Early to sustained impacts of lethal radiation on circulating miRNAs in a minipig model.

Early diagnosis of lethal radiation is imperative since its intervention time windows are considerably short. Hence, ideal diagnostic candidates of radiation should be easily accessible, enable to inform about the stress history and objectively triage subjects in a time-efficient manner. Therefore, the small molecules such as metabolites and microRNAs (miRNAs) from plasma are legitimate biomarker candidate for lethal radiation. Our objectives were to comprehend the radiation-driven molecular pathogenesis and thereby determine biomarkers of translational potential. We investigated an established minipig model of LD70/45 total body irradiation (TBI). In this pilot study, plasma was collected pre-TBI and at multiple time points post-TBI. The majority of differentially expressed miRNAs and metabolites were perturbed immediately after TBI that potentially underlined the severity of its acute impact. The integrative network analysis of miRNA and metabolites showed a cohesive response; the early and consistent perturbations of networks were linked to cancer and the shift in musculoskeletal atrophy synchronized with the comorbidity-networks associated with inflammation and bioenergy synthesis. Subsequent comparative pipeline delivered 92 miRNAs, which demonstrated sequential homology between human and minipig, and potentially similar responses to lethal radiation across these two species. This panel promised to retrospectively inform the time since the radiation occurred; thereby could facilitate knowledge-driven interventions.

Mitigation of total body irradiation-induced mortality and hematopoietic injury of mice by a thrombopoietin mimetic (JNJ-26366821).

The threat of a nuclear attack has increased in recent years highlighting the benefit of developing additional therapies for the treatment of victims suffering from Acute Radiation Syndrome (ARS). In this work, we evaluated the impact of a PEGylated thrombopoietin mimetic peptide, JNJ-26366821, on the mortality and hematopoietic effects associated with ARS in mice exposed to lethal doses of total body irradiation (TBI). JNJ-26366821 was efficacious as a mitigator of mortality and thrombocytopenia associated with ARS in both CD2F1 and C57BL/6 mice exposed to TBI from a cobalt-60 gamma-ray source. Single administration of doses ranging from 0.3 to 1 mg/kg, given 4, 8, 12 or 24 h post-TBI (LD70 dose) increased survival by 30-90% as compared to saline control treatment. At the conclusion of the 30-day study, significant increases in bone marrow colony forming units and megakaryocytes were observed in animals administered JNJ-26366821 compared to those administered saline. In addition, enhanced recovery of FLT3-L levels was observed in JNJ-26366821-treated animals. Probit analysis of survival in the JNJ-26366821- and saline-treated cohorts revealed a dose reduction factor of 1.113 and significant increases in survival for up to 6 months following irradiation. These results support the potential use of JNJ-26366821 as a medical countermeasure for treatment of acute TBI exposure in case of a radiological/nuclear event when administered from 4 to 24 h post-TBI.

Whole blood gene expression within days after total-body irradiation predicts long term survival in Gottingen minipigs.

Gottingen minipigs mirror the physiological radiation response observed in humans and hence make an ideal candidate model for studying radiation biodosimetry for both limited-sized and mass casualty incidents. We examined the whole blood gene expression profiles starting one day after total-body irradiation with increasing doses of gamma-rays. The minipigs were monitored for up to 45 days or time to euthanasia necessitated by radiation effects. We successfully identified dose- and time-agnostic (over a 1-7 day period after radiation), survival-predictive gene expression signatures derived using machine-learning algorithms with high sensitivity and specificity. These survival-predictive signatures fare better than an optimally performing dose-differentiating signature or blood cellular profiles. These findings suggest that prediction of survival is a much more useful parameter for making triage, resource-utilization and treatment decisions in a resource-constrained environment compared to predictions of total dose received. It should hopefully be possible to build such classifiers for humans in the future.

Investigating the Multifaceted Nature of Radiation-Induced Coagulopathies in a Göttingen Minipig Model of Hematopoietic Acute Radiation Syndrome.

