As the disease progressed, higher bacterial and cytokine levels were detected. More extensive pathology was observed, with multifocal lesions seen in the lungs, liver and spleen. Disease progression in the common marmoset appears to be consistent with human clinical and pathological features of tularaemia, indicating that this may be a suitable animal model for the investigation of novel medical interventions such as vaccines or therapeutics

Hematological Changes as Prognostic Indicators of Survival: Similarities Between Gottingen Minipigs, Humans, and Other Large Animal Models

The animal efficacy rule addressing development of drugs for selected disease categories has pointed out the need to develop alternative large animal models. Based on this rule, the pathophysiology of the disease in the animal model must be well characterized and must reflect that in humans. So far, manifestations of the acute radiation syndrome (ARS) have been extensively studied only in two large animal models, the non-human primate (NHP) and the canine. We are evaluating the suitability of the minipig as an additional large animal model for development of radiation countermeasures. We have previously shown that the Gottingen minipig manifests hematopoietic ARS phases and symptoms similar to those observed in canines, NHPs, and humans.We establish here the LD50/30 dose (radiation dose at which 50% of the animals succumb within 30 days), and show that at this dose the time of nadir and the duration of cytopenia resemble those observed for NHP and canines, and mimic closely the kinetics of blood cell depletion and recovery in human patients with reversible hematopoietic damage (H3 category, METREPOL approach). No signs of GI damage in terms of diarrhea or shortening of villi were observed at doses up to 1.9 Gy. Platelet counts at days 10 and 14, number of days to reach critical platelet values, duration of thrombocytopenia, neutrophil stress response at 3 hours and count at 14 days, and CRP-to-platelet ratio were correlated with survival. The ratios between neutrophils, lymphocytes and platelets were significantly correlated with exposure to irradiation at different time intervals.As a non-rodent animal model, the minipig offers a useful alternative to NHP and canines, with attractive features including ARS resembling human ARS, cost, and regulatory acceptability. Use of the minipig may allow accelerated development of radiation countermeasures

Identification of Hub Genes Related to the Recovery Phase of Irradiation Injury by Microarray and Integrated Gene Network Analysis

BACKGROUND: Irradiation commonly causes long-term bone marrow injury charactertized by defective HSC self-renewal and a decrease in HSC reserve. However, the effect of high-dose IR on global gene expression during bone marrow recovery remains unknown. METHODOLOGY: Microarray analysis was used to identify differentially expressed genes that are likely to be critical for bone marrow recovery. Multiple bioinformatics analyses were conducted to identify key hub genes, pathways and biological processes. PRINCIPAL FINDINGS: 1) We identified 1302 differentially expressed genes in murine bone marrow at 3, 7, 11 and 21 days after irradiation. Eleven of these genes are known to be HSC self-renewal associated genes, including Adipoq, Ccl3, Ccnd1, Ccnd2, Cdkn1a, Cxcl12, Junb, Pten, Tal1, Thy1 and Tnf; 2) These 1302 differentially expressed genes function in multiple biological processes of immunity, including hematopoiesis and response to stimuli, and cellular processes including cell proliferation, differentiation, adhesion and signaling; 3) Dynamic Gene Network analysis identified a subgroup of 25 core genes that participate in immune response, regulation of transcription and nucleosome assembly; 4) A comparison of our data with known irradiation-related genes extracted from literature showed 42 genes that matched the results of our microarray analysis, thus demonstrated consistency between studies; 5) Protein-protein interaction network and pathway analyses indicated several essential protein-protein interactions and signaling pathways, including focal adhesion and several immune-related signaling pathways. CONCLUSIONS: Comparisons to other gene array datasets indicate that global gene expression profiles of irradiation damaged bone marrow show significant differences between injury and recovery phases. Our data suggest that immune response (including hematopoiesis) can be considered as a critical biological process in bone marrow recovery. Several critical hub genes that are key members of significant pathways or gene networks were identified by our comprehensive analysis

Cytokines to treat casualties involved in nuclear & radiological events: gold standard and new perspectives

Counteracting the hematopoietic syndrome following accidental or intentional irradiation remains an important therapeutic challenge. However, the pathophysiology of accidental irradiation has been recently revisited. Indeed irradiation has to be considered as a global illness that is more complex than the juxtaposition of single syndromes. This was especially illustrated in the Tokaï-Mura case where the two highly irradiated victims died from a complex multiorgan distress then failure syndrome (MODS/MOFS) in spite of transient hematopoietic chimerism following hematopoietic stem cell transplantation. Radiation burns were especially involved in this MOF development. In fact, the pathophysiology of MODS strongly overlaps the systemic inflammatory response syndrome that is mainly the consequence of extensive endothelial cell damage. Clearly, there is a need to develop new therapeutic strategies to counter such radiation-induced extrahematological toxicity, in addition to correcting hematopoietic disorders. Regarding hematopoiesis, the rationale of cytokine/hematopoietic growth factor use is the heterogeneity of bone marrow damage in most documented accidents. Today, early administration of granulopoietic factors is recommended to stimulate residual hematopoiesis in victims irradiated at intermediate dose levels. In complement our group proposes the emergency antiapoptotic cytokine (EACK) therapy in case of high dose irradiation, which consists in preservating and stimulating residual hematopoietic stem and progenitor cells following irradiation. We selected the stem cell factor + Flt-3 ligand + thrombopoietin + interleukin-3 + PegG-CSF antiapoptotic combination that is capable of abrogating thrombocytopenia and reducing neutropenia when given as an early single administration in highly irradiated monkeys (7 Gy gamma) 2 hrs after total body irradiation. Regarding extrahematological toxicity, injection of Keratinocyte growth factor or erythropoietin could be a flexible cytokine complementary/alternative approach to cell therapy (i.e. mesenchymal stem cells grafting). We recently re-evaluated the benefit of using erythropoietin (Epo) as a pleiotropic cytokine to counteract hematologic and extra-hematologic toxicity following lethal irradiation. B6D2F1 mice were globally exposed to 9 Gy gamma (LD90%/30days) and then injected with SFT3 at 2 hours + 24 hours with or without Epo (1000-3000 UI/kg) at 2 hours + 8 days. Epo synergized with SFT3 to rescue lethally irradiated mice from radiation-induced death (60%, 95% and 5% respectively for SFT3, SFT3+Epo and controls at 30 days) whereas Epo alone exhibited no protective effect. Interestingly, hematopoietic parameters did not significantly differ between SFT3 and SFT3+Epo groups. This suggests predominant extra-hematological targets for Epo. Ongoing studies aim at improving EACK strategy in terms of tolerance and efficacy