Coagulopathies are well documented after acute radiation exposure at hematopoietic doses, and radiation-induced bleeding is notably one of the two main causes of mortality in the hematopoietic acute radiation syndrome. Despite this, understanding of the mechanisms by which radiation alters hemostasis and induces bleeding is still lacking. Here, male Göttingen minipigs received hematopoietic doses of 60Co gamma irradiation (total body) and coagulopathies were characterized by assessing bleeding, blood cytopenia, fibrin deposition, changes in hemostatic properties, coagulant/anticoagulant enzyme levels, and markers of inflammation, endothelial dysfunction, and barrier integrity to understand if a relationship exists between bleeding, hemostatic defects, bone marrow aplasia, inflammation, endothelial dysfunction and loss of barrier integrity. Acute radiation exposure induced coagulopathies in the Göttingen minipig model of hematopoietic acute radiation syndrome; instances of bleeding were not dependent upon thrombocytopenia. Neutropenia, alterations in hemostatic parameters and damage to the glycocalyx occurred in all animals irrespective of occurrence of bleeding. Radiation-induced bleeding was concurrent with simultaneous thrombocytopenia, anemia, neutropenia, inflammation, increased heart rate, decreased nitric oxide bioavailability and endothelial dysfunction; bleeding was not observed with the sole occurrence of a single aforementioned parameter in the absence of the others. Alteration of barrier function or clotting proteins was not observed in all cases of bleeding. Additionally, fibrin deposition was observed in the heart and lungs of decedent animals but no evidence of DIC was noted, suggesting a unique pathophysiology of radiation-induced coagulopathies. These findings suggest radiation-induced coagulopathies are the result of simultaneous damage to several key organs and biological functions, including the immune system, the inflammatory response, the bone marrow and the cardiovasculature.

Late Health Effects of Partial Body Irradiation Injury in a Minipig Model Are Associated with Changes in Systemic and Cardiac IGF-1 Signaling.

Clinical, epidemiological, and experimental evidence demonstrate non-cancer, cardiovascular, and endocrine effects of ionizing radiation exposure including growth hormone deficiency, obesity, metabolic syndrome, diabetes, and hyperinsulinemia. Insulin-like growth factor-1 (IGF-1) signaling perturbations are implicated in development of cardiovascular disease and metabolic syndrome. The minipig is an emerging model for studying radiation effects given its high analogy to human anatomy and physiology. Here we use a minipig model to study late health effects of radiation by exposing male Göttingen minipigs to 1.9-2.0 Gy X-rays (lower limb tibias spared). Animals were monitored for 120 days following irradiation and blood counts, body weight, heart rate, clinical chemistry parameters, and circulating biomarkers were assessed longitudinally. Collagen deposition, histolopathology, IGF-1 signaling, and mRNA sequencing were evaluated in tissues. Our findings indicate a single exposure induced histopathological changes, attenuated circulating IGF-1, and disrupted cardiac IGF-1 signaling. Electrolytes, lipid profiles, liver and kidney markers, and heart rate and rhythm were also affected. In the heart, collagen deposition was significantly increased and transforming growth factor beta-1 (TGF-beta-1) was induced following irradiation; collagen deposition and fibrosis were also observed in the kidney of irradiated animals. Our findings show Göttingen minipigs are a suitable large animal model to study long-term effects of radiation exposure and radiation-induced inhibition of IGF-1 signaling may play a role in development of late organ injuries.

Gene Expression Profiles from Heart, Lung and Liver Samples of Total-Body-Irradiated Minipigs: Implications for Predicting Radiation-Induced Tissue Toxicity.

In the event of a major accidental or intentional radiation exposure incident, the affected population could suffer from total- or partial-body exposures to ionizing radiation with acute exposure to organs that would produce life-threatening injury. Therefore, it is necessary to identify markers capable of predicting organ-specific damage so that appropriate directed or encompassing therapies can be applied. In the current work, gene expression changes in response to total-body irradiation (TBI) were identified in heart, lungs and liver tissue of Göttingen minipigs. Animals received 1.7, 1.9, 2.1 or 2.3 Gy TBI and were followed for 45 days. Organ samples were collected at the end of day 45 or sooner if the animal displayed morbidity necessitating euthanasia. Our findings indicate that different organs respond to TBI in a very specific and distinct manner. We also found that the liver was the most affected organ in terms of gene expression changes, and that lipid metabolic pathways were the most deregulated in the liver samples of non-survivors (survival time <45 days). We identified organ-specific gene expression signatures that accurately differentiated non-survivors from survivors and control animals, irrespective of dose and time postirradiation. At what point did these radiation-induced injury markers manifest and how this information could be used for applying intervention therapies are under investigation.

Morphological and functional impairment in the gut in a partial body irradiation minipig model of GI-ARS.

Purpose: Göttingen minipig (G-MP) displays classic gastrointestinal acute radiation syndrome (GI-ARS) following total body irradiation (TBI) at GI doses which are lethal by 10-14 days. In collaboration with BARDA, we are developing a hemi-body/partial body irradiation (PBI) model by exposing only the abdomen and lower extremities to study GI structure/function impairment, natural history of injury and recovery, as well as correlative biomarkers out to 30 days.Materials and methods: Twenty-four G-MP were exposed to either 12 or 16 Gy (LINAC Elekta); head, forelimbs, and thorax were outside the irradiation field, sparing ∼50% of the bone marrow. Animals were followed for 30 days with euthanasia scheduled at pre-set intervals to study the time course of GI injury and recovery. Hematological profiles, clinical symptoms, gross- and histo-pathology including markers of proliferation and apoptosis in the small intestines, gut function parameters (food tolerance, digestion, absorption, citrulline production), and levels of two biomarkers, CRP and IGF-1, were evaluated.Results: PBI at 16 Gy yielded higher lethality than 12 Gy. Unlike TBI, PBI did not cause severe pancytopenia or external hemorrhage, as expected, and allowed to focus the injury on GI organs while sparing the radiation sensitive heart and lung. Compromised animals showed inactivity, anorexia, vomiting, diarrhea, and weight loss. Histology revealed that in 12 Gy irradiated animals, lesions recovered overtime. In 16 Gy irradiated animals, lesions were more pronounced and persistent. BrdU and Ki67 labelling demonstrated dose-dependent loss of crypts and subsequent mucosal ulceration which recovered over time. Minimal apoptosis was observed at both doses. Reductions in food tolerance, digestion, absorption, and citrulline production were time and dose-dependent. Loss of citrulline reached a nadir between 6-12 days and then recovered partially. CRP and IGF-1 were upregulated following PBI at GI doses.Conclusions: This lower hemi-body irradiation model allowed for extended survival at GI-specific ARS doses and development of a well-controlled GI syndrome with minimal hematopoietic injury or confounding mortality from cardiopulmonary damage. A dose-dependent impairment in the intestinal structure resulted in overall decreased gut functionality followed by a partial recovery. However, while the structure appeared to be recovered, not all functionality was attained. PBI induced systemic inflammation and altered the IGF-1 hormone indicating that these can be used as biomarkers in the minipig even under partial body conditions. This PBI model aligns with other minipig models under BARDA's large animal consortium to test medical countermeasure efficacy against a less complex GI-specific ARS injury.

NPC1161B, an 8-Aminoquinoline Analog, Is Metabolized in the Mosquito and Inhibits Plasmodium falciparum Oocyst Maturation.

Malaria is a major global health threat, with nearly half the world's population at risk of infection. Given the recently described delayed clearance of parasites by artemisinin-combined therapies, new antimalarials are needed to facilitate the global effort toward elimination and eradication. NPC1161 is an 8-aminoquinoline that is derived from primaquine with an improved therapeutic profile compared to the parent compound. The (R)-(-) enantiomer (NPC1161B) has a lower effective dose that results in decreased toxic side effects such as hemolysis compared to the (S)-(+)-enantiomer, making it a promising compound for consideration for clinical development. We explored the effect of NPC1161B on Plasmodium falciparum oocyst and sporozoite development to evaluate its potential transmission-blocking activity viz. its ability to cure mosquitoes of an ongoing infection. When mosquitoes were fed NPC1161B 4 days after P. falciparum infection, we observed that total oocyst numbers were not affected by NPC1161B treatment. However, the sporozoite production capacity of the oocysts was impaired, and salivary gland sporozoite infections were completely blocked, rendering the mosquitoes non-infectious. Importantly, NPC1161B did not require prior liver metabolism for its efficacy as is required in mammalian systems, suggesting that an alternative metabolite is produced in the mosquito that is active against the parasite. We performed liquid chromatography-mass spectrometry (LC-MS)/MS analysis of methanol extracts from the midguts of mosquitoes fed on an NPC1161B (434.15 m/z)-treated blood meal and identified a compound with a mass of 520.2 m/z, likely a conjugate of NPC1161B or an oxidized metabolite. These findings establish NPC1161B, and potentially its metabolites, as transmission-blocking candidates for the treatment of P. falciparum.

Protein O-Fucosyltransferase 2 Is Not Essential for Plasmodium berghei Development.

Thrombospondin type I repeat (TSR) domains are commonly O-fucosylated by protein O-fucosyltransferase 2 (PoFUT2), and this modification is required for optimal folding and secretion of TSR-containing proteins. The human malaria parasite Plasmodium falciparum expresses proteins containing TSR domains, such as the thrombospondin-related anonymous protein (TRAP) and circumsporozoite surface protein (CSP), which are O-fucosylated. TRAP and CSP are present on the surface of sporozoites and play essential roles in mosquito and human host invasion processes during the transmission stages. Here, we have generated PoFUT2 null-mutant P. falciparum and Plasmodium berghei (rodent) malaria parasites and, by phenotyping them throughout their complete life cycle, we show that PoFUT2 disruption does not affect the growth through the mosquito stages for both species. However, contrary to what has been described previously by others, P. berghei PoFUT2 null mutant sporozoites showed no deleterious motility phenotypes and successfully established blood stage infection in mice. This unexpected result indicates that the importance of O-fucosylation of TSR domains may differ between human and RODENT malaria parasites; complicating our understanding of glycosylation modifications in malaria biology.

Ribosomal/nucleolar stress induction regulates tert-Butyl hydroperoxide (tBHP) mediated oxidative stress in Anopheles gambiae midguts.

OBJECTIVE: A fundamental understanding of redox homeostasis in Anopheles gambiae midgut cells under different oxidative conditions is missing. Such knowledge can aid in the development of new malaria transmission-blocking strategies aimed at disrupting natural homeostatic processes in the mosquito during Plasmodium parasite uptake (i.e. blood feeding). The aim of this study was to understand how the An. gambiae midgut regulates oxidative stress to reactive oxygen species (ROS), especially to a potent ROS-inducer such as tert-Butyl hydroperoxide (tBHP). RESULTS: Initial studies using quantitative immunoblot indicated that the expression of the classical antioxidant protein An. gambiae thioredoxin-1 (AgTrx-1) remained unchanged across challenges with different concentrations of tBHP suggesting that additional mechanisms to regulate ROS may be involved. We therefore conducted a global proteomic survey, which revealed that An. gambiae midguts under low (50 µM) and high (200 µM) tBHP concentrations were enriched in proteins indicative of ribosomal/nucleolar stress. Ribosomal stress is an inherent cellular response to an imbalance in ribosomal proteins (RPs) due to cellular stress such as oxidative stress. Our data suggest that ribosomal/nucleolar stress is the primary cellular response in An. gambiae midguts under tBHP challenge. Considering these results, we discuss harnessing the ribosomal stress response as a potential malaria transmission-blocking strategy.

Neulasta Regimen for the Hematopoietic Acute Radiation Syndrome: Effects Beyond Neutrophil Recovery.

PURPOSE: Understanding the physiopathology underlying the acute radiation syndrome (ARS) and the mechanism of action of drugs known to ameliorate ARS is expected to help identify novel countermeasure candidates and improve the outcome for victims exposed to radiation. Granulocyte colony-stimulating factor (G-CSF) has been approved by the US Food and Drug Administration for treatment of hematopoietic ARS (H-ARS) because of its ability to alleviate myelosuppression. Besides its role in hematopoiesis, G-CSF is known to protect the cardiovascular and neurologic systems, to attenuate vascular injury and cardiac toxicity, to preserve gap junction function, and to modulate inflammation and oxidative stress. Here, we characterized the protective effects of G-CSF beyond neutrophil recovery in minipigs exposed to H-ARS doses. METHODS AND MATERIALS: Twenty male Göttingen minipigs were exposed to total body, acute ionizing radiation. Animals received either pegylated G-CSF (Neulasta) or dextrose at days 1 and 8 after irradiation. Survival was monitored over a 45-day period. RESULTS: Neulasta decreased mortality compared with the control, reduced nadir and duration of neutropenia, and lowered prevalence of organ hemorrhage and frank bleeding episodes. Neulasta also increased plasma concentration of IGF-1 hormone, activated the cardiovascular protective IGF-1R/PI3K/Akt/eNOS/NO pathway, and enhanced membrane expression of VE-cadherin in the heart, improving vascular tone and barrier function. Expression of the acute phase protein CRP, a mediator of cardiovascular diseases and a negative regulator of the IGF-1 pathway, was also induced but at much lower extent compared with IGF-1. Activity of catalase and superoxide dismutase (SOD-1) was only marginally affected, whereas activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was downregulated. CONCLUSIONS: In addition to a neutrophilic effect, amelioration of endothelial homeostasis and barrier function and reduction in NADPH oxidase contribute to the beneficial effects of Neulasta for the treatment of H-ARS.

Selective Insulin-like Growth Factor Resistance Associated with Heart Hemorrhages and Poor Prognosis in a Novel Preclinical Model of the Hematopoietic Acute Radiation Syndrome.

Although bone marrow aplasia has been considered for the past decades as the major contributor of radiation-induced blood disorders, cytopenias alone are insufficient to explain differences in the prevalence of bleeding. In this study, the minipig was used as a novel preclinical model of hematopoietic acute radiation syndrome to assess if factors other than platelet counts correlated with bleeding and survival. We sought to determine whether radiation affected the insulin-like growth factor-1 (IGF-1) pathway, a growth hormone with cardiovascular and radioprotective features. Gottingen and Sinclair minipigs were exposed to ionizing radiation at hematopoietic doses. The smaller Gottingen minipig strain was more sensitive to radiation; differences in IGF-1 levels were minimal, suggesting that increased sensitivity could depend on weak response to the hormone. Radiation caused IGF-1 selective resistance by inhibiting the anti-inflammatory anti-oxidative stress IRS/PI3K/Akt but not the pro-inflammatory MAPK kinase pathway, shifting IGF-1 signaling towards a pro-oxidant, pro-inflammatory environment. Selective IGF-1 resistance associated with hemorrhages in the heart, poor prognosis, increase in C-reactive protein and NADPH oxidase 2, uncoupling of endothelial nitric oxide synthase, inhibition of nitric oxide (NO) synthesis and imbalance between the vasodilator NO and the vasoconstrictor endothelin-1 molecules. Selective IGF-1 resistance is a novel mechanism of radiation injury, associated with a vicious cycle amplifying reactive oxygen species-induced damage, inflammation and endothelial dysfunction. In the presence of thrombocytopenia, selective inhibition of IGF-1 cardioprotective function may contribute to the development of hemostatic disorders. This finding may be particularly relevant for individuals with low IGF-1 activity, such as the elderly or those with cardiometabolic dysfunctions